O-(ethoxycarbonyl)-N-(1-methyl-2-oxo-2-phenylethylidene)hydroxylamine

Endpoint summary

Description of key information

Skin Sensitisation: DPRA, Fleet (2017)

Under the conditions of this study the test material was positive in the DPRA (moderate reactivity class).

Skin Sensitisation: h-CLAT, Roth (2018)

Under the conditions of this study, the test material activated THP-1 cells, therefore the test material is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Skin Sensitisation: LLNA, Pooles (2018)

Under the conditions of this study the test material is sensitising to skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 November 2017 to 30 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

July 2016

Deviations:

yes

Remarks:

The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry. Proficiency substances have been tested at the facility, and demonstrated that this has no impact on the results.

Principles of method if other than guideline:

In case of a very different result between the cytotoxicity of the dose range finder and the main experiment, due to the deviation, the test material concentrations for the main experiments are adjusted accordingly.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need for in vivo testing. One of the validated in vitro skin sensitisation tests is the h-CLAT, which is recommended in international guidelines (e.g. OECD).

Specific details on test material used for the study:

- On the day of the experiment (immediately prior to start) the test material was stably suspended/dispersed in culture medium.
- The maximum concentration of test material was 625 μg/mL in culture medium, as tested by a solubility test.
- For the XTT test (dose finding assay) eight concentrations of the test material were analysed. For this, dilutions were prepared by 1:2 serial dilutions from 625 μg/mL culture medium.

Details on the study design:

TEST SYSTEM
- Reasons for the Choice of THP-1 Cells: THP-1 cells (Human monocytic leukaemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers.
- THP-1 Cell Cultures: Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of the testing facility (aliquots of cells in freezing medium at 1 × 10^6 to 2 × 10^6 cells/mL) allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly. The cell density should not exceed 1 × 10^6 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30). The passage numbers of the used THP-1 cells were 17 and 13 in the XTT assay, and 19 and 16 in the h-CLAT for runs 1 and 2, respectively.
- Culture Medium: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1 % (v/v) sodium pyruvate, 1 % (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) is used to culture the cells during the assay. Medium with supplements has to be stored at 2 – 8 °C and used within one month. The culture medium has to be warmed to room temperature just before use.
- Preparation and Seeding of THP-1 Cells: On the day of the cytotoxicity experiment (XTT) directly before the application of the test material, solvent and medium control, a volume of 100 μL with a cell density of 0.9 - 1 × 106 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate. For the main experiment (h-CLAT) 0.9 - 1 × 10^6 cells/well in a volume of 500 μL were seeded in a 24-well plate before the treatment.


EXPERIMENTAL DESIGN AND PROCEDURES OF XTT
- Dose Finding Assay (XTT Test): The test material concentrations investigated in the main experiment (h-CLAT) were determined with two XTT tests.
- The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [= (sodium 3'-(1-phenylaminocarbonyl) - (3,4 - tetrazolium) – bis - (4 – methoxy – 6 - nitro) - benzenesulfonic acid hydrate)] to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. Two independent cytotoxicity experiments were performed with different cell cultures and on different days to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT).
- XTT Labelling Mixture: The XTT labelling mixture consists of two components, a XTT buffer solution and the substrate solution. Both components were mixed right before application at a ratio of 1:100.
-Treatment: After the cell seeding, 100 μL of the test material dilutions, the medium and solvent controls, respectively, were added to the cells. All dose groups were tested in 7 replicates for each XTT test. At the end of the incubation period of 24 ± 0.5 hours, the cell cultures were microscopically evaluated for morphological alterations.
- XTT Labelling and Measurement: At the end of the incubation period, 50 μL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wavelength 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).
- Evaluation of the XTT results: A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. The relative absorbance (= viability in [%]) as compared to the solvent control is calculated using this formula:

Relative absorbance [%] = 100 x [(mean absorbance of test material – absorbance of chemical blank) / (mean absorbance of solvent control – absorbance of chemical blank)]

To calculate the concentration of toxicant needed to reduce the relative absorbance to 75 % of the solvent control (CV75) the following formula is used:

CV75 = Conc.>75 – [[(Conc.>75 – Conc.<75) x (%>75 – 75)] / (%>75 - %<75)]

a) Conc. >75 = max. measured concentration with the % of solvent control >75 %
b) Conc. <75 = min. measured concentration with the % of solvent control <75 %
c) % >75 = relative absorbance at a) in %
d) % <75 = relative absorbance at b) in %

