Ethyl enantate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

23.03.2015 - 27.04.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

skin sensitisation: in chemico

Remarks:

in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16.03.2015 - 23.03.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

TEST-SUBSTANCE PREPARATION
- Stock solution: 100 mM
- Vehicle: acetonitrile
- Reason for the vehicle: The test substance is soluble in acetonitrile.

CONTROLS
- Negative control: vehicle control: acetonitrile
- Concurrent control: vehicle control with the peptides
- Co-elution control: buffer and test substance without the peptide
- Positive control: ethylene glycol dimethacylate (50 nM solution in acetonitrile)

PEPTIDES
- Synthetic 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).
- Stock solution:
-- C-containing peptide: 0.667 mM of peptide in pH 7.5 phosphate buffer
-- K-containing peptide: 0.667 mM of peptide in pH 10.2 ammonium acetate buffer
- Ratios
- C-containing peptide: ratio of 1:10 (0.5 mM peptide, 5mM test substance)
- K-containing peptide: ratio of 1:50 (0.5 mL peptide, 25 mM test substance)

EXPERIMENTAL PROCEDURE
- Replicates: 3 for each peptide
- Determination remaining non-depleted peptide concentration: HPLC at 220nm: HPLC analysis started 24 hours after sample preparation and the analysis time was 30 hours.
- Calibration samples: samples of a known peptide concentration are measured in parallel

PREPARATIONS SAMPLES
- Calibration sample was prepared from the peptide stock solution in 20% acetonitrile in the respective buffer using serial concentration: 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 or 0.000 mM peptide
- Test-substance samples: samples were incubated at 25°C +/- 2.5°C in the dark for 24 +/- 2 hours and visually investigated for any precipitate that may occur during the exposure period.
- Preparation of vehicle control: in triplicated in the same way as the test-substance samples described above but with acetonitrile instead of the test subtance.
- Preparation of positive control: Ethylene glycol dimethacrylate (EGDMA) prepared as a 50 mM solution in acetonitrile.
- Preparation of co-elution control: in the same way as the test-substance sampled described above but without the peptide, instead buffer was used.

MEASUREMENT PEPTIDE CONCENTRATIONS
- Method: HPLC Agilent PH 1100
- Column: Phenomenex Luna 3µ C18(2), 100 mm x 2 mm with guard column "Security Guard" C18, 4 mm x 2 mm
- Wavelength: 220 nm and 258 nm
- Detection: Diode Array Detector
- Mobile phase
A: 0.1% (v/v) trifluoroacetic acid in de-ionized water, HPLC grade
B: 0.085% (v/v) trifluoroacetic acid in acetonitrile, HPLC grade
- Flow: 0.35 mL/min
- Gradient: 0 min: 10%B; 10 min: 25%B; 11 min: 90%B; 13 min: 90%B, 13.5 min: 10%B; 25 min: 10%B`
- Injection volume: 2 µL
- Software: Dionex Chromeleon

DATA EVALUATION
- Calculation of the peptide concentrations: peptide concentration (mM) = [peak area (mAU * s) - b] / m with b: axis intercept and m: slope
- Calculation of the peptide depletion: peptide depletion of sample : [ 1 - (peptide concentration of sample (mM) / mean peptide concentration of NC (mM))] * 100%
- Mean peptide depletion for each peptide: C-containing peptide depletion of a test substance (%) = mean (C-containing peptide depletion of samples 1-3) (%)
- Mean peptide depletion (%) = (C-containing peptide depletion (%) + K-containing peptide depletion (%)) / 2

ACCEPTANCE CRITERIA
- The positive control causes depletion of both peptides comparable to historic data.
- The standard calibration curve should have an r2 > 0.99.
- The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
- The CV of nine vehicle controls B and C should be < 15%
- The variability should be acceptable low (SD < 14.9% for cysteine depletion and < 11.6% for lysine depletion)

EVALUATION RESULTS
Chemical reactivity was determined by mean peptide depletion [%] and was rated as
- high: mean peptide depletion > 42.47
- moderate: mean peptide depletion > 22.62 ≤ 42.47
- low: mean peptide depletion > 6.38 ≤ 22.62
- minimal: mean peptide depletion ≤ 6.38
High, moderate and low reactivity are evaluated as positive.

