Registration Dossier

Administrative data

Description of key information

skin sensitisation (OECD 429): sensitising (EC3 value = 2.7%)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
18 Aug - 23 Dec 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
other: CBA/Jcr
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 15.7 - 25.8 g
- Housing: 1 to 5 animals per cage in polycarbonate boxes with bedding
- Diet: Formulab #5008 (PMI Feeds Inc.), ad libitum
- Water: municipal water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 23
- Humidity (%): 27 - 98
- Air changes (per hr): minimum 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Experiment 1
Test substance: 25, 50 and 100%
Positive control: 100%

Experiment 2
Test substance: 2.5, 5, 10 and 25%
No. of animals per dose:
5
Details on study design:
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: A stimulation index (SI) was calculated for each group using the activity of each test group divided by the activity of the vehicle control group. The criterion for a positve response is that at least one concentration of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance on Day 1. The application was repeated on Day 2 and 3. Three days after the third application on Day 6 an injection of 250 µl phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (³HTdR) was made into the tail vein of each experimental mouse. Approximately five hours later, following injection of ³HTdR, the mice were sacrificed and draining auricular lymph nodes were excised and pooled for each individual animal. A single cell suspension was prepared by gentle separation through a 200 mesh stainless steel gauze. The cell suspensions were washed two times with an excess of PBS and precipitated with 5% trichloroacetic acid at 4 °C for 18 h. The pellets were resuspended in 1 mL of trichloroacetic acid and transferred to 10 mL of scintillation fluid.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A one-way parametric analysis of variance (ANOVA) with Dunnett's Multiple Comparisons Test, using GraphPad InStat version 3.06, was performed in DPM counts.
If test groups showed a SI >3, then an extrapolated EC3 value was calculated from SI values at low% and either mid% or high% concentrations.
If at least one concentration shows SI < 3, then the formula is: EC3 = [(3-d)/(b-d)] x (a-c) + c
Positive control results:
The SI value calculated for the positive control was 11.0.
Key result
Parameter:
EC3
Value:
2.7
Parameter:
SI
Value:
10.3
Test group / Remarks:
100% test substance
Remarks on result:
other: experiment 1
Parameter:
SI
Value:
6.9
Test group / Remarks:
50% test substance
Remarks on result:
other: experiment 1
Parameter:
SI
Value:
5.7
Test group / Remarks:
25% test substance
Remarks on result:
other: experiment 1
Parameter:
SI
Value:
6.2
Test group / Remarks:
25% test substance
Remarks on result:
other: experiment 2
Parameter:
SI
Value:
4.7
Test group / Remarks:
10% test substance
Remarks on result:
other: experiment 2
Parameter:
SI
Value:
3.9
Test group / Remarks:
5% test substance
Remarks on result:
other: experiment 2
Parameter:
SI
Value:
2.9
Test group / Remarks:
2.5% test substance
Remarks on result:
other: experiment 2
Cellular proliferation data / Observations:
EC3 CALCULATION
The EC3 value was calculated based on the results from Experiment 2 using the SI values at 2.5 and 5% test substance concentration. The EC3 value was calculated to be 2.7%.

CLINICAL OBSERVATIONS
All animals appeared normal for the duration of the study.

BODY WEIGHTS
Two to three animals each of the treatment groups in Experiment 2 lost or failed to gain weight during the study.

Table 1: Body weights and DPM counts.

 

Animal

Body weight (g)

DPM count

Mean DPM ± SD

Day 1

Day 6

Experiment 1

Vehicle Control Group

1

15.7

22.5

128

616 ± 327

2

17.2

23.8

500

3

15.7

22.5

702

4

16.0

24.8

743

5

16.4

23.9

1006

Test Group I - 25%

1

21.5

21.9

4715

3846 ± 978

2

22.7

22.8

3820

3

24.8

24.9

2477

4

23.7

24.3

3382

5

22.9

23.6

4837

Test Group II - 50%

1

21.8

22.6

5357

4271 ± 1087

2

21.8

22.6

2871

3

22.5

23.7

3448

4

24.0

24.9

4474

5

22.5

23.1

5205

Test Group III - 100%

1

21.4

23.3

7715

6322 ± 1932

2

23.5

23.7

6937

3

23.7

23.8

4944

4

21.3

21.8

3713

5

22.2

22.7

8299

Positive Control Group

1

21.8

22.6

3734

6767 ± 4685

2

21.0

22.1

6230

3

23.6

24.4

3838

4

23.3

23.9

5086

5

21.4

22.4

14947

Experiment 2

Vehicle Control Group

1

19.7

16.2

254

639 ± 416

2

19.1

18.9

351

3

23.9

24.3

511

4

22.9

23.3

791

5

23.4

24.4

1288

Test Group IV – 2.5%

1

24.1

23.2

1968

1863 ± 292

2

22.7

22.7

2283

3

24.0

24.9

1520

4

23.8

24.1

1870

5

22.2

22.4

1673

Test Group V - 5%

1

22.9

23.4

1995

2502 ± 641

2

23.9

22.5

2935

3

23.1

22.7

1707

4

24.2

24.5

3245

5

23.1

22.2

2630

Test Group VI - 10%

1

25.1

23.7

2294

3019 ± 799

2

22.7

23.7

2805

3

24.4

24.0

2305

4

24.2

23.1

3611

5

23.7

21.3

4079

Test Group VII – 25%

 1

20.8

21.0

3318

3635 ± 1169

 2

24.9

23.0

5577

 3

25.8

24.7

3067

 4

22.5

22.3

3701

 5

24.0

24.4

2510

Statistics

The treatment groups I - III (25, 50 and 100%) and IV – VII (2.5, 5, 10 and 25%) were compared to the vehicle control group of Experiment 1 and 2, respectively. In Experiment 1, statistically significant mean DPM counts (P < 0.01, ANOVA) were observed at 100% test substance concentration and for the positive control group. In Experiment 2, statistically significant mean DPM counts were observed at 5, 10 and 25% test substance concentration.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the test the test substance revealed sensitising properties. The EC3 value was calculated to be 2.7%.
Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
23 Mar - 22 Jun 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
adopted 4 February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Type of study:
direct peptide binding assay
Test material information:
Composition 1
Details on study design:
TEST SYSTEM
- Source: GenScript, Piscataway, US and RS Synthesis, Louisville, US
- Specification of the peptides:
Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)