- Calculation of the h-CLAT Test Material Concentrations: Two independent cytotoxicity experiments were performed with different cell cultures and on different days to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT). Eight final concentrations (μg/mL) were used for the test material in the main experiment (h-CLAT). The highest concentration used was 1.2 × CV75 and seven further concentrations of the test material were prepared by serial 1:1.2 dilution.
- Acceptability of the Assay:
The XTT test is considered to be acceptable if it meets the following criteria: Mean absorbance of the medium control is ≥ 0.5 and mean viability of the solvent control is ≥ 90 % in comparison to the medium control.


EXPERIMENTAL DESIGN AND PROCEDURES OF h-CLAT
- The test material was tested in two independent runs.
- Treatment of the Cells: For the test material exposure the highest concentration calculated from the XTT assay was prepared corresponding to 1.2 × CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. An appropriate volume (500 μL) of the dilutions of the test material, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations. Each concentration of the test material, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).
- Staining of the Cells: The triplicates of each test material-treated and not test material-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1 % (w/v) BSA). hereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control). All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures. The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).
- Sample Preparation for Measurement: After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added.
- Flow Cytometry Acquisition: Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions. The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.
The following acquisition plots were prepared:
- 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
- Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such that the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson GmbH). The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.
- Acquisition: Dead cells were determined by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50 % (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).

- Data Analysis and Interpretation
Flow Cytometry Analysis: The RFI is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical:

RFI [%] = [(MFI of test material treated cells) – (MFI of test material treated isotype control cells)] / [(MFI of solvent control cells) – (MFI of solvent isotype control cells)] x 100

MFI = Geometric Mean Fluorescence Intensity (GeoMean)
The cell viability of the h-CLAT experiment is calculated as follows for each concentration of every chemical:

Cell Viability [%] = (Mean cytotox of solvent control cells / Mean cytotox of test material treated cells) x 100

Where the mean cytotox is the mean of GeoMean (7-AAD) isotype control, GeoMean(7-AAD) CD54 and GeoMean(&-AAD) CD86.

ACCEPTANCE CRITERIA
The following acceptance criteria should be met when using the h-CLAT method:
- Cell viability of medium control is adjusted to 100 % and the cell viability of the DMSO control should be more than 90 % in comparison to the medium control.
- In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %).
- For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105 %.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the cell viability should be > 50 % in at least one concentration of the two tested positive control concentrations.
- For the test material, the cell viability should be more than 50 % in at least four tested concentrations in each run.
- Negative results are acceptable only for test materials exhibiting a cell viability of < 90 % at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90 % the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test material, a negative result is acceptable even if the cell viability > 90 % (OECD 442E guideline).

PREDICTION MODEL
For CD86/CD54 expression measurement, each test material is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥ 150 % at any tested concentration (with cell viability ≥ 50 %);
− The RFI of CD54 is ≥ 200 % at any tested concentration (with cell viability ≥ 50 %).
Otherwise, the h-CLAT prediction is considered NEGATIVE.
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline).
- Test materials with a Log Pow > 3.5 tend to produce false negative results. Therefore negative results with test chemicals with a Log Pow > 3.5 should not be considered (according to the OECD guideline). However, positive results obtained with test materials with a Log Pow > 3.5 could still be used to support the identification of the test chemical as a skin sensitiser (OECD 442E guideline).

Positive control results:

At 2.0 µg positive control /mL the RFI CD54 antibody was 232.4 % and the RFI CD 86 antibody was 542.7 %.
At 3.0 µg positive control /mL the RFI CD54 antibody was 277.6 % and the RFI CD 86 antibody was 534.8 %.

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %) and the cell viability was >50 %.

Run / experiment:

other: XTT tests mean value (µg/mL)

Parameter:

other: CV75

Value:

328.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

other: CD86 first run (%)

Parameter:

other: RFI

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

other: CD54 first run (%)

Parameter:

other: RFI

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

other: CD86 second run (%)

Parameter:

other: RFI

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

RESULTS OF THE DOSE FINDING ASSAY (XTT TEST)
- Results of the first XTT test for the test material can be seen in Table 1. The mean viability of the solvent control in comparison to the medium control was 106.34 %.The CV75 value of the first XTT test was 412.6 μg/mL
- Results of the second XTT test for the test material can be seen in Table 2. The mean viability of the solvent control in comparison to the medium control was 108.85 %. The CV75 value of the second XTT test was 244.4 μg/mL
- The mean CV75 value of both XTT tests: 328.5 μg/mL

RESULTS OF THE h-CLAT TESTS
- Results of the first and second runs of the h-CLAT tests can be seen in Table 3 and 4, respectively.