In case the mean peptide depletion cannot be determined due to invalid K-peptide depletion the evaluation is performed as follows:
- high: mean peptide depletion > 98.24
- moderate: mean peptide depletion > 23.09 ≤ 98.24
- low: mean peptide depletion > 13.89 ≤ 23.09
- minimal: mean peptide depletion ≤ 13.89
High, moderate and low reactivity are evaluated as positive.

Run / experiment:

other: C-containing peptide (mM)

Parameter:

other: Peptide depletion

Value:

-0.52

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: K-containing peptide (mM)

Parameter:

other: Peptide depletion

Value:

-0.04

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Parameter:

other: Mean peptide depletion

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
Solubility: The test substance was soluble in acetonitrile. The samples of the test substance with the Cpeptide were solutions immediately after preparation and after 24 hours. The samples with the K-peptide were homogeneous emulsions immediately after preparation and after 24 hours.

ACCEPTANCE OF RESULTS:
A study is considered acceptable if the positive control causes depletion of both peptides comparable to historic data.
The standard calibration curve should have an r² >0.99.
The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
The CV of the nine vehicle controls B and C should be < 15%.
Since the mean peptide depletion for each peptide is determined from the mean of three single samples, the variability between these samples should be acceptably low (SD <14.9% for % cysteine depletion and <11.6% for % lysine depletion).

1. Results Cysteine-containing peptide:

DPRA: Peak area, peptide concentration and peptide depletion of NC, PC and test substance for cysteine-peptide
Reaction with cysteine-peptide	Peak area (mAU*s) at 220 nm			Peptide concentration (mM)
	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3	Mean	SD
NC: ACN	857.6	843.8	852.0	0.482	0.474	0.479	0.478	0.004
Test item	860.0	852.3	854.4	0.483	0.479	0.480	0.481	0.002
PC: EGDMA in ACN	431.6	380.4	337.1	0.242	0.214	0.189	0.215	0.027
Reaction with cysteine-peptide	Peptide depletion (%)
	Sample 1	Sample 2	Sample 3	Mean	SD
NC: ACN	-0.76	0.86	-0.10	0.00	0.82
Test item	-1.04	-0.13	-0.38	-0.52	0.47
PC: EGDMA in ACN	49.34	55.36	60.46	55.05	5.56

2. Results Lysine-containing peptide:

DPRA: Peak area, peptide concentratio and peptide depletion of NC, PC and test substance for lysine-peptide
Reaction with Lysine-peptide	Peak area (mAU*s) at 220 nm			Peptide concentration (mM)
	Sample 1	Sample 2	Sample 3	Sample 1	Sample 2	Sample 3	Mean	SD
NC: ACN	745.4	748.7	747.6	0.496	0.498	0.497	0.497	0.001
Test item	760.3	742.4	739.9	0.506	0.494	0.492	0.497	0.007
PC: EGDMA in ACN	650.0	639.2	627.8	0.432	0.425	0.417	0.425	0.007
Reaction with Lysine-peptide	Peptide depletion (%)
	Sample 1	Sample 2	Sample 3	Mean	SD
NC: ACN	0.25	-0.20	-0.05	0.00	0.23
Test item	-1.74	0.65	0.99	-0.04	1.49
PC: EGDMA in ACN	13.03	14.46	16.0	14.5	1.48

3. Mean peptide depletion:

For the test substance the mean peptide depletion as average of cysteine and lysine-peptide depletions is calculated. Negative depletions were considered to be zero for calculation:

	Cysteine-peptide		Lysine-peptide		Mean of both depletions (%)
	Mean depletion (%)	SD	Mean depletion (%)	SD
Test substance	-0.54	0.47	-0.04	1.49	0.00
PC: EGDMA in ACN	55.05	5.56	14.5	1.48	34.77

4. Evaluation criteria of DPRA:

Cysteine 1:10 / Lysine 1:50 prediction model

Mean peptide depletion (%)	Reactivity	Evaluation
>42.47	high reactivity	positive
>22.62 ≤ 42.47	moderate reactivity	positive
> 6.38 ≤ 22.62	low reactivity	positive
≤ 6.38	minimal or no reactivity	negative

5. Acceptance criteria:

The acceptance criteria were met with the exception of the vehicle control of the K-peptide samples which was not available due to a technical error. However, the test is considered to be valid, as all other control samples met the criteria.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance shows no chemical reactivity in the DPRA test under the current conditions. Therefore, the test item is not a sensitiser.

Executive summary:

In the current study the skin sensitisation effect of the test item was assessed in an in chemico assay according to OECD 442C.

This method quantifies the remaining concentrations of cysteine- or lysine-containing peptides after 24 hours incubation with the test item at 25 +/-2.5 °C. The percentage of depletion of cysteine- and lysine-peptides is calculated and used in a prediction model to assign the test chemical to one of four reactivity classes and in this way discriminate between sensitisers and non-sensitisers.

The test item was dissolved at a 100 mM concentration in acetonitrile (ACN) and 3 samples of the test item were incubated with a typical concentration of 0.667 mM of cysteine in a pH 7.5 phosphate buffer or 0.667 mM of lysine in a pH 10.2 ammonium acetate buffer. Incubation was done for 24 hours in the dark at 25 +/- 2.5°C. The remaining non-depleted concentrations were determined by HPLC with gradient elution and UV-detector at 220 nm.

The mean C-peptide depletion, caused by the test substance was determined to be -0.52%.

The mean K-peptide depletin, caused by the test substance was determined to be -0.04%.

Negative depletions are considered to be "zero" for the calculation of the mean peptide depletion, resulting in 0.0% for the test item. Based on the results and the prediction model, it is concluded that the test substance shows no chemical reactivity in the DPRA test under the current conditions. In conclusion, the test item is not a sensitiser.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

23.04.2015 - 28.08.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 442E h-CLAT

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Ashikaga, T. 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.

Qualifier:

according to guideline

Guideline:

other: Sakaguchi H, 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.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

TEST SYSTEM
- Cell line: THP-1 cells; human monocytic leukemia cell line
- Source: American Type Culture Collection Manassas, USA (ATCC, TIB-202)

PRE-TEST
- Goal: determination of the concentrations for the main test
- Setup: cells were exposed to 10 concentrations of the test substance preparation in the range of 0.5 to 5000 µg/mL. Cytotoxicity was determined by propidium iodide intercalation into the DNA. Determination of the CV75 value (= estimated concentration that affords 75% cell viability) by linear regression from the concentration response curve.
- Selection of maximum concentration in the main test: (1.2)^2-fold of the CV75 value. Additional concentrations by a 1:1.2 serial dilution of the maximum concentration.

TEST-SUBSTANCE PREPARATION
- Concentrations: 1st experiment: 2027, 1689, 1408, 1173, 977, 815, 679 and 566 µg/mL; 2nd experiment: 1173, 977, 815, 679, 566, 471, 393 and 327 µg/mL
- Stock: 2x concentration of the highest concentration stock solution
- Vehicle: culture medium
- Reason for vehicle: The h-CLAT was performed in an aqueous test system.