TEST METHOD
The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 h incubation with the test substance at 25 ± 2.5 °C in the dark. The synthetic peptides contain phenylalanine to aid in the detection and either cysteine or lysine as the reactive center. 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 a prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

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

VEHICLE CONTROL
- Substance: acetonitrile

POSITIVE CONTROL
- Substance: ethylene glycol dimethacrylate
- Concentration: 50 mM in acetonitrile

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM preparation in acetonitrile. The solvent was chosen because the test substance was soluble in the vehicle.

VEHICLE CONTROL PREPARATION
Several acetonitrile controls were prepared in triplicates in the same way as the test substance samples but with acetonitrile instead of the test substance. Set A was analysed together with the calibration samples without incubation and serves as a performance control. Another three sets (two sets B and set C) were prepared and incubated with the samples. Sets B were placed at the very start and ending of the sample list and serve as stability control of the peptide over the analysis time. Set C was analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in acetonitrile.

INCUBATION CONDITIONS
- Peptide ratios: C-containing peptide: 1:10 (0.5 mM peptide, 5 mM test substance); K-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance)
- Temperature used during treatment / exposure: 25 ± 2.5 °C
- Duration of treatment / exposure: 24 ± 2 h

NUMBER OF REPLICATES
for each peptide in triplicates for test substance and controls

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Agilent HP 1100 with DAD (Software: Dionex Chromeleon)
- Column: Phenomenex Luna 3µ C18 (2), 100 mm x 2 mm with guard column „Security Guard“ C18, 4 mm x 2 mm
- Analytical balance: Accuracy 0.1 mg
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid (99+%) in de-ionized water, HPLC grade
B: 0.085% (v/v) trifluoracetic acid (99+%) in acetonitrile, HPLC grade
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 25
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 and 258 nm
- Injection volume: 2 µL
Key result
Parameter:
other: % depletion of cysteine-containing peptide
Remarks:
mean value of 3 replicates
Run / experiment:
24 h incubation
Value:
7.13
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Parameter:
other: % depletion of lysine-containing peptide
Remarks:
mean value of 3 replicates
Run / experiment:
24 h incubation
Value:
-0.76
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Other effects / acceptance of results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance was soluble in acetonitrile. The samples of the test substance with the peptides were homogeneous emulsions immediately after preparation. After 24 hours precipitates were noticed in the samples of the C-containing peptide. The samples of the K-containing peptide were visually homogeneous emulsions after 24 hours.
- Other confounding effects: No co-elution of the test substance and peptides occurred.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The acceptance criteria were met with the exception of the vehicle control A of the K-containing peptide samples which is not available due to a technical error. However, the test is considered to be valid despite the lack of the performance control, as all other control samples (samples B and C) met the acceptance criteria.
- Acceptance criteria met for positive control: The positive control caused depletion of both peptides comparable to historic data.

Table 1. Depletion of c-containing peptide (%)

 

Sample 1

Sample 2

Sample 3

Mean ± SD

Vehicle control

-0.76

0.86

-0.10

0.00 ± 0.82

Positive control

49.34

55.36

60.46

55.05 ± 1.20

Test substance

8.10

7.50

5.79

7.13 ± 1.20

 

Table 2. Depletion of k-containing peptide (%)

 

Sample 1

Sample 2

Sample 3

Mean ± SD

Vehicle control

0.25

-0.20

-0.05

0.00 ± 0.23

Positive control

13.03

14.46

16.00

14.50 ± 1.48

Test substance

-1.45

-0.72

-0.11

-0.76 ± 0.67

Negative depletions were considered to be 0 for calculation of the mean peptide depletion, which was thus calculated to be 0.00%.

