Ethanol, 2-(ethenylthio)-

Endpoint summary

Description of key information

The test substance is not peptide reactive, activates keratinocytes and activates dendritic cells. Therefore the test substance is considered to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-02-01 to 2016-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

Principles of method if other than guideline:

For data evaluation a borderline range was included as an amendment to the evaluation given in the OECD guideline.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 230-2015

Details on the study design:

Skin sensitisation (In chemico test system)
Synthetic peptides used:
- Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
- Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

Controls used:
- Negative (vehicle) control: acetonitrile
- Positive control: Ethylene glycol dimethacrylate (EGDMA) 50 mM in acetonitrile
- Co-elution control: Sample prepared of the respective peptide buffer and the test
substance but without peptide.

Test substance preparation:
The test substance was prepared as a 100 mM preparation in acetonitrile. After short stirring the test substance was soluble in the vehicle.

Peptide stock solution preparation:
Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide).

Experimental procedure:
Three samples of the test substance in acetonitrile were incubated with each peptide for 24h at room temperature. The incubation tubes were sealed. Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

Sample preparation:
The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).

Vehicle control preparation:
Several vehicle controls were prepared in triplicates in the same way as the test-substance samples but with vehicle instead of the test substance: One set (set A) was analyzed 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 vehicle.

Co-elution control preparation:
The co-elution control was prepared in the same way as the test substance samples but without the peptide. Instead the respective peptide buffer was used.

HPLC conditions:
- Prior to HPLC analysis the samples were visually investigated for any precipitate that may have formed during the exposure period. If the samples were visually turbid or displayed precipitates they were centrifuged or filtered prior to injection into the HPLC in order to remove any unsolved particles. The HLPC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.
- Device: Liquid chromatograph Thermo Scientific, Dionex Ultimate 3000
- Column: ZORBAX SB-C18 2.1 x 100 mm, 3.5 μm with guard column SecurityGuard Ultra Cartridges, UHPLC C18 for 4.6 mm ID (Phenomenex)
- Mobile phase A: de-ionized water/ acetonitrile/ triflouracetic acid 950/50/1 V/V/V
- Mobile phase B: acetonitrile/ de-ionized water/ triflouracetic acid 950/50/0.85 V/V/V
- Flow rate: 0.5 mL/min
- Wavelength: 220 and 258 nm
- Injection volume: 2 µL

Calculation and data evaluation:
The mean peptide depletion for each of the two peptides is calculated as the mean value of the three samples conducted for each peptide and test substance (C-containing and K- containing peptide depletion).
When samples were additionally analyzed by measuring UV absorbance at 258 nm, the area ratio 220 nm / 258 nm was calculated and served as a measure of peak purity. The ratio of a pure peptide peak should be consistent over all samples (100% ± 10% of the mean of the vehicle controls). However, due to small peak areas calculation of the area ratio may not be possible for all samples.

Acceptance criteria:
- 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).
- In addition the positive control should cause depletion of both peptides comparable to historic data.

Evaluation of results:
Chemical reactivity was determined by mean peptide depletion [%] and was rated as high, moderate, low, or minimal.
A mean peptide depletion of > 42.47 % is regarded as high reactivity and positive for peptide depletion (indication for sensitization potential). A moderately reactive test substance (22.62 % < mean peptide depletion ≤ 42.47 %) and a test substance of low reactivity (8.11% < mean peptide depletion ≤ 22.62 %) are regarded as positive for peptide depletion (indication for sensitization potential). A mean peptide depletion of > 4.65 % ≤ 8.11 % is considered no to low reactivity and borderline for sensitization, while ≤ 4.65 % is a minimal to no reactivity and negative for peptide depletion (no indication for sensitization potential).

Positive control results:

The positive control caused depletion of both peptides comparable to historic data.

Key result

Run / experiment:

mean

Parameter:

other: combined peptide depletion [%]

Value:

0.37

Vehicle controls validity:

valid

Remarks:

0.00 %

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

29.84 %

Remarks on result:

no indication of skin sensitisation

Run / experiment:

mean

Parameter:

other: cysteine peptide depletion [%]

Value:

0.74

Vehicle controls validity:

valid

Remarks:

0.00 %

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

47.61 %

Remarks on result:

no indication of skin sensitisation

Run / experiment:

mean

Parameter:

other: lysine peptide depletion [%]

Value:

-0.03

Vehicle controls validity:

valid

Remarks:

0.00 %

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

12.06 %

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The test substance was dissolved in acetonitrile. The samples of the test substance with the peptides were solutions. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. No co-elution of the test substance and peptides occurred.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Interpretation of results:

GHS criteria not met

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-02-01 to 2016-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD: Draft Proposal for a New Guideline on In Vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT)

Version / remarks:

accessed on 01 Sep 2014

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation Human Cell Line Activation Test (h-CLAT))

Version / remarks:

2016

Deviations:

not specified

Principles of method if other than guideline:

In addition, the h-CLAT was performed according to the methods described in the following publications:
- Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa , Ito Y, Nishiyama N, Itagaki H. (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 H, Ryan C, Ovigne JM, Schroeder KR, Ashikaga T. (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.

For data evaluation a borderline range was included as an amendment to the evaluation given in the OECD guideline

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 230-2015

Details on the study design:

Skin sensitisation (In vitro test system)
Cell line used: THP-1 cells, The human monocytic leukemia cell line was obtained from “American Type Culture Collection, Manassas, USA” (ATCC, TIB-202).

Technical material and conditions:
- For cell culture standard laboratory equipment was used.
- Flow cytometer: FC 500 MPL (Beckman Coulter) with CXP Software (Beckman Coulter) and Guava easyCyteTM HT (Millipore) with guavaSoft software InCyte
(Millipore) (pre-test only)
- Culture medium: RPMI 1640: with L-glutamine, 25mM HEPES + 10% FBS inactivated + 1% Penicillin/Streptomycin + 0.05 mM 2-Mercaptoethanol
- Buffer: Phosphate Buffered Saline (DPBS) without Ca2+ / Mg2+ + 0.1% BSA
- Blocking Solution 0.01% Globulins Cohn fraction II,III
- Antibodies: FITC anti-human CD54 (DAKO/DAK-F714301); FITC mouse IgG1 (DAKO/DAK-X092701); FITC anti-human CD86 (BD Pharmingen 555657)
- Antibody working solutions: for CD86 6 μL Antibody + 44 μL Buffer; for CD54 and lgG1 3 μL Antibody + 47 μL Buffer
- Reagent for cytotoxicity test: Propidium iodide

Controls used:
- Negative control: Lactic acid, 1000 μg/mL in culture medium
- Vehicle control: culture medium
- Positive control: 1- chloro-2,4-dinitrobenzene (DNCB, CAS no.: 97-00-7), 4.0 μg/mL in 0.2% DMSO in 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.

Test substance preparation:
The test substance was weighed and topped up with the chosen vehicle (culture medium ) to achieve the required 2x concentration of the highest concentration (stock solution). Further concentrations were prepared as 2x concentrations by serial 1:1.2 dilution according to the planned concentrations. The test-substance preparations were prepared by stirring.

Selection of concentrations
In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 10 concentrations of the test-substance preparation (0.5 μg/mL up to 5006 μg/mL) corresponding to final test substance concentrations of 0.5 μg/mL up to 5000 μg/mL (taking the purity into account) and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The 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 2455 μg/mL. The highest tested concentration in the 1st main experiment was 1.22 fold of the CV75 value. The additional concentrations were obtained by a 1:1.2 serial dilution series of the maximum concentration.

Experimental procedure
Two independent, valid experiments were performed. In each experiment, duplicates of each test-substance concentration were tested.

- Cell preparation:
THP-1 cells from the working cell bank were thawed and cultured in suspension using the cell medium 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).
Prior to use of the cells for a study, a reactivity check is performed with each new-thawed cells, as proposed in the OECD draft test guideline, using Nickel(II)sulfate hexahydrate, lactic acid and 1- chloro-2,4-dinitrobenzene in order to demonstrate qualification of the cells for the assay.

- Test substance application for MTT and luciferase assay:
Treatment was performed by adding 500 μL of test-substance preparation to the cells, thus diluting the 2x concentrated test substance preparations to their final concentration and the cells to 1.0 x 10^6 cells/mL. After test substance application the plates were sealed with semipermeable plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 24 hours.
Each test substance concentration was visually inspected directly after application and after the exposure period of 24 hours in order to detect test-substance precipitates.

- 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 buffer. Cells were incubated with 600 μL of 0.01% Globulins Chon fraction II,III at 4°C for 15 min 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, supernatant was discarded and 50 μL working antibody solution was added to each pellet. Cell staining was performed at 4°C for 30 min in the dark. After staining the cells were washed twice with 200 μL buffer and finally re-suspended in 200 μL buffer. Before analysis in flow cytometer the cells were stained with 5 μL of PI (50 μg/mL diluted in buffer) to yield a final concentration of 1.22 μg/mL PI.