CONTROLS
- Negative control (NC): Lactic acid (LA); 1000 µg/mL in culture medium
- Vehicle control (VC): Culture medium
- Isotype control: In order to help distinguish non-specific staining from specific antibody staining each test-substance concentration and control is additionally incubated with mouse IgG1
- Positive control: 1-chloro-2-4-dinitrobenzene (DNCB); 4.0 µg/mL in 0.2% DMSO in culture medium

MEDIUM
- Culture medium: RPMI 1640: with L-glutamine, 25mM HEPES (Gibco) + 10% FBS inactivated + 1% Penicillin/Streptomycin + 0.05 mM 2-Mercaptoethanol (Gibco)
- FACS Buffer: Phosphate Buffered Saline (DPBS) without Ca2+ / Mg2+ (Gibco) + 0.1% BSA (Sigma A7030)
- Blocking Solution: 0.01% Globulins Cohn fraction II,III (Sigma G2388) with DPBS (without Ca2+ / Mg2+)
- Reagent for cytotoxicity test: Propidium iodide (Sigma)

EXPERIMENTAL PROCEDURE
- 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 penicilin, 100 µg/mL streptomycin and 0.05 mM 2-mercaptoethanol at 37°C, ca. 5% CO2, >= 90% humidity for 5-30 passages prior to testing. For substance incubation, cells were seeded in 24-well plates: 500 µL of 2.0E+06 cells/mL suspensions.
- Test substance application:
Addition of 500 µL of test substance preparation to the cells (= diluting the 2x concentrated test substance preparations to their final concentration and the cells to 1.0E+06 cells/mL. Incubation of the plates under standard culture conditions for 24 hours. Visual inspection for test substance precipitates directly after application and after 24h exposure period. 2 Independent experiments, 2 replicates.
- Cell staining and flow cytometric analysis:
Cells are transferred into safe-lock tubes, collected by centrifugation and washed twice with 1 mL FACS buffer. Incubation of cells with 600 µL of 0.01% Globulins Chon faction II,III at 4°C for 15 min. Cells were divided into 3 aliquotes (approx. 0.3E+06 cells/180 µL/group) in 96-well microtiter plates. Cells were centrifuged and the supernatant was discarded. Addition of 50 µL working antibody solution to each pellet. Cell staining for 30 min at 4°C in the dark. After staining, cells were washed twice with 200 µL FACS buffer and subsequently resuspended in 200 µL FACS buffer. Before analysis in the flow cytometer the cells were stained with 5 µL of PI (50 µg/mL in FACS buffer) to yield a final concentration of 1.25 µg/mL PI.


DATA EVALUATION
- CV75 calculation: Relative survival rate is calculated by linear extrapolation. This value is the substance concentration at which cell viability is 75% compared to the vehicle control.
- Relative cell viability: % relative cell viability = (cell viability of test substance treated cells / mean cell viability of vehicle control treated cells) * 100
- Relative fluorescence intensity: RFI (%) = ( MFI of chemical-treated cells - MFI of chemical-treated isotype control cells) / (MFI of vehicle control cells - MFI of vehicle isotype control cells) * 100
- Calculation of EC 150% and EC 200%: If applicable, the concentration resulting in a positive response (RFI of 150% (CD86) or 200% (CD54) and viability >50%) was calculated for each cell surface marker from each experiment. The calculation is performed by linear regression from the two concentrations directly above and below the EC 150% / EC 200% concentration.

ACCEPTANCE CRITERIA
- A tested concentration is not to be further evaluated when viability is less than 50%
- Cell viability of vehicle control cells must yield at least 90%
- In the positive control (DNCB), RFI (relative fluorescence intensity) values of both CD86 and CD54 should be exceed the positive criteria (RFI CD86≥ 150% and CD54 ≥ 200%) and cell viability should be ≥ 50%.
- For all vehicle control, the MFI (mean fluoresence intensity) ratio of both CD86 and CD54 to isotype controls should be ≥ 105%.
- A study is considered to be acceptable if the positive, negative and vehicle control data lies within the range of the historic data.