Interpretation of results:
other: inconclusive
Remarks:
according to OECD TG 442C
Conclusions:
Based on the observed results it was concluded that the test substance shows a minimal chemical reactivity in the Direct Peptide Reactivity Assay under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance the samples with both peptides were emulsions and that the result could therefore be under-predictive. According to OECD Guideline 442C a negative result should be considered inconclusive in this case.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Apr-Jun 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose:
reference to same study
Reference:
Composition 1
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 442E (In Vitro Skin Sensitisation: h-CLAT)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Type of study:
activation of dentritic cells
Test material information:
Composition 1
Details on study design:
THP-1 cells. The human monocytic leukemia cell line was obtained from “American Type Culture Collection, Manassas, USA” (ATCC, TIB-202).
Details on test animals and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.
Vehicle:
other: culture medium (RPMI 1640: with L-glutamine, 25mM HEPES (Gibco) + 10% FBS inactivated + 1% Penicillin/Streptomycin + 0.05 mM 2-Mercaptoethanol (Gibco))
Concentration / amount:
Pre-test (cytotoxicity): 0.5, 1.0, 5.0, 10, 51, 102, 511, 1021, 2043, and 5107 µg/mL
Main experiment 1: 65, 78, 93, 112, 134, 161, 193, 232 µg/mL
Main experiment 2: 18, 22, 26, 31, 37, 45, 54, 65 µg/mL
Main experiment 3: 26, 31, 37, 45, 54, 65, 78, 93 µg/mL
Vehicle:
other: culture medium (RPMI 1640: with L-glutamine, 25mM HEPES (Gibco) + 10% FBS inactivated + 1% Penicillin/Streptomycin + 0.05 mM 2-Mercaptoethanol (Gibco))
Concentration / amount:
Main experiment 1: 65, 78, 93, 112, 134, 161, 193, 232 µg/mL
Main experiment 2: 18, 22, 26, 31, 37, 45, 54, 65 µg/mL
Main experiment 3: 26, 31, 37, 45, 54, 65, 78, 93 µg/mL
No. of animals per dose:
Duplicates of each treatment
Details on study design:
SELECTION OF DOSES/CONCENTRATIONS FOR THE h-CLAT
- to determine the concentrations suitable for the main experiment a pre-test was performed.
- cells were exposed to 10 concentrations of the test-substance preparation and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA.
- CV75 value (= estimated concentration that affords 75% cell viability) of the test substance was determined by linear regression from the concentration response curve to be 2603 μg/mL.
- due to the extreme cytotoxicity observed at 5000 g/mL in the pre-test the highest tested concentration in the 1st main experiment was 1.2 fold of the CV75 value, only. The additional concentrations were obtained by a 1:1.2 serial dilution series of the maximum concentration.
- due to high cytotoxicity in the 1st experiment lower concentrations were chosen for the 2nd experiment. A dosage in between the 1.2-fold dilution step was conducted in order to better investigate the relevant field below cytotoxicity.

CONTROLS
- negative control (NC): lactic acid (1000 μg/mL)
- positive control (PC): 1- chloro-2,4-dinitrobenzene (4.0 μg/mL)
- vehicle control: culture medium (RPMI 1640: with L-glutamine, 25mM HEPES (Gibco) + 10% FBS inactivated + 1% Penicillin/Streptomycin + 0.05 mM 2-Mercaptoethanol (Gibco)
- isotype control: in order to help distinguish non-specific (“background”) staining from specific antibody staining each test-substance concentration and control is additionally incubated with mouse IgG1.

TEST SUBSTANCE PREPARATION
- test substance preparations were prepared within 4 hours of application
- test substance was weighed and topped up with the vehicle to achieve the required 2x concentration of the highest concentration (stock solution, produced in glass vials).
- further concentrations were prepared as 2x concentrations by serial 1:1.2 dilution according to the planned doses in culture medium (RPMI 1640: with L-glutamine, 25mM HEPES (Gibco) + 10% FBS inactivated + 1% Penicillin/Streptomycin + 0.05 mM 2-Mercaptoethanol (Gibco)).

- test-substance preparation was performed on a weight per volume basis by stirring.
- visual inspection of each dilution step was performed
- verification of the stability of the test substance in the vehicle was not required.

EXPERIMENTAL PROCEDURE
1) Preparation of the cells:
- THP-1 cells from the working cell bank were thawed and cultured in suspension using complete RPMI 1640 medium supplemented with 10% fetal bovine serum (heat inactivated), 100 U/mL penicillin, 100 μg/mL streptomycin and 0.05 mM 2-mercaptoethanol under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) until for 5 passages but not longer than passage 30 prior to testing.
- for substance incubation, cells were seeded in 24-well plates (500 μL of 2.0 x 10^6 cells/mL cell suspensions).
- at least two independent experiments were performed. In each experiment, duplicates of each treatment were tested.
- further experiments were conducted if no cytotoxicity was obtained in the case of a negative result.

2) Test-substance application:
- 500 μL of test-substance preparation was added to the cells, thus diluting the 2x concentrated test-substance preparations to their final concentration and the cells to 1.0 x 106 cells/mL.
- plates were placed into the incubator under standard culture conditions for the exposure period of 24 hours.

3) Visual inspections:
- visual inspection for test-substance precipitates were performed in each test-substance concentration directly after application.
- each well was inspected under a microscope after the exposure period of 24 hours in order to detect test-substance precipitates.

4) Cell staining and flow cytometric analysis:
- after visual inspection the cells were transferred into safe-lock tubes, collected by centrifugation and washed twice with 1 mL FACS buffer.
- cells were incubated with 600 μL of 0.01% Globin Chon fraction II,III at 4°C for 15 minutes to block FC receptors (FcR).
- after FcR blocking, cells of each treatment condition were divided into 3 aliquotes (approximately 0.3 x 10^6 cells/180 μL/group) in 96-well microtiter plates.
- cells were centrifuged and supernatant was discarded
- 50 μL working antibody solution was added to each pellet (please refer for information on preparation of the antibody working solutions to table 1 in the field "Any other information on materials and methods incl. tables" below)
- cell staining was performed at 4°C for 30 minutes in the dark.
- after staining the cells were washed twice with 200 μL FACS buffer and finally re-suspended in 200 μL FACS buffer.
- before analysis in flow cytometer the cells were stained with 5 μL of propidium iodide (PI) (50 μg/mL diluted in PBS) to yield a final concentration of 1.25 μg/mL PI.

DATA EVALUATION
1) CV75 calculation:
- CV75-value (relative survival rate) was calculated by linear extrapolation. This value is the substance concentration at which cell viability is 75% compared to the vehicle control.