DISCUSSION
- This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of the test material stably suspended/dispersed in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test material concentration for the main experiment (h-CLAT) was previously determined by two XTT tests.
- Cytotoxic effects were observed following incubation with the test material starting with the concentration of 625 μg/mL in the first XTT test and starting with the concentration of 312.5 μg/mL up to the highest tested concentration (625 μg/mL) in the second XTT test (threshold of cytotoxicity: < 75 %). The mean CV75 value of both XTT tests was calculated as 328.5 μg/mL.
- The following concentrations of the test material (stably suspended/dispersed in culture medium) were tested in the main experiment (h-CLAT): 110, 132, 158, 190, 228, 274, 328 and 394 μg/mL.
- The test material with a log Pow of 2.8 was tested in 2 independent runs. The RFI of CD86 and CD54 was greater than 150 and 200 %, respectively, in at least one concentration of the first run and the RFI of CD86 was greater than 150 % in at least one concentration of the second run. Therefore the h-CLAT prediction is considered positive for the tested test material in this h-CLAT.
- In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %) and the cell viability was >50 %.
- This human cell line activation test can be used as part of a testing battery (including e.g. the Direct Peptide Reactivity Assay, DPRA, and/or ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

CONCLUSION
- The test material with a log Pow of 2.8 activated THP-1 cells under the test conditions of this study. Therefore the test material is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Table 1: Results of the first XTT test for the Test Material

		Microscopic Evaluation	Photometric Evaluation
Test Group	Concen-tration [µg/mL]	Cytotoxicity	Mean Ab-sorbance*	Standard-Deviation	Chem. Blank	Mean Ab-sorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.709	0.040	0.225	0.484	94.04
Solvent Control	-	no	0.739	0.060	0.225	0.514	100.00
Test Item	4.9	no	0.755	0.044	0.225	0.530	103.04
	9.8	no	0.794	0.056	0.226	0.568	110.44
	19.5	no	0.757	0.044	0.223	0.534	103.73
	39.1	no	0.798	0.074	0.232	0.566	110.03
	78.1	no	0.778	0.062	0.222	0.555	107.97
	156.3	no	0.764	0.079	0.221	0.543	105.58
	312.5	no	0.656	0.061	0.192	0.464	90.12
	625	no	0.439	0.080	0.219	0.221	42.90

Shaded test groups: cytotoxic effects occurred in the photometric evaluation (< 75 % cell viability).

* = mean absorbance (absolute) of 7 wells

** = relative absorbance [rounded values]

Table 2: Results of the second XTT test for the Test Material

		Microscopic Evaluation	Photometric Evaluation
Test Group	Concen-tration [µg/mL]	Cytotoxicity	Mean Ab-sorbance*	Standard-Deviation	Chem. Blank	Mean Ab-sorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.600	0.016	0.237	0.363	91.87
Solvent Control	-	no	0.633	0.013	0.237	0.396	100.00
Test Item	4.9	no	0.615	0.012	0.234	0.382	96.45
	9.8	no	0.622	0.017	0.236	0.385	97.43
	19.5	no	0.634	0.026	0.235	0.399	100.86
	39.1	no	0.648	0.021	0.230	0.418	105.79
	78.1	no	0.642	0.030	0.236	0.406	102.73
	156.3	no	0.614	0.058	0.241	0.374	94.46
	312.5	no	0.480	0.072	0.243	0.237	59.96
	625	no	0.296	0.055	0.221	0.075	18.96

Shaded test groups: cytotoxic effects occurred in the photometric evaluation (< 75 % cell viability).

* = mean absorbance (absolute) of 7 wells

** = relative absorbance [rounded values]

Table 3: Results of the first h-CLAT test for the Test Material

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	100.0
DMSO Control	-	100.0	100.0	100.0
Positive Control (DNCB)	2.0	232.4*	542.7*	67.2
	3.0	277.6*	534.8*	70.0
Test Material	110	94.6	83.0	89.1
	132	108.1	116.7	88.8
	158	115.4	123.2	84.3
	190	118.8	124.6	80.3
	228	136.9	127.2	73.9
	274	155.0	159.8*	71.8
	328	147.7	208.0*	69.6
	394	222.1*	288.8*	58.8

* = RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥150 % and CD54≥200 %).