EVALUATION RESULTS
- Positive result: A test is considered to be positive when the dendritic cells are activated meaning that CD86 expression is increased ≥150% and/or CD54 expression increased ≥200% in relation to vehicle control in at least 2 independent experiments.
- Negative result: A test is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (=5000 µg/mL for the vehicle culture medium or 2000 µg/mL for 0.2% DMSO in culture medium).

Positive control results:

Positive controls achieved RFI values of both CD86 and CD54 over the positive criteria (CD86= 311.9% and CD54 = 368.1%) and cell viability was 82.1%.

Key result

Parameter:

other: dendritic cell activation

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

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 induces dendritic cell activation. For a full overview of the experimental results, please see below.

Result of the preliminary cytotoxicity assessment:

The CV75 value was determined by linear regression from the concentration response curve to be 1408 µg/mL.

Hence, the highest tested concentration is 1408 * (1.2)^2 = 2027 µg/mL.

Mean value and standard deviation for 1st experiment:

Concentration (test substance) µg/mL	RFI CD86		RFI CD54		Rel. Viability (%)	SD of viability
	mean	SD	mean	SD
566	169.9	20.3	320.5	6.8	89.0	2.8
679	180.6	29.0	288.5	62.3	75.4	1.3
815	160.8	13.2	395.5	16.5	67.6	4.8
977	150.9	0.8	333.3	22.8	14.6	1.4
1173	221.5	1.8	418.7	106.4	53.0	23.8
1408	198.4	28.8	582.5	72.4	63.7	9.8
1689	218.4	19.5	607.1	78.6	53.0	3.3
2027	78.0	114.5	506.4	38.1	39.3	2.4
VC	100.0	-	100.0	-	100.0	0.2
LA 1000 µg/mL	79.5	10.7	124.1	25.5	99.6	0.2
DNCB 4µg/mL	311.9	88.5	368.1	70.2	82.1	3.7

Mean value and standard deviations fo 2nd experiment:

Concentration (test substance) µg/mL	RFI CD86		RFI CD54		Rel. Viability (%)	SD of viability
	mean	SD	mean	SD
327	102.1	4.6	240.7	15.3	100.4	0.3
393	96.1	0.1	236.4	41.8	100.3	0.3
471	106.3	3.4	382.3	47.8	100.0	0.1
566	100.2	1.3	364.9	189.4	99.7	0.1
679	107.2	4.7	422.1	66.3	98.3	0.8
815	127.0	9.8	557.8	89.0	78.5	3.9
977	130.0	12.2	737.3	65.9	71.0	0.9
1173	129.1	5.7	592.7	150.1	56.2	1.7
VC	100.0	-	100.0	-	100.0	100.0
LA 1000 µg/mL	66.9	1.4	104.9	14.2	100.3	0.2
DNCB 4µg/mL	300.0	19.7	392.6	0.9	84.4	1.1

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Based on the results and the prediction model, it is concluded that the test substance induces dendritic cell activation.

Executive summary:

In the current study the skin sensitisation potential of the test item was assessed according to the new OECD 442E TG.

The potential of the test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the human Cell Line Test (h-CLAT). The test substance was incubated with the human pro-monocytic cell line THP-1 for 24 hours at 37°C and the membrane markers expression was measured by flow cytometry through staining with FITC labeled anti-human-CD86/anti human CD54 antibody and propidium iodide.

In order to determine the concentration suitable for the main test a pre-test was performed.

The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

In the main experiment the test substance was tested at concentrations of 566 μg/mL onwards (1st experiment: 2027, 1689, 1408, 1173, 977, 815, 679 and 566 µg/mL; 2nd experiment: 1173, 977, 815, 679, 566, 471, 393 and 327 µg/mL). No precipitates were noticed in any concentration after 24 hours.

Relative fluorescence intensity and concurrent relative viability were determined in 2 main experiments. Calculation of an EC150 (the concentration resulting in a RFI of 150) for CD86 and an EC200 (the concentration resulting in a RFI of 200) for CD54 was not applicable.