2) Relative cell viability:
- cell viability was determined by propidium iodide (PI) staining.
- mean is calculated from the independent replicates of a test substance concentration
- relative cell viability was calculated as follows: % relative cell viability = (cell viability of test substance treated cells/mean cell viability of vehicle control treated cells) x 100

3) Relative fluorescence intensity:
- analysis of the membrane markers were performed in 10,000 living cells, determined by PI staining.
- concentrations inducing viability less than 50% were not considered for further assessment of dendritic cell activation.
- for data analysis, the CXP software (Beckman Coulter) was used.
- data evaluation was performed with mean fluorescence intensity (MFI) of chemical treated cells among the viable cells, with systematic isotype control use to quantify and remove nonspecific antibody binding.
- after subtracting the MFI of the isotype control, the RFI of each surface marker on the treated cells as compared with the vehicle control cells was calculated.
- results were expressed as relative fluorescence intensity (RFI) of % CD86 pos. or % CD54 pos. expression compared to the respective vehicle control.
- RFI of CD86 or CD54 was calculated by the following equation:
RFI = ((MFI of chemical - treated cells - MFI of chemical - treated isotype control cells)/(MFI of vehicle control cells - MFI of vehicle isotype control cells)) x 100

ACCEPTANCE CRITERIA
If the acceptance criteria mentioned below were not met, repetition of the test was considered:
- a tested concentration is not to be further evaluated when relative viability is less than 50%.
- cell viability of vehicle control cells must yield at least 90%.
- positive control (1-chloro-2,4-dinitrobenzene (4.0 μg/mL)): RFI values of both CD86 and CD54 should be over the positive criteria (CD86 ≥150 and CD54 ≥200) and cell viability should be >50%.
- negative control (lactic acid): RFI values of both CD86 and CD54 should not exceed the positive criteria (RFI CD86 ≥150 and RFI CD54 ≥200) and cell viability should be >50%.
- all vehicle controls: MFI ratio of both CD86 and CD54 to isotype controls should be >105%.
- a study is considered acceptable if the positive and negative and vehicle control data lies within the range of the historical data.

EVALUATION
- test substance is predicted to activate dendritic cells when CD86 expression was increased >150 and/or CD54 expression increased >200 in relation to vehicle control in at least two independent experiments.
- primary point of consideration is the biological relevance of the results.
Positive control substance(s):
yes
Remarks:
1- chloro-2,4-dinitrobenzene (4.0 μg/mL)
Positive control results:
Please refer to the field "Any other information on results incl. tables" below.
Parameter:
other: CD86 Relative Fluorescence Intensity (%)
Run / experiment:
Exp2 (max value at 22 µg/mL)
Value:
90
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: below threshold for positive reaction of 150%
Parameter:
other: CD86 Relative Fluorescence Intensity (%)
Run / experiment:
Exp3 (max value at 54 µg/mL
Value:
209
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: above threshold for positive reaction of 150%
Parameter:
other: CD54 Relative Fluorescence Intensity (%)
Run / experiment:
Exp2 (max value at 65 µg/mL)
Value:
475
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: above threshold for positive reaction of 200%
Parameter:
other: CD54 Relative Fluorescence Intensity (%)
Run / experiment:
Exp3 (max value at 65 µg/mL)
Value:
2 801
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: above threshold for positive reaction of 200%

RESULTS

PRELIMINARY CYTOTOXICITY ASSESSMENT

Table 1: Results of preliminary cytotoxicity assessment

Concentration (active ingredient µg/mL

Concentration (test substance) µg/mL

%PI negative cells replicate 1

%PI negative cells replicate 2

mean viability of duplicates

rel. Viability mean [%]

Vehicle control

Vehicle control

97.2

97.0

97.1

100.0

0.5

0.5

97.2

97.2

97.2

100.1

1.0

1.0

97.1

96.9

97.0

99.9

5.0

5.0

97.3

96.8

97.0

100.0

10

10

97.1

96.9

97.0

99.9

50

51

96.3

95.7

96.0

98.9

100

102

82.3

86.4

84.4

86.9

500

511

5.3

3.3

4.3

4.5

1000

1021

2.9

1.5

2.2

2.3

2000

2043

3.5

1.1

2.3

2.3

 5000

 5107

4.5

1.8

3.1

3.2 

PI = propidium iodide

The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 161 μg/mL (test substance as provided by the sponsor).

Table 2: RFI CD86, RFI CD54 and rel. viability. Mean values and standard deviations of 1st experiment.

Concentration (test substance) µg/mL

RFI CD86 mean

SD

RFI CD54 mean

SD

rel. viability mean

SD

65

183.6

1.9

2274.4

1203.4

47.3

3.1

78

161.0

66.2

3019.7

643.4

35.2

4.2

93

203.6

5.0

2738.6

618.6

15.9

6.1

112

235.8

24.7

984.7

587.3

4.9

1.3

34

188.5

27.3

429.7

22.1

3.8

0.5

161

175.4

0.4

467.9

9.5

4.4

0.9

193

402.6

39.0

771.7

236.1

4.7

1.8

232

217.4

0.4

1059.5

243.9

6.6

2.2

Vehicle control

100.0

---

100.0

---

100.0

0.3

Lactic acid (1000 µg/mL)

62.8

1.2

97.8

8.2

99.9

0.3

1- chloro-2,4-dinitrobenzene (4 µg/mL)

246.1

8.7

520.2

82.6

86.7

4.9

*Single RFI CD54 values: 278.1 and 613.0

Table 3: RFI CD86, RFI CD54 and rel. viability. Mean values and standard deviations of 2nd experiment.