Table 4: Results of the second h-CLAT test for the Test Material

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	100.0
DMSO Control	-	100.0	100.0	100.0
Positive Control (DNCB)	2.0	210.6*	531.2*	73.0
	3.0	210.6*	490.2*	74.2
Test Material	110	92.2	92.0	90.5
	132	102.0	98.5	94.7
	158	93.2	88.7	86.4
	190	105.4	95.2	82.7
	228	102.9	99.0	79.5
	274	145.9	168.2*	68.7
	328	144.9	137.6	72.4
	394	194.1	259.1*	65.7

* = RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %).

Interpretation of results:

other: Positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP)

Conclusions:

Under the conditions of this study, the test material activated THP-1 cells, therefore the test material is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 422E, under GLP conditions.

The in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of the test material stably suspended/dispersed in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test material concentration for the main experiment (h-CLAT) of the test material was previously determined by two XTT tests.

Cytotoxic effects were observed following incubation with the test material starting with the concentration of 625 µg/mL in the first XTT test and starting with the concentration of 312.5 µg/mL up to the highest tested concentration (625 µg/mL) in the second XTT test (threshold of cytotoxicity: < 75 %). The mean CV75 value of both XTT tests was calculated as 328.5 µg/mL.

The following concentrations of the test material (stably suspended/dispersed in culture medium) were tested in the main experiment (h-CLAT): 110, 132, 158, 190, 228, 274, 328 and 394 µg/mL

The test material, with a log Pow of 2.8, was tested in 2 independent runs. The RFI of CD86 and CD54 was greater than 150 and 200 %, respectively, in at least one concentration of the first run and the RFI of CD86 was greater than 150 % in at least one concentration of the second run. Therefore the h-CLAT prediction is considered positive for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %) and the cell viability was >50 %.

Under the conditions of this study, the test material activated THP-1 cells, therefore the test material is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 March 2018 to 22 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: The animals were housed in suspended solid floor polypropylene cages furnished with softwood wood flakes.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: 30 – 70 %
- Air changes: At least fifteen changes per hour
- Photoperiod: Lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

50, 25 and 10 % w/w

No. of animals per dose:

Four females per dose.

Details on study design:

TEST MATERIAL PREPARATION
- For the purpose of the study, the test material was freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.
- The test material was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

PRE-SCREEN TESTS:
- As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test material at a concentration of 50 % w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included as Annex 5. Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
- The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25 % was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN STUDY
- Groups of four mice were treated with test material or the test material at concentrations of 50, 25 or 10 % w/w in acetone/olive oil 4:1. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
- A further group of four mice received the vehicle alone in the same manner.
- 3H-methyl thymidine administration: Five days following the first topical application of the test material or vehicle (Day 6) all mice were injected via the tail vein with 0.25 mL (250 µL) of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations:
- Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Ear Thickness and Daily Irritation observations: The thickness of each ear was measured pre dose on Day 1, post dose on Day 3 and on Day 6. Daily irritation observations were performed.
- Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
- Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.
- Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re pelleted. To precipitate out the radioactive material, the pellet was re suspended in 3 mL of 5 % Trichloroacetic acid (TCA).
- Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

DATA EVALUATION
- The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
- The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitiser".
- The EC3 value was also calculated. The EC3 value is the concentration of test material expected to cause a 3 fold increase in 3HTdR incorporation. - For chemicals, where the lowest concentration tested resulted in a stimulation index of greater than 3, an EC3 value is extrapolated from the two lowest doses utilised. The extrapolated EC3 value was calculated by log linear interpolation between the two points on a plane where the α axis represents the dose level and the γ axis represents the stimulation index. The point with the higher stimulation index was denoted (a, b) and the point with the lower stimulation index was denoted (c, d). The formula for the extrapolated EC3 value was as follows:

EC3 = 2^ {log2(c) + (3-d)/(b-d) x [log2(a) - log2(c)]}

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann Whitney U test procedures were used.
Probability values (p) are presented as follows:
P<0.001 = ***
P<0.01 = **
P<0.05 = *
P>0.05 = (not significant)

Positive control results:

At a concentration of 25 % (v/v) in acetone/olive oil (4:1) the stimulation index was 5.54, this result was positive and therefore the positive control was considered to be a sensitiser under the conditions of the test.