After 24 hours of exposure, the test substance induced CD54 expression in THP-1 cells with 83% viability in two independent experiments. Therefore, it was concluded that the test substance induces dendritic cell activation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Ramirez, T. et al; LuSens: A keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification. Toxicol In Vitro. 2014; 28(8).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl hexanoate

EC Number:

204-640-3

EC Name:

Ethyl hexanoate

Cas Number:

123-66-0

Molecular formula:

C8H16O2

IUPAC Name:

ethyl hexanoate

In vitro test system

Details on the study design:

TEST SYSTEM
- Cell line: Human transgenic keratinocyte cell line derived from HaCaT cells
- Source: prepared in collaboration with Christoph J. Wruck, RWTH Aachen

PRE-TEST
- Goal: determination of the concentrations for the final test.
- Setup: cells were exposed to 9 concentrations of the test substance preparation in a range of 0.5 to 2000 µg/mL. Cytotoxicity was determined by MTT assay. Determination of the CV75 value (= estimated concentration that affords 75% cell viability) by linear regression from the concentration response curve.
- Selection of concentrations for the main experiment: maximum concentration = (1.2)^3-fold of the CV75 value; additional concentrations are a 1:1.2 dilution series of this maximum concentration.

TEST-SUBSTANCE PREPARATION
- Concentrations: 1941, 1618, 1348, 1123, 936, 780, 650 and 542 µg/mL
- Stock: 4x concentration of the highest concentration
- Vehicle: 4% DMSO in culture medium 3
- Reason for vehicle: 4% DMSO in culture medium 3 was used because good homogeneity of the preparation was achieved

CONTROLS
- Negative control: DL-Lactic acid (LA, CAS n° 50-21-5), 450 µg/mL in 1% DMSO in culture medium 3
- Vehicle control (VC): 1% DMSO in culture medium 3;
- Blank control: Culture medium 3 without cells;
- Basal control: Culture medium 3 with cells
- Positive control: Ethylene glycol dimethacrylate (EGDMA, CAS no. 97-90-5), 18 μg/mL in 1% DMSO in culture medium 3

MEDIUM
- Culture medium 1: D-MEM (Cat. No. Biochrom FG 0445)+ 10 % FBS + 1 % Penicillin / Streptomycin, Puromycin dihydrochloride 25 μL (Sigma P9620- 10mL)
- Culture medium 2: D-MEM (Cat. No. Biochrom FG 0445) + 10 % FBS
- Culture medium 3: D-MEM (Cat. No. Biochrom FG 0445) + 1 % FBS
- Buffer: Phosphate Buffered Saline (PBS) w/o Ca2+ / Mg2++ 0.05 % EDTA Phosphate Buffered Saline (PBS) w/o Ca2+ / Mg2+ Phosphate Buffered Saline (PBS) with Ca2+ / Mg2+

EXPERIMENTAL PROCEDURE
- Preparation of the cells:
LuSens cells from the working cell bank were thawed and cultured using culture medium1 (37°C, ca 5%CO2, >= 90% humidity) for >= 5 but < 15 passages prior to testing. Before substance incubation, cells were seeded in 96-well microtiter plates (120 µL of 0.83E+05 cells/mL cell suspensions) using culture medium 2 for incubation for 24h.
- MTT and luciferase assay:
After cell adaptation for 24h, cell culture medium 2 was aspirated and replaced with 150 µL of medium 3. 50 µL of test substance preparation or dilution was then applied to the cells. The final DMSO concentration in the test medium is 1%. Plates were sealed with semi-permeable plate sealers, and placed in the incubater for 48h. For the luciferase assay a luminescence compatible plate (white plate) was used. Additionally, a clear plate was treated in parallel for the determination of cell viability.
Visual inspections: on each test substance concentration to check for precipitates.
Luciferase assay: supernatant is removed from the white plate. Cells are washed 2x with 300 µL PBS (with Ca2+/Mg2+). Subsequent addition of 200 µL Steady-Glo preparation (= 100 µL PBS (without Ca2+/Mg2+)) per well. Cells were shaken for 10 min at room temperature in darkness. After incubation, the luminescence is recorded.
Cell viability assay MTT: cell culture medium was aspirated from the wells and the cells were washed twice with 300 µL PBS (with Ca2+/Mg2+). Addition of 200 µL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution to each well and further incubation of the sealed plates for 2 hours. Analysis of the cells by aspiration of the medium and cell lysis using 100 µL of lysis solution. Absorbance measured at 570 nm with reference wavelength 690 nm.
- Experiments: 3
- Replicates: 3