Concentration (test substance) µg/mL

RFI CD86 mean

SD

RFI CD54 mean

SD

rel. viability mean

SD

18

98.0

4.9

105.5

7.7

100.2

0.3

22

90.0

1.2

108.5

29.6

100.2

0.4

26

95.6

3.6

177.7

37.4

100.1

0.2

31

85.0

4.1

118.5

20.2

100.3

0.2

37

90.2

13.9

146.8

10.4

99.8

0.3

45

73.0

4.5

173.2

9.3

99.4

0.3

54

77.8

12.8

262.1

64.3

98.3

0.3

65

89.8

14.4

475.4

37.7

91.2

0.4

Vehicle control

100.0

---

100.0

---

100.0

0.3

Lactic acid (1000 µg/mL)

53.0

0.1

99.3

2.5

100.0

1.5

1- chloro-2,4-dinitrobenzene (4 µg/mL)

244.1

33.7

335.7

16.9

86.9

1.3

The EC200 (the concentration resulting in a RFI of 200) for CD54 was calculated by linear regression from the results of the 45 μg/mL and the 54 μg/mL concentration to be 48 μg/mL.

Table 4: RFI CD86, RFI CD54 and rel. viability. Mean values and standard deviations of 3rd experiment.

Concentration (test substance) µg/mL

RFI CD86 mean

SD

RFI CD54 mean

SD

rel. viability mean

SD

26

143.1

8.8

191.4

29.6

98.1

0.3

31

129.0

12.7

330.2

40.9

94.9

1.0

37

188.4

20.7

437.0

10.5

86.4

2.1

45

205.0

22.2

551.4

79.2

79.6

0.9

54

209.3

8.8

1355.4

52.6

62.7

1.9

65

194.6

29.5

2800.7

381.5

50.6

1.8

78

225.1

22.3

3786.6

109.7

32.9

5.0

93

253.4

61.1

3348.4

258.1

18.1

1.1

Vehicle control

100.0

---

100.0

---

100.0

0.3

Lactic acid (1000 µg/mL)

67.5

0.8

118.4

21.2

99.8

0.2

1- chloro-2,4-dinitrobenzene (4 µg/mL)

306.2

45.5

425.8

59.1

84.8

0.8

The EC150 (the concentration resulting in a RFI of 150) for CD86 was calculated by linear regression from the results of the 31 μg/mL and the 37 μg/mL concentration to be 33 μg/mL. The EC200 (the concentration resulting in a RFI of 200) for CD54 was calculated by linear regression from the results of the 26 μg/mL and the 31 μg/mL concentration to be 26 μg/mL.

HISTORICAL CONTROL DATA

Table 4: Historical control data of h-CLAT. Data shown of test period Mar. 2015 until Apr. 2015.

Positive control 

(Lactic acid 1000 µg/mL)

CD86 RFI mean (%)

CD54 RFI 1 mean (%)

Rel. viability mean (%)

Min

61.5

81.9

99.8

Max

99.6

156.1

101.2

Mean

75.7

104.7

100.3

SD

8.9

16.5

0.4

 n 27       

Positive control

(1-chloro-2,4-dinitrobenzene 4.0 µg/mL)

CD86 RFI mean

CD54 RFI 1 mean

Rel. viability mean

Min

173.5

207.6

64.8

Max

357.1

787.7

100.2

Mean

274.7

383.3

87.1

SD

44.6

147.7

6.9

 n 27       

Vehicle control

(DMSO)

Rel. viability mean

Min

95.9

Max

98.3

Mean

97.0

SD

0.9

 n      27  

MFI = mean fluorescence intensity

RFI = relative fluorescence intensity

Interpretation of results:
other: Test substance induces activation of dendritic cells.
Conclusions:
In summary, after 24 hours of exposure to test substance CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that test substance Palisandal induces dendritic cell activation.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Mar-May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted Feb 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Type of study:
activation of keratinocytes
Test material information:
Composition 1
Details on study design:
Human transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen
Details on test animals and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.
Vehicle:
other: D-MEM (Cat. No. Biochrom FG 0445) + 1 % FBS
Concentration / amount:
Pre-test (cytotoxicity): 0.5, 1.0, 5.0, 10, 51, 102, 511, 1021, and 2043 µg/mL
Main experiment (exp 1 results not available due to technical error): Exp2 35, 41, 50, 60, 72, 86, 103, 124; Exp3 17, 20, 24, 29, 35, 41, 50, 60; Exp4 24, 29, 35, 41, 50, 60, 72, 86; Exp5 35, 41, 50, 60, 72, 86, 103, 124, Exp6 35, 41, 50, 60, 72, 86, 103, 124 µg/mL
Vehicle:
other: D-MEM (Cat. No. Biochrom FG 0445) + 1 % FBS
Concentration / amount:
Pre-test (cytotoxicity): 0.5, 1.0, 5.0, 10, 51, 102, 511, 1021, and 2043 µg/mL
Main experiment (exp 1 results not available due to technical error): Exp2 35, 41, 50, 60, 72, 86, 103, 124; Exp3 17, 20, 24, 29, 35, 41, 50, 60; Exp4 24, 29, 35, 41, 50, 60, 72, 86; Exp5 35, 41, 50, 60, 72, 86, 103, 124, Exp6 35, 41, 50, 60, 72, 86, 103, 124 µg/mL
No. of animals per dose:
three replicates of each treatment
Details on study design:
TEST SYSTEM
- Cell line: LuSens (transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen)

SELECTION OF DOSES/CONCENTRATIONS FOR THE LuSENS
- to determine the concentrations suitable for the main experiment a pre-test was performed.
- cells were exposed to 9 concentrations of the test-substance preparation
- cytotoxicity was determined by MTT assay.
- no decrease in cell viability below 75% was observed.
- as no cytotoxicity was observed the maximum concentration was chosen to be 2004 µg/mL. The additional concentrations were obtained by a 1:1.2 serial dilution series of the maximum concentration.