Key result

Parameter:

SI

Value:

12.36

Test group / Remarks:

10 % w/w

Key result

Parameter:

SI

Value:

15.57

Test group / Remarks:

25 % w/w

Key result

Parameter:

SI

Value:

15.28

Test group / Remarks:

50 % w/w

Key result

Parameter:

EC3

Value:

0.69

Cellular proliferation data / Observations:

PRE-SCREEN TEST
- No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25 % increase in mean ear thickness were noted.
- Based on this information the dose levels selected for the main test were 50, 25 and 10 % w/w in acetone/olive oil 4:1.

MAIN TEST
Estimation of the Proliferative Response of Lymph Node Cells
- The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:
10 % (w/w) in acetone/olive oil (4:1): SI = 12.36 (Positive)
25 % (w/w) in acetone/olive oil (4:1): SI = 15.57 (Positive)
50 % (w/w) in acetone/olive oil (4:1): SI = 15.28 (Positive)

Clinical Observations and Mortality Data
- There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Body Weight
- Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Calculation of EC3 Value
EC3 = 2^ {log2(c) + (3-d)/(b-d) x [log2(a) - log2(c)]}
a (mid concentration giving a stimulation index >3) = 25
b (actual stimulation index caused by ‘a’) = 15.57
c (lowest concentration giving a stimulation index of >3) = 10
d (actual stimulation index caused by ‘c’) = 12.36

EC3 = 2^ {log2 (10) + (3 - 12.36)/(15.57 - 12.36) x [log2 (25) - log2(10)]} = 0.69
The concentration of test material expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 0.69 %.

Table 1: Individual Disintegrations per Minute and Stimulation Index

Concentration (% w/w) in acetone/olive oil 4:1	Animal Number	^dpm/ Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle	1-1	725.56	784.78 (±81.10)	na	na
	1-2	892.40
	1-3	718.91
	1-4	802.26
10	2-1	8883.95	9696.54* (±3557.14)	12.36	Positive
	2-2	14938.42
	2-3	7401.79
	2-4	7562.00
25	3-1	10083.35	12215.22* (±2161.01)	15.57	Positive
	3-2	12527.07
	3-3	11161.61
	3-4	15088.84
50	4-1	17827.59	11991.83* (±4573.22)	15.28	Positive
	4-2	11864.65
	4-3	11625.16
	4-4	6649.90

dpm = Disintegrations per minute

a = Total number of lymph nodes per animal is 2

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

* = Significantly different from the control group p<0.05

Interpretation of results:

other: EU Criteria: Category 1A (H317: May cause an allergic skin reaction)

Conclusions:

Under the conditions of this study the test material is sensitising to skin.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 429, under GLP conditions in the Local Lymph Node Assay (LLNA).

The skin sensitisation potential of the test material was investigated in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50 % w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of test material as a solution in acetone/olive oil 4:1 at concentrations of 50, 25 or 10 % w/w. A further group of four animals was treated with acetone/olive oil 4:1 alone.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 10 % (w/w): SI = 12.36 (Positive), 25 % (w/w): SI = 15.57 (Positive) and 50 % (w/w): SI = 15.28 (Positive).

The concentration of test material expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 0.69 %.

Under the conditions of this study the test material is sensitising to skin.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 May 2017 to 18 May 2017

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Recommended test system in the international OECD guidelines.

Details on the study design:

PEPTIDE AND POSITIVE CONTROL
- Synthetic peptide containing Cysteine (Ac-RFAACAA-COOH), Batch number: 1556171
- Synthetic peptide containing Lysine (Ac-RFAAKAA-COOH), Batch number: 1556172
- Cinnamic Aldehyde (Positive control), Batch number: MKBR2427V

APPARATUS
- High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector.

ASSESSMENT OF TEST MATERIAL SOLUBILITY
- The solubility of the test material in acetonitrile was assessed at a concentration of 100 mM.

PREPARATION OF PEPTIDE STOCK SOLUTIONS
- Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

PREPARATION OF PEPTIDE CALIBRATION STANDARDS
- Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and they each contained the peptides at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.

PREPARATION OF STABILITY CONTROLS AND PRECISION CONTROLS
- Six stability controls and precision controls of both peptides were prepared at a concentration of 0.5 mM.