Calculation and data evaluation:
The CV75-value (relative survival rate) was calculated by linear regression. The relative cell viability (PI staining) was calculated as a mean of the independent replicates. Analysis of the membrane markers was 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) is used. Data evaluation is performed with mean fluorescence intensity (MFI) of chemical treated cells among the viable cells, with systematic isotype control use to quantify and remove non-specific 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 is calculated. The results
are expressed as relative fluorescence intensity (RFI) of % CD86 pos. or % CD54 pos. expression compared to the respective vehicle control. 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 conducted. The calculation was performed by linear regression from the two concentrations directly above and below the EC150% / EC200% concentration.

Acceptance criteria:
- 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%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86≥ 150% and CD54 ≥ 200%) and cell viability should be ≥ 50%.
- In the negative control (LA), 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%.
- For all vehicle controls, the MFI ratio of both CD86 and CD54 to isotype controls should be ≥ 105%.
- The reactivity check of new thawed cells should produce a positive response in CD86 and CD54 for NiSO4 and DNCB and a negative response in CD86 and CD54 for LA.
- Positive, negative and vehicle control data should lie within the range of the historic data.

Evaluation of results:
The test substance is considered positive for activation of monocytic THP-1 cells (indication for sensitization potential) when CD86 expression is increased ≥ 150% and/or CD54 expression increased ≥ 200% at any concentration in relation to vehicle control that do not reduce viability below 50% and reproduced in the same cell surface marker in at least two independent experiments and at least one concentration must lie above the borderline area (> 170% (CD86) or 220% (CD54)).

If those conditions were not met and at least one of these concentrations was below the borderline area (< 130% (CD86) or < 180% (CD54)), the test substance was considered negative. For consideration as negative the use of sufficiently high concentrations had to be demonstrated by at least one concentration displaying viability below 50% or the application of the maximum (applicable) concentration. In addition the borderline criteria must not be met.
A borderline result was obtained when RFI was within the historic margin of deviation of ± 20% around the cut off value (130% - 170% for CD86; 180% - 220% for CD54) at concentrations that did not reduce viability below 50% of two independent experiments.
After conduction of at least two and a maximum of three independent valid and evaluable experiments, the result was inconclusive if no final evaluation was possible (e.g. one experiment positive, negative and borderline).

Positive control results:

The results of the positive control were within the historical control range.

Key result

Run / experiment:

other: Experiment 1 [µg/mL]

Parameter:

other: EC200% (CD54)

Remarks:

(the concentration resulting in a RFI of 200%)

Value:

1 093

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 2 [µg/mL]

Parameter:

other: EC200 % (CD54)

Remarks:

(the concentration resulting in a RFI of 200%)

Value:

1 351

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 was not applicable as fold inductions above 150% were obtained in all tested concentrations in experiment 1 (viability ≥50%) and up to the lowest concentration in experiment 2 (viability ≥ 50%).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Concentration (test substance) μg/mL	1st experiment			Concentration (test substance) μg/mL	2nd experiment
	RFI CD86 mean [%]	RFI CD54 mean [%]	Viability rel. Viability [%]		RFI CD86 mean [%]	RFI CD54 mean [%]	Viability rel. Viability [%]
987	183.2	193.4	99.2	987	16.3	170.2	98.1
1184	177.6	205.6	98.5	1184	175.6	164.2	95.9
1421	188.5	209.5	96.2	1421	163.9	214.8	95
1705	179.4	301.2	95.5	1705	154.8	235.9	92.9
2046	181.9	310.7	92.9	2046	152.7	248.4	88.8
2455	182.4	332.9	86	2455	158.9	246.3	75.5
2946	192.4	595	66.1	2946	111.8	433.8	59.1
3535	179.5	502.6	41.2	3535	85.9	108.4	31.4
VC	100	100	100	VC	100	100	100
LA (1000 µg/mL)	67.1	111	99.9	LA (1000 µg/mL)	98.5	98.5	99.7
DNCB (4 µg/mL)	249.9	641.6	85.4	DNCB (4 µg/mL)	294.6	294.6	89

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-02-01 to 2016-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

Principles of method if other than guideline:

For data evaluation a borderline range was included as an amendment to the evaluation given in the OECD guideline.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 230-2015

Details on the study design:

Skin sensitisation (In vitro test system)
Cell line used: LuSens, Human transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen

Technical material and conditions:
- For cell culture standard laboratory equipment was used.
- Luminometer: TriStar² Multimode reader LB 942 (Berthold)
- Spectralphotometer: TriStar² Multimode reader LB 942 (Berthold)
- Culture medium 1: D-MEM + 10 % FBS + 1 % Penicillin /Streptomycin, Puromycin dihydrochloride 25 μL
- Culture medium 2: D-MEM + 10 % FBS
- Culture medium 3: D-MEM + 1 % FBS
- All buffers consisted of PBS containing Ca2+ / Mg2+. 0.05 % EDTA was added for one buffer.
- Lysisbuffer: 10 g SDS, 99.6 mL DMSO, 0.4 mL glacial acetic acid
- Luciferace reagent : SteadyGloLuciferase Assay, (Cat. No Promega E2520)
- MTT Solution: Thiazolyl Blue Tetrazolium Bromide 5 mg/mL with PBS without Mg2+/Ca2+; diluted 1 :10 with medium 3 for the assay

Controls used:
- Negative control: DL-Lactic acid (LA), 450 μg/mL in 1% DMSO in culture medium 3
- Vehicle control: 1% DMSO in culture medium 3
- Positive control: Ethylene glycol dimethacrylate (EGDMA) 18 μg/mL in 1% DMSO in culture medium 3
- Blank control: Culture medium 3 without cells
- Basal control: Culture medium 3 with cells

Test substance preparation:
The test substance was weighed and topped up with the chosen vehicle (4% DMSO in culture medium 3) to achieve the required 4x concentration of the highest concentration (stock solution). Further concentrations were prepared as 4x concentrations by serial 1:1.2 dilution according to the planned concentrations (master plate).The test substance preparations were prepared by stirring.

Selection of concentration
In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 9 concentrations of the test-substance preparation (0.5 μg/mL up to 2002 μg/mL) corresponding to final test substance concentrations of 0.5 μg/m up to 2000 μg/mL taking the purity into account) and cytotoxicity was determined by MTT assay. The 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 209 μg/. The highest tested concentration in the 1st main experiment was 1.2^3 fold of the CV75 value. The additional concentrations were obtained by a 1:1.2 serial dilution series of the maximum concentration.
As no positive effect and no relative cell viability below 70% was observed in the 1st experiment, higher concentrations were chosen for the 2nd and 3rd experiment using 16 concentrations and two test plates.

Experimental procedure
Three independent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested.
- Cell preparation:
LuSens cells from the working cell bank were thawed and cultured using culture medium 1, 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 2 for incubation for 24 hours.

- Test-substance application for MTT and luciferase assay:
After cell adaption for 24 hours cell culture medium 2 was aspirated and replaced with 150 μL medium 3. Each test substance preparation of the dilution plate was applied in a ratio of 1:4 (50 μL) to the cells (final DMSO concentration in the test medium = 1%).
After test substance application the plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance. The 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.
Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 hours in order to detect test-substance precipitates.

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

- Cell viability assay MTT:
Cell culture medium was aspirated from all wells. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Thereafter 200 μL of a 0.5 mg/mL MTT solution 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. Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectralphotometer.

Calculation and data evaluation:
The CV75-value (relative survival rate) was calculated by linear regression. The relative cell viability and luciferase fold induction was calculated as a mean of the three independent replicates. The statistical evaluation of luciferase fold induction was performed using the EXCEL-function T.TEST. The concentration resulting in a positive response (1.50 fold induction of statistical significance and viability >70%) was calculated from each experiment conducted. The calculation was performed by linear regression from the two concentrations directly above and below the EC1.50 concentration.

Acceptance criteria:
- A tested concentration was not further evaluated when relative viability was less than 70%.
- The cell viability of VC cells must yield at least 85%.
- The mean of the positive control EGDMA should achieve ≥2.50 fold induction and the mean of the LA <1.50 and the mean of the viability must be ≥70%.
- The CV [%] of the luminescence in the vehicle control wells for each plate should be below 20%.
- The mean of the basal expression of the cells must be <1.50 as compared to the solvent control.
- In addition, positive, negative and vehicle control data should lie within the range of the historic data.