DATA EVALUATION
- CV 75 Calculation: 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.
- % relative cell viability = (absorbance of test substance treated cells - absorbance of blank) / (absorbance of mean vehicle control treated cells - absorbance of blank) * 100
- Luciferase fold induction = (test substance treated cells - background without cells) / (mean vehicle control treated cells - background without cells).
- Statistical analyses: for the luciferase fold induction, the excel-function T.TEST was used.

ACCEPTANCE CRITERIA
- The cells viability of vehicle control must be at least 90%
- The mean of the positive control EGDMA should achieve ≥ 2.50 fold induction
- The mean of the LA (negative control) < 1.50
- The mean of the viability must be ≥ 70%
- The cell viability (%) of luminescence in the vehicle control wells for each plate should be < 20%
- The mean of the basal expression of the cells must be < 1.50 as compared to the solvent control.
- The positive, negative and vehicle control data must lie within the range of the historic data.

EVALUATION OF RESULTS
A test substance is concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds a 1.50 fold induction of statistical significance with respect to the vehicle control at concentrations that do not reduce viability below 70% in at least consecutive concentrations 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).

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Mean of 3 experiments

Parameter:

other: relative cell viability

Value:

70

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Parameter:

other: EC1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

After 48h of exposure to the test substance, luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. For a full overview of the experimental results, please see below.

Any other information on results incl. tables

1. Preliminary cytotoxicity assessment:

Results of preliminary cytotoxicity assessment:
Concentration (active ingredient) µg/mL	Concentration (test substance) µg/mL	mean OD 570-690 of 3 replicates	Mean rel. Viability (%)
VC	VC	0.392	100.0
0.5	0.5	0.335	85.5
1	1	0.326	83.1
5	5	0.353	90.0
10	10	0.359	91.7
50	50	0.327	83.3
100	100	0.319	81.4
500	501	0.328	83.8
1000	1001	0.321	82.0
2000	2002	0.098	24.9

The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 1123 µg/mL.

Hence, the highest tested concentration is 1123 * (1.2)^3 = 1941 µg/mL.

2. Experiment 2: Mean values and standard deviations of luciferase induction and rel. viability as well as p-value of t-test. Concentration with fold induction above 1.50 with rel. viabilty ≥ 70% and with statistical significance are indicated in bold.

Concentration (test substance) µg/mL	Fold induction		Rel. Viability (%)		t-test
	mean	SD	mean	SD	p-value	markers
542	1.22	0.10	90.9	5.3	0.024	*
650	1.15	0.21	97.9	6.2	0.176	n.s.
780	1.16	0.19	103.8	4.2	0.145	n.s.
936	1.06	0.12	97.3	9.4	0.252	n.s.
1123	1.11	0.13	91.7	8.6	0.142	n.s.
1348	1.04	0.04	96.4	8.7	0.107	n.s.
1618	1.12	0.06	88.0	7.5	0.022	*
1941	1.17	1.00	30.4	22.6	0.398	n.s.
VC	1.00	0.09	100.0	6.8	-	-
EGDMA (18 µg/mL)	8.12	0.91	96.8	4.3	0.000	**
LA (450 µg/mL)	0.83	0.08	101.1	6.7	0.002	**

3. Experiment 3: Mean value and standard deviations of luciferase induction and rel. viability as well as p-values of t-test. Concentration with fold inductions above 1.50 with rel. viabilty ≥ 70% and with statistical significance are indicated in bold.