PREPARATION OF POSITIVE CONTROL SOLUTION
- The positive control chemical (Cinnamic aldehyde) was prepared at a concentration of 100 mM in acetonitrile.

PREPARATION OF POSITIVE CONTROL AND CYSTEINE PEPTIDE DEPLETION SAMPLES AND CO-ELUTION CONTROLS
- Acetonitrile solutions of the test material and the positive control were diluted with the Cysteine peptide to prepare solutions (each in triplicate) containing 0.5 mM Cysteine and 5 mM of either the test material or the positive control. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

PREPARATION OF POSITIVE CONTROL AND LYSINE PEPTIDE DEPLETION SAMPLES AND CO-ELUTION CONTROLS
- Acetonitrile solutions of the test material and the positive control were diluted with the Lysine peptide to prepare solutions (each in triplicate) containing 0.5 mM Lysine and 25 mM of either test material or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

INCUBATION
- The appearance of the test material and positive control samples in the HPLC vials was documented following preparation and then the vials were placed into the autosampler of the HPLC set at 25 °C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to run initiation, the appearance of the samples in the vials was assessed and documented again.

ANALYSIS
- The concentration of each peptide in the presence of the test material and the associated positive controls were quantified by HPLC using UV detection as detailed in the chromatographic section.

INSTRUMENTATION PARAMETERS
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30 °C
Sample temperature: 25 °C
Mobile Phase (MP) A: 0.1 % TFA in Water
Mobile Phase (MP) B: 0.085 % TFA in ACN
Gradient:
0 minutes: 90 % MP A, 10 % MP B
20 minutes: 75 % MP A, 25 % MP B
21 minutes: 10 % MP A, 90 % MP B
23 minutes: 10 % MP A, 90 % MP B
23.5 minutes: 90 % MP A, 10 % MP B
30 minutes: 90 % MP A, 10 % MP B
Flow rate: 0.35 mL/minute
Stroke volume 25 μL
Detector wavelength: UV, 220 nm
Injection volume: 2 μL (slow draw rate)
Run time: 30 minutes
Approximate retention time (Cysteine): 11 minutes
Approximate retention time (Lysine): 7 minutes

CALCULATIONS
- The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:

% Peptide depletion = 100 – [Peptide peak area in replicate depletion samples / Mean Peptide peak area of reference control samples B] x 100

Positive control results:

All analytical acceptance criteria for each peptide run were met: Positive control depletion values were 69.7 and 58.7 % for cysteine and lysine, respectively.

Key result

Run / experiment:

other: Cysteine

Parameter:

other: Mean % Depletion

Value:

45.3

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

other: Lysine

Parameter:

other: Mean % Depletion

Value:

-2 898.4

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Co-elution/interference occurred with the Lysine peptide hence the negative result.

Other effects / acceptance of results:

SOLUBILITY ASSESSMENT
- The solubility of the test material in acetonitrile at a nominal concentration of 100 mM was confirmed.

REACTIVITY ASSESSMENT
- All analytical acceptance criteria for each peptide run were met.
- The depletion of peptide in the presence of the test material was 45.3 % for Cysteine, and -2898.4 % for Lysine. Co-elution/interference occurred with the Lysine peptide hence the negative result.
- As there was co-elution with the Lysine peptide, the cysteine result only is reported by application of the cysteine 1:10 reactivity prediction model (co-eluting peaks) below. Based on this model, reactivity is classed as “moderate”, hence the DPRA prediction is positive and the test material is therefore a potential skin sensitiser.
- Negative:
0 % ≤ Cys % depletion ≤ 13.89 % (No or minimal reactivity)
- Positive:
13.89 % < Cys % depletion ≤ 23.09 % (Low reactivity)
23.09 % < Cys % depletion ≤ 98.24 % (Moderate reactivity)
98.24 % < Cys % depletion ≤ 100 % (High reactivity)

Table 1: Cysteine Peptide Depletion

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion^2(%)	Mean Depletion (%)	CV (%)
Positive control	266290	112.82	69.9	69.7	1.35
	264668	112.12	79.0
	271551	115.09	69.3
Test Material	481146	205.30	45.5	45.3	0.50
	485947	207.36	45.0
	482856	206.03	45.3

CV Coefficient of Variation

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

2 Calculated against a mean reference control peak area of 883360 µV.sec (n=6)

 