CONTROLS
- negative control (NC): 450 μg/mL, DL-Lactic acid
- positive control (PC): 18 μg/mL, Ethylene glycol dimethacrylate
- vehicle control: 1% DMSO in culture medium D-MEM (Cat. No. Biochrom FG 0445) + 1 % FBS
- blank control: Medium D-MEM (Cat. No. Biochrom FG 0445) + 1 % FBS without cells
- basal control: Medium D-MEM (Cat. No. Biochrom FG 0445) + 1 % FBS with cells

TEST SUBSTANCE PREPARATION
- test substance was weighed and topped up with the vehicle (4% DMSO in D-MEM + 1% FBS culture medium) to achieve the required 4x concentration of the highest concentration (stock solution, produced in glass vials).
- further concentrations were prepared as 4x concentrations by serial 1:1.2 dilution according to the planned doses (master plate, produced in glass vials).
- test-substance preparations were prepared by stirring.
- visual inspection of each dilution step was performed.
- no verification of the stability of the test substance in the vehicle was required.

EXPERIMENTAL PROCEDURE
1) Preparation of the cells:
- LuSens cells from the working cell bank were thawed and cultured using culture medium D-MEM ((Cat. No. Biochrom FG 0445)+ 10 % FBS + 1 % Penicillin/Streptomycin, Puromycin dihydrochloride 25 μL (Sigma P9620-10mL)), under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) for at least passage ≥5 but not longer than 15 passages prior to testing.
- before substance incubation, cells were seeded in 96-well microtiter plates (120 μL of 0.83 x 10^5 cells/mL cell suspensions), using culture medium D-MEM ((Cat. No. Biochrom FG 0445) + 10 % FBS) for incubation for 24 hours.
- at least two independent experiments were performed. In each experiment, three replicates of each treatment were tested. In case of contradictory results further experiments were conducted. Further experiments were also conducted if no cytotoxicity was obtained in the case of a negative result.

2) Test-substance preparation and application of MTT and Luciferase assay:
- after cell adaption for 24 hours cell culture medium D-MEM ((Cat. No. Biochrom FG 0445) + 10 % FBS) was aspirated and replaced with 150 μL medium D-MEM ((Cat. No. Biochrom FG 0445) + 1 % FBS).
- test substance was prepared as described above.
- each preparation of the master plate was then applied in a ratio of 1:4 (50 μL) to the cells.
- DMSO final concentration was 1%.
- plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance.
- plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours.
- for the luciferase assay a white plate (luminescence compatible plate) was used. In addition a clear plate was treated in parallel for the determination of cell viability.

3) Visual inspection:
- visual inspection for test-substance precipitates was performed for each test-substance concentration directly after application.
- each well was inspected under a microscope after the exposure period of 48 hours in order to detect test-substance precipitates.

4) Luciferase assay:
- after visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded.
- cells were washed twice with 300 μL PBS (with Ca2+/Mg2+).
- 200 μL of Steady-Glo-preparation (= 100 μL Steady-Glo- Mix and 100 μL PBS (without Ca2+/Mg2+)) per well was added
- cells were shaken on a plate shaker for 10 minutes at room temperature in darkness.
- after the incubation the luminescence was measured in the luminometer.

5) Cell viability assay MTT:
- cell culture medium was aspirated from all wells.
- cells were washed twice with 300 μL PBS (with Ca2+/Mg2+).
- 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and medium D-MEM ((Cat. No. Biochrom FG 0445) + 1 % FBS)) was added to each well of the 96-well microtiter plate and incubated for further 2 hours after sealing the plates in the incubator.
- for analysis: medium was aspirated and cells were lysed by adding 100 μL of lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength 690 nm using the SunriseTM Absorbance Reader.

DATA EVALUATION
1) CV75 calculation:
- CV75-value (relative survival rate) was calculated by linear extrapolation. This value is the substance concentration at which relative cell viability is 75% compared to the vehicle control.

2) Cell viability:
- cell viability is calculated as follows:
Absorbance = absorbance at 570 nm- absorbance at 690 nm
% relative cell viability = ((absorbance of test substance treated cells - absorbance of blank)/(absorbance of mean vehicle control treated cells - absorbance of blank)) x 100
- from the three independent replicates a mean is calculated.

3) Luciferase fold induction:
- fold induction is calculated as follows
fold induction = (test substance treated cells - background without cells)/(mean vehicle control treated cells - backgroundwithout cells)
- from the independent replicates a mean is calculated.

ACCEPTANCE CRITERIA
- a tested concentration was not further evaluated when relative viability was less than 70%.
- cell viability of untreated cells must yield at least 90%.
- positive control ethylene glycol dimethacrylate should be >2.5 fold induction and lactic acid <1.5 and viability ≥70%.
- average standard of the variability in the vehicle control wells for each plate should be below 20%.
- if any of the acceptance criteria mentioned above was not met, repetition of the test was considered.
- a study was considered acceptable if the positive and negative and vehicle control data lied within the range of the historical data.