Evaluation of results:
The test substance is considered positive for keratinocyte activation (indication for sensitization potential) when a statistically significant induction of luciferase activity ≥ 1.50 with respect to the vehicle control was determined in at least two consecutive concentrations that did not reduce viability below 70% of two independent experiments and at least one of these concentrations was above the borderline area (> 1.65).
If those conditions were not met and at least one of these concentrations was below the borderline area (< 1.35), the test substance was considered negative. For consideration as negative the use of sufficiently high concentrations had to be demonstrated by at least one concentration displaying viability below 70% or the application of the maximum (applicable) concentration. In addition the borderline criteria must not be met.
A borderline result was obtained when induction of luciferase activity with respect to the vehicle control was in the historic margin of deviation of the cut off value 1.50 (1.35 – 1.65) in at least two consecutive concentrations that did not reduce viability below 70% of two independent experiments.
After conduction of at least two and a maximum of three independent valid and evaluable experiments, the result was inconclusive if no final evaluation was possible (e.g. one experiment positive, negative and borderline).

Positive control results:

The results of the positive control were within the historical control range.

Key result

Run / experiment:

other: Experiment 2 [µg/mL]

Parameter:

other: EC1.50

Remarks:

(the concentration resulting in a 1.50-fold luciferase induction)

Value:

204

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 3 [µg/mL]

Parameter:

other: EC1.50

Remarks:

(the concentration resulting in a 1.50-fold luciferase induction)

Value:

336

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 2 plate 1, 521 µg/mL

Parameter:

other: fold induction

Value:

1.68

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: and relative viability 73.8 %

Run / experiment:

other: Experiment 2 plate 1, 626 µg/mL

Parameter:

other: fold induction

Value:

1.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: and relative viability 75.4 %

Run / experiment:

other: Experiment 2 plate 2, 209 µg/mL

Parameter:

other: fold induction

Value:

1.55

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: and relative viability 93.6 %

Run / experiment:

other: Experiment 2 plate 2, 302 µg/mL

Parameter:

other: fold induction

Value:

1.51

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: and relative viability 101 %

Run / experiment:

other: Experiment 3 plate 1, 434 µg/mL

Parameter:

other: fold induction

Value:

1.73

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: and relative viability 76.3 %

Run / experiment:

other: Experiment 3 plate 1, 521 µg/mL

Parameter:

other: fold induction

Value:

1.97

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: and relative viability 73.9 %

Run / experiment:

other: Experiment 3 plate 2, 362 µg/mL

Parameter:

other: fold induction

Value:

1.51

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: and relative viability 78.0 %

Other effects / acceptance of results:

The 1st experiment was not useful for evaluation as the concentrations selected were too low (no viability < 70% or keratinocyte activating effect were observed).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Experiment 1

Concentration (test substance) μg/mL	2nd experiment plate 1				Concentration (test substance) μg/mL	2nd experiment plate 2
	fold induction mean	rel. viability [%] mean	t-test p-value	markers		fold induction mean	rel. viability [%] mean	t-test
								p-value	markers
434	1.49	80.3	0.041	*	101	1.14	87	0.036	*
521	1.68	73.8	0	**	121	1.28	86.9	0.024	*
626	1.71	75.4	0	**	145	1.28	87.5	0.032	*
751	1.51	65.2	0.022	*	175	1.21	87.1	0.081	n.s.
901	2.14	62.4	0	**	209	1.55	93.6	0.009	**
1081	2.07	53.1	0.005	**	251	1.37	101.3	0.022	*
1297	2.27	41.9	0.006	**	302	1.51	101	0.038	*
1557	2.18	34.9	0	**	362	1.48	97.6	0.021	*
VC	1	100	-	-	VC	1	100	-	-
EGDMA (18 µg/mL)	5.17	95.6	0	**	EGDMA (18 µg/mL)	4.24	90.5	0	**
LA (450 µg/mL)	0.9	93.2	0.109	n.s.	LA (450 µg/mL)	0.96	91.4	0.238	n.s.

 

Experiment 2

Concentration (test substance) μg/mL	3rd experiment plate 1				Concentration	3rd experiment plate 2
	fold induction mean	rel. viability [%] mean	t-test		(test substance) μg/mL	fold induction mean	rel. viability [%] mean	t-test
			p-value	markers				p-value	markers
434	1.73	76.3	0.012	*	101	1.17	96.2	0.064	n.s.
521	1.97	73.9	0.001	**	121	1.19	89	0.006	**
626	1.84	69	0.001	**	145	1.37	90.8	0	**
751	1.83	61.7	0.003	**	175	1.26	87.3	0.02	*
901	2.05	56.6	0.006	**	209	1.4	84.2	0.038	*
1081	2.16	51.5	0.004	**	251	1.42	84.6	0.3	*
1297	2.67	40.4	0.006	**	302	1.48	83.8	0.02	*
1557	2.94	32.6	0.01	**	362	1.51	78	0	**
VC	1	100	-	-	VC	1	100	-	-
EGDMA (18 µg/mL)	4.82	90.3	0	**	EGDMA (18 µg/mL)	4.37	97.1	0	**
LA (450 µg/mL)	0.82	100	0	**	LA (450 µg/mL)	0.78	99.3	0.005	**