Table 2: Lysine Peptide Depletion

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion^2(%)	Mean Depletion (%)	CV (%)
Positive control	310176	159.13	59.1	58.7	1.67
	309876	158.98	59.2
	319096	163.71	57.9
Test Material	22280819	11426	-2836.2	-2898.4	1.93
	22830913	11708	-2908.7
	23148238	11871	-2950.5

CV Coefficient of Variation

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

2 Calculated against a mean control peak area of 758830 µV.sec (n=6)

Table 3: Analytical Acceptance Criteria

	Peptide	Linearity	Positive control depletion (%)	Reference controls	Test item
Acceptance criteria	Cysteine	r^2 >0.99	60.8-100 (CV <14.9 %)	0.45-0.55 Mm (CV <15 %)	CV <14.9 %
	Lysine	r^2 >0.99	40.2-69.0 (CV <11.6 %)	0.45-0.55 mM (CV <15 %)	CV<11.6 %
Achieved results	Cysteine	r^2 >0.99	69.7 (CV, 1.35 %, n=3)	0.503 mM (CV 0.59 %, n=6)	CV 0.50 % (n=3)
	Lysine	r^2 >0.99	58.7 (CV, 1.67 %, n=3)	0.501 mM (CV 0.80 %, n=6)	CV 1.93 % (n=3)

Interpretation of results:

other: Positive in the DPRA (moderate reactivity class)

Conclusions:

Under the conditions of this study the test material was positive in the DPRA (moderate reactivity class).

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442C, under GLP conditions with the Direct Peptide Reactivity Assay (DPRA).

The solubility of the test material in acetonitrile at a nominal concentration of 100 mM was confirmed. All analytical acceptance criteria for each peptide run were met in the study.

The test material was examined in the Cysteine containing synthetic peptide only. There was co-elution in the Lysine peptide. 45.3 % mean cysteine depletion was observed.

Under the conditions of this study the test material was positive in the DPRA (moderate reactivity class).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

DPRA, Fleet (2017)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442C, under GLP conditions with the Direct Peptide Reactivity Assay (DPRA). The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The solubility of the test material in acetonitrile at a nominal concentration of 100 mM was confirmed. All analytical acceptance criteria for each peptide run were met in the study.

The test material was examined in the Cysteine containing synthetic peptide only. There was co-elution in the Lysine peptide. 45.3 % mean cysteine depletion was observed.

Under the conditions of this study the test material was positive in the DPRA (moderate reactivity class).

h-CLAT, Roth (2018)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 422E, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of the test material stably suspended/dispersed in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test material concentration for the main experiment (h-CLAT) of the test material was previously determined by two XTT tests.

Cytotoxic effects were observed following incubation with the test material starting with the concentration of 625 µg/mL in the first XTT test and starting with the concentration of 312.5 µg/mL up to the highest tested concentration (625 µg/mL) in the second XTT test (threshold of cytotoxicity: < 75 %). The mean CV75 value of both XTT tests was calculated as 328.5 µg/mL.

The following concentrations of the test material (stably suspended/dispersed in culture medium) were tested in the main experiment (h-CLAT): 110, 132, 158, 190, 228, 274, 328 and 394 µg/mL

The test material, with a log Pow of 2.8, was tested in 2 independent runs. The RFI of CD86 and CD54 was greater than 150 and 200 %, respectively, in at least one concentration of the first run and the RFI of CD86 was greater than 150 % in at least one concentration of the second run. Therefore the h-CLAT prediction is considered positive for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %) and the cell viability was >50 %.

Under the conditions of this study, the test material activated THP-1 cells, therefore the test material is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

LLNA, Pooles (2018)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 429, under GLP conditions in the Local Lymph Node Assay (LLNA). The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The skin sensitisation potential of the test material was investigated in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50 % w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of test material as a solution in acetone/olive oil 4:1 at concentrations of 50, 25 or 10 % w/w. A further group of four animals was treated with acetone/olive oil 4:1 alone.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows: 10 % (w/w): SI = 12.36 (Positive), 25 % (w/w): SI = 15.57 (Positive) and 50 % (w/w): SI = 15.28 (Positive).

The concentration of test material expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 0.69 %.

Under the conditions of this study the test material is sensitising to skin.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance is classified as Category 1A (H317: May cause an allergic skin reaction).