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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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).

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

265-967-5

EC Name:

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

Cas Number:

65894-76-0

Molecular formula:

C12H13NO4

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Off white powder

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.

In vitro test system

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).

Results and discussion

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 %.

In vitro / in chemico

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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).

Any other information on results incl. tables

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 %).

Applicant's summary and conclusion

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).