 

Statistical significance of luciferase fold induction is indicated by asterisk (* for p < 0.05, ** for p < 0.01; n.s. = no statistical significance).

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

For the evaluation of the skin sensitisation potential of the test substance a test battery of non in vivo experiments was performed, as a single in virto/in chemico test is not sufficient for adequate judgment. Together the experiments cover all 3 key events of skin sensitisation. The protein reactivity is addressed with the Direct Peptide Reactivity Assay (DPRA). The Keratinocyte Activation Assay LuSens assay evaluates keratinocyte activation and the Human Cell Line Activation Test (h- CLAT) determines activation of dendritic cells. An evaluation of all tests in a Weight of Evidence approach is used to reach a final conclusion on the skin sensitisation potential of the test substance and its need for classification.

 

DPRA

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)- containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA) according to OECD 442C. The test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover, the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. No co-elution of test substance and peptides was noticed. The mean C-peptide depletion, caused by the test substance was determined to be 0.74%. The mean K-peptide depletion, caused by the test substance was determined to be -0.06%. Negative depletions were considered to be “zero” for calculation of the mean combined peptide depletion, which was thus calculated to be 0.37%. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows a minimal chemical reactivity in the DPRA under the test conditions chosen and was considered to be not skin sensitising.

 

LuSens

The keratinocyte activating potential of test substance was evaluated in the LuSens assay. For this purpose, the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to 9 concentrations of the test substance and 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 to be 209 μg/mL (corresponding to test substance as provided by the sponsor). In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 3 valid experiments were performed. However, in the 1st experiment the concentrations selected were too low (no viability < 70% or keratinocyte activating effect were observed) and the experiment was not used for evaluation. The test substance was soluble in 4% DMSO in culture medium 3 (4 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours. Relative luciferase fold induction and concurrent relative viability were determined in the main experiments. The EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was calculated to be 204 μg/mL (experiment 2) and 336 μg/mL (experiment 3), respectively. After 48 hours of exposure to test substance luciferase activity in LuSens cells was induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance has a keratinocyte activating potential. Thereby a potential for skin sensitization is indicated.

 

h-CLAT

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h- CLAT). For this purpose, the test substance was incubated with human monocytic leukemia cell line THP-1 for ca. 24 hours at 37°C and membrane marker expression (CD86 / CD54) was measured by flow cytometry. In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to 10 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 2455 μg/mL (corresponding to test substance as provided by the sponsor). In the main test after 24-hour exposure THP-1 cells were stained with FITC labeled anti-human- CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 2 valid experiments were performed. The following results were observed: At concentrations used in the main experiment the test substance was soluble in culture medium (2 x stock preparations and final concentrations). No precipitates were noticed in any concentration after 24 hours. Relative fluorescence intensity and concurrent relative viability were determined in the main experiments. Calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 was not applicable as fold inductions above 150% were obtained in all tested concentrations in experiment 1 (viability≥50%) and up to the lowest concentration in experiment 2 (viability≥50%). The EC200% (the concentration resulting in a RFI of 200%) for CD54 was calculated to be 1093 μg/mL (experiment 1) and 1351 μg/mL (experiment 2). In summary, after 24 hours of exposure to test substance CD86 and 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 and shows a potential for skin sensitisation.

 

Conclusion

For protein reactivity in the Direct Peptide Reactivity Assay (DPRA) a negative result was determined which indicated no skin sensitsation potential of the test substance. The Keratinocyte Activation Assay LuSens assay concluded a positive result for keratinocyte activation. As those two tests resulted in contradictory effects an additional assay addressing the third key event was conducted to reach a final conclusion. This Human Cell Line Activation Test (h- CLAT) showed an activation of dendritic cells indicating skin sensitisation potential. Therefore an overall conclusion was determined that the test substance is predicted to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218. As a result the substance is considered to be classified for skin sensitisation Category 1 (H317: May cause an allergic skin reaction).