EVALUATION
- a test substance is concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds a 1.5 fold induction with respect to the vehicle control at concentrations that do not reduce viability below 70% of two independent experiments.
- a test substance is considered to be “negative” when the criteria mentioned above are not met up to the maximum concentration (= 2000 μg/mL
Positive control substance(s):
yes
Remarks:
Ethylene glycol dimethacrylate (18 μg/mL)
Positive control results:
Please refer to the field "Any other information on results incl. tables" below.
Parameter:
other: Relative luciferase Induction
Run / experiment:
Exp2 (max. value of two consecutive conc. >=1.5fold)
Value:
1.74 <
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: above threshold of 1.5fold for two consecutive concentrations
Parameter:
other: Relative luciferase Induction
Run / experiment:
Exp3 (max. value of two consecutive conc. >=1.5fold)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: below threshold of 1.5fold for two consecutive concentrations
Parameter:
other: Relative luciferase Induction
Run / experiment:
Exp4 (max. value of two consecutive conc. >=1.5fold)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: below threshold of 1.5fold for two consecutive concentrations
Parameter:
other: Relative luciferase Induction
Run / experiment:
Exp5 (max. value of two consecutive conc. >=1.5fold)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: below threshold of 1.5fold for two consecutive concentrations
Parameter:
other: Relative luciferase Induction
Run / experiment:
Exp6 (max. value of two consecutive conc. >=1.5fold)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: below threshold of 1.5fold for two consecutive concentrations

RESULTS

PRELIMINARY CYTOTOXICITY ASSESSMENT

Table 1: Results of preliminary cytotoxicity assessment

Concentration

(active ingredient) µg/mL

Concentration

(test substance) µg/mL

Mean OD570-690 of 3 replicates

rel. viability [%]

Vehicle control

Vehicle control

0.313

100.0

0.5

0.5

0.305

97.5

1.0

1.0

0.299

95.4

5.0

5.0

0.317

101.2

10

10

0.295

94.4

50

51

0.264

84.2

100

102

0.192

61.2

500

511

0.001

0.3

1000

1021

0.001

0.4

2000

2043

0.001

0.4

Exp 1

Results of the 1st experiment are not available due to a technical error

Exp 2

Table 2: Mean values and standard deviations of luciferase induction and rel. viability

Concentration

(test substance)

µg/mL

experiment 2

Fold induction

 

Rel. viability

 

mean

SD

mean

SD

35

1.69

0.17

76.5

5.9

41

1.71

0.30

88.5

4.9

50

1.74

0.30

55.3

12.8

60

0.01

0.14

0.2

1.0

72

0.05

0.10

1.3

0.0

86

-0.05

0.07

0.8

0.9

103

-0.06

0.07

1.1

0.0

124

-0.11

0.03

0.5

0.8

Vehicle control

1.00

0.16

100.0

3.4

Ethylene glycol dimethacrylate

(18 µg/mL)

9.18

1.01

99.6

2.3

Lactic acid

(450 µg/mL)

0.92

0.12

106.3

4.9

Exp 3

Table 3: Mean values and standard deviations of luciferase induction and rel. viability

Concentration

(test substance)

µg/mL

experiment 3

Fold induction

Rel. viability

mean

SD

mean

SD

17

0.95

0.11

96.2

3.2

20

1.07

0.08

90.3

0.9

24

0.95

0.09

99.4

1.5

29

0.96

0.07

99.4

3.6

35

1.09

0.12

99.6

4.6

41

1.09

0.19

102.0

6.6

50

1.03

0.00

102.8

2.9

60

1.06

0.16

103.6

6.4

Vehicle control

1.00

0.13

100.0

4.0

Ethylene glycol dimethacrylate

(18 µg/mL)

6.88

0.51

105.3

7.6

Lactic acid

(450 µg/mL)

0.95

0.17

116.0

7.5

Exp 4

Table 4: Mean values and standard deviations of luciferase induction and rel. viability

Concentration

(test substance)

µg/mL

experiment 4

Fold induction

Rel. viability

mean

SD

mean

SD

24

1.25

0.20

103.3

6.8

29

1.21

0.24

98.3

5.1

35

1.13

0.13

102.1

4.7

41

1.07

0.14

103.0

4.3

50

1.07

0.07

101.0

6.7

60

1.21

0.21

101.0

5.6

72

1.15

0.18

100.7

10.0

86

0.94

0.05

99.1

6.5

Vehicle control

1.00

0.14

100.0

4.1

Ethylene glycol dimethacrylate

(18 µg/mL)

8.35

0.52

104.1

5.3

Lactic acid

(450 µg/mL)

0.99

0.17

115.9

5.6

Exp 5

Table 5: Mean values and standard deviations of luciferase induction and rel. viability

Concentration

(test substance)

µg/mL

experiment 5

Fold induction

Rel. viability

mean

SD

mean

SD

35

1.01

0.06

97.0

5.1

41

1.14

0.11

98.0

6.4

50

1.06

0.07

99.0

7.3

60

1.14

0.16

97.7

6.4

72

1.03

0.14

98.6

9.6

86

1.10

0.11

90.2

18.3

103

1.23

0.40

68.9

13.0

124

-0.03

0.07

0.7

1.0

Vehicle control

1.00

0.13

100.0

4.8

Ethylene glycol dimethacrylate

(18 µg/mL)

6.76

0.42

111.0

6.4

Lactic acid

(450 µg/mL)

0.81

0.09

107.6

3.2

Exp 6

Table 6: Mean values and standard deviations of luciferase induction and rel. viability

Concentration

(test substance)

µg/mL

experiment 6

Fold induction

Rel. viability

mean

SD

mean

SD

35

1.02

0.20

104.4

5.2

41

1.05

0.13

95.4

6.0

50

1.02

0.13

114.1

7.9

60

1.10

0.11

103.3

7.0

72

1.54

0.07

92.0

6.1

86

1.35

0.21

60.3

16.3

103

1.36

0.24

16.7

15.4

124

-0.09

0.05

0.4

0.2

Vehicle control

1.00

0.12

100.0

5.9

Ethylene glycol dimethacrylate

(18 µg/mL)