Concentration (test substance) µg/mL	Fold induction		Rel. Viability (%)		t-test
	mean	SD	mean	SD	p-value	markers
542	1.23	0.14	83.6	2.4	0.046	*
650	1.48	0.17	77.8	3.5	0.015	*
780	1.43	0.09	87.3	5.7	0.001	**
936	1.22	0.13	82.8	3.2	0.044	*
1123	1.47	0.13	81.4	4.6	0.008	**
1348	1.08	0.08	86.7	4.2	0.112	n.s.
1618	1.23	0.10	88.8	5.8	0.019	*
1941	1.46	0.19	78.4	3.2	0.022	*
VC	1.00	0.14	100.0	5.3	-	-
EGDMA (18 µg/mL)	7.52	0.27	105.9	7.5	0.000	**
LA (450 µg/mL)	0.86	0.16	107.5	4.1	0.060	n.s.

4. Experiment 4: Mean value and standard deviations of luciferase induction and rel. viability as well as p-values of t-test. Concentration with fold inductions above 1.50 with rel. viabilty ≥ 70% and with statistical significance are indicated in bold.

Concentration (test substance) µg/mL	Fold induction		Rel. Viability (%)		t-test
	mean	SD	mean	SD	p-value	markers
542	1.12	0.12	88.5	8.5	0.109	n.s.
650	1.12	0.16	84.0	8.7	0.168	n.s.
780	1.22	0.25	92.4	1.7	0.134	n.s.
936	1.24	0.07	86.4	6.7	0.002	**
1123	1.22	0.23	93.0	5.4	0.118	n.s.
1348	1.22	0.07	86.8	6.4	0.002	**
1618	1.25	0.04	81.2	7.8	0.000	**
1941	1.25	0.10	45.0	13.9	0.017	*
VC	1.00	0.18	100.0	9.1	-	-
EGDMA (18 µg/mL)	6.99	0.76	101.4	8.1	0.000	**
LA (450 µg/mL)	1.11	0.12	110.4	6.1	0.067	n.s.

Mean of 3 experiments

Concentration (test substance) µg/mL	Fold induction		Rel. Viability (%)
	mean	SD	mean	SD
542	1.19	0.06	87.67	3.72
650	1.25	0.20	86.57	10.29
780	1.27	0.14	94.5	8.45
936	1.17	0.10	88.83	7.55
1123	1.27	0.18	88.7	6.36
1348	1.11	0.10	89.97	5.57
1618	1.20	0.07	86.00	4.18
1941	1.29	0.15	50.6	23.51

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item does not have keratinocyte activating potential. Therefore, the test substance is to be considered as a non-sensitiser.

Executive summary:

In the current study the skin sensitising potential of the test item was assessed according to OECD 442D without significant deviations and GLP.

The keratinocyte activating potential of test substance was evaluated in the LuSens assay. The test substance was incubated with a luciferase reporter cell line (LuSens cells) for 48 hours at 37°C and the antioxidant response element (ARE) dependent luciferase activity was measured.

In order to determine the concentrations suitable for the main experiment a pre-test was performed in which cells were exposed to 9 concentrations and the cytotoxicity was determined by MTT assay. The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

In the main test the luciferase activity was measured and in parallel a MTT assay was performed to assess the cytotoxicity of the test substance. The experiment was done in triplicate. The test substance was dissolved in 4% DMSO and in the end 1% DMSO was present in culture at all concentrations. No precipitates were noticed in any preparations.

The acceptance criteria were met.

Exposure to the test substance did not induce a statistical significant 1.5 fold increase in luciferase activity in LuSens cells while 70% viability was reached. In other words, the test item did not show any keratinocyte activating potential.