5.21

0.88

96.9

5.9

Lactic acid

(450 µg/mL)

0.96

0.07

97.0

7.9

HISTORICAL CONTROL DATA

Table 7: Historical control data of LuSens. Data shown of test period Oct. 2014 until Jun. 2015

Negative control

(Lactic acid 450 µg/mL)

Luminescence

(RLU)

Fold ind.

rel. viability

[%]

Min

236.6

0.75

90.8

Max

488.4

1.38

123.2

Mean

349.3

0.97

108.4

SD

59.8

 

0.10

6.6

n

98

Positive control (EGDMA 18 µg/mL)

Luminescence

(RLU)

 

Fold ind.

rel. viability

[%]

Min

848.2

4.03

77.1

Max

2659.2

8.94

136.1

Mean

1539.0

6.84

104.4

SD

372.6

 

1.07

9.7

n

98

Vehicle control (1% DMSO)

Luminescence

(RLU)

 

Fold ind.

rel. viability

[%]

Min

241.1

1.00

100.0

Max

517.4

1.00

100.0

Mean

358.1

1.00

100.0

SD

66.9

 

0.00

0.0

n

98

Basal

expression

Luminescence

(RLU)

 

Fold ind.

rel. viability

[%]

Min

217.0

0.56

96.2

Max

507.3

1.10

170.8

Mean

319.4

0.81

137.9

SD

68.2

 

0.13

12.8

n

98

Interpretation of results:
other: test substance has no keratinocyte activating potential
Conclusions:
In one experiment a relevant ARE-dependent luciferase activity induction above 1.5-fold was observed at two consecutive concentrations, however, this result could not be reproduced in 4 further experiments. It has to be concluded that test substance does not have a keratinocyte activating potential.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

According to Basketter et al. (2005) & ECHA guidance R.7a (2014), due to the complexity of the mechanisms of skin sensitisation, a single test is not be able to replace the currently required animal procedures. However, a combination of several in chemico / in vitro tests, covering several relevant mechanistic steps of skin sensitisation (i.e., the ability to (i) react with protein, (ii) activate keratinocytes, and (iii) activate dendritic cells) into a test battery could lead to replacement of the in vivo tests.

 

Hence, the following in chemico / in vitro studies (addressed to a specific mechanistic step of skin sensitization) were conducted with the test item under consideration:

(i)            In chemico skin sensitisation: Direct Peptide Reactivity Assay (DPRA) (OECD 442C)

(ii)           In vitro skin sensitisation: ARE Reporter Assay (LuSens) (OECD 442D) in accordance with Bauch et al. (2012),Maxwell et al. (2011) and Bauch et al. (2011)

(iii)          In vitro skin sensitisation: Human Cell Line Activation Test (h-CLAT) (OECD 442E) in accordance with Bauch et al. (2012), Bauch et al. (2011), Maxwell et al. (2011), Ashikaga et al. (2010) and Sakaguchi et al. (2010).

In case of conflicting results a pragmatic weight of evidence approach may be used in according to Bauch et al. 2012. The authors' approach is to classify the respective substance as non-sensitiser if two of these three tests performed, resulting in a negative outcome. Using this approach, 47 out of 50 or 44 out of 53 test substances were predicted correctly if compared to human or LLNA, respectively.

The substance showed a minimal chemical reactivity in the Direct Peptide Reactivity Assay under the test conditions chosen. However, it should be noted that due to the limited solubility of the test substance the samples with both peptides were emulsions and that the result could therefore be under-predictive. In line with OECD Guideline 442C the result was considered inconclusive in this case.

In the LuSens keratinocyte activation test, in one experiment a relevant ARE-dependent luciferase activity induction above 1.5 -fold was observed at two consecutive concentrations. However, this result could not be reproduced in 4 further experiments. Therefore, it was concluded that test substance does not have a keratinocyte activating potential.

In the h-CLAT, after 24 hours of exposure to test substance CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it was concluded that test substance induces dendritic cell activation.

As the test battery resulted in 1 inconclusive, 1 negative and 1 positive result, the 2 out of 3 approach cannot be applied to this substance. Therefore it was decided to perform an LLNA.

Under the conditions of the LLNA the test substance revealed sensitising properties. The EC3 value was calculated to be 2.7%.

Therefore, in weighing all data from the in silico, in vitro and in vivo experiments, the substance has to be considered to have skin sensitising properties.

References:

- ECHA (2014): Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a: Endpoint specific guidance, v 3.0

- Bauch et al. (2012): Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials, Regul Toxicol Pharmacol, 63(3):489-504.

- Bauch et al. (2011): Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals. Toxicology in Vitro 25, 1162-1168, 2011.

- Maxwell et al. (2011): Skin sensitisation: the Colipa strategy for developing and evaluating nonanimal test methods for risk assessment.ALTEX 28(1): 50-5, 2011.

- Ashikaga et al. (2010): A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern Lab Anim. 38(4):275-84.

- Sakaguchi et al. (2010): Predicting skin sensitization potential and inter-laboratory reproducibility of a human Cell Line Activation Test (h-CLAT) in the European Cosmetics Association (COLIPA) ring trials. Toxicol In Vitro. 24(6):1810-20.

- Basketter et al. (2005): 3.4. Skin Sensitisation, ATLA 33, Suppl. 1, 83–103, 2005

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Justification for selection of respiratory sensitisation endpoint:
Study not required according to Annex VII-X of Regulation (EC) No 1907/2006.

Justification for classification or non-classification

The available data on sensitisation of the test substance meet the criteria for classification as Skin Sens. 1B; H317 according to Regulation (EC) 1272/2008.