Reaction mass of Ethyl 3-(cyclohex-1-en-1-yl) propanoate and Ethyl 3-(cyclohex-2-en-1-yl) propanoate

Endpoint summary

Description of key information

An alternative method has been developed for the evaluation of the skin sensitisation potential by measuring phenotypic changes, such as CD86 and CD54 expression on dendritic cells. The human leukemia cell line THP-1 is used as surrogate for human myeloid dendritic cells, since these cells show also enhanced CD86 and/or CD54 expression when treated with sensitisers.

The purpose of the Human Cell Line Activation Test (h-CLAT) will be to assess the skin sensitising potential of FRET 11-0078 in an appropriate solvent (DMSO, saline or culture medium) when administered to THP-1 cells for 24 hours. The dose for the main experiment (h-CLAT) of FRET 11-0078 will be determined by a XTT test.

This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the skin sensitising potential of FRET 11-0078 dissolved in culture medium when administered to THP-1 cells for 24 ± 1 hours. The dose for the main experiment (h-CLAT) of FRET 11-0078 was previously determined by two XTT tests.

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 1250 µg/mL in the first XTT test and 625 µg/mL in the second XTT up to the highest tested concentration (2500 µg/mL). The mean CV75 value of both XTT tests was calculated as 576 µg/mL.

The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT):

193, 231, 278, 333, 400, 480, 576 and 691 µg/mL

The test item with a log10 Pow: 4.08 to 4.19 was tested in 3 independent runs. The RFI of CD86 was greater than 150% in at least one dose of 2 out of 3 independent run data. In addition, the RFI of CD54 was greater than 200% in three test item concentrations of the second run. Therefore the test item is considered to be a sensitiser.

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

In conclusion, the test item FRET 11-0078 with a log10 Pow: 4.08 to 4.19 was found to be positive under the test conditions of this study. Therefore, this h-CLAT test indicates a skin sensitizing potential of the test item, being one indicator of the testing battery for the assessment of the skin sensitisation potential.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 30 November 2016 and 10 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 442E using the Human Cell Line Activation Test (h-CLAT) and in compliance with GLP

Qualifier:

according to guideline

Guideline:

other: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), July 2016.

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

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

An alternative method has been developed for the evaluation of the skin sensitisation potential by measuring phenotypic changes, such as CD86 and CD54 expression on dendritic cells. The human leukemia cell line THP-1 is used as surrogate for human myeloid dendritic cells, since these cells show also enhanced CD86 and/or CD54 expression when treated with sensitisers.
The purpose of the Human Cell Line Activation Test (h-CLAT) will be to assess the skin sensitising potential of FRET 11-0078 in an appropriate solvent (DMSO, saline or culture medium) when administered to THP-1 cells for 24 hours. The dose for the main experiment (h-CLAT) of FRET 11-0078 will be determined by a XTT test.
This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

Specific details on test material used for the study:

Identification : FRET 11-0078
Chemical Name: 1-Cyclohexene-1-propanoic acid, ethyl ester (primary isomer)
2-Cyclohexene-1-propanoic acid, ethyl ester (secondary isomer)
CAS No .: 65173-43-5 (primary isomer)
109976-49-0 (secondary isomer)
Batch : NG342-12
Purity : 96.6%, dose calculation was not adjusted to purity
Partition coefficient:
n-octanol/water : log10 Pow: 4.08 to 4.19
Water solubility : 81.7 mg/L at 20 °C
Appearance : Clear colorless, liquid
Expiry Date : 01 September 2017
Stability in Solvent: Not indicated by the Sponsor
Storage Conditions: In the refrigerator, under protection of light
Purpose of Use: Fragrance chemical

Details on the study design:

TEST SYSTEM AND SUPPORTING INFORMATION
Reasons for the Choice of THP-1 Cells
THP-1 cells (Human monocytic leukemia 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 Envigo CRS GmbH (aliquots of cells in freezing medium at 1  106 to 2  106 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  106 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 was 14 in both XTT assays and 15, 16 and 10 in the h CLAT for runs 1, 2 and 3, 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.
reparation and Seeding of THP-1 Cells
On the day of the cytotoxicity experiment (XTT) directly before the application of the test item, solvent and medium control, a volume of 100 µL with a cell density of 0.9 - 1 x 10^6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate.
For the main experiment (h-CLAT) 0.9 - 1x10^6 cells/well in a volume of 500 µL was seeded in a 24-well plate before the treatment.

Experimental Design and Procedures of XTT
Dose Finding Assay (XTT Test)
The doses 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. This method was first described 1988 by SCUDIERO et al. and improved in subsequent years by several other investigators.
Two independent cytotoxicity experiments were performed with different cell cultures 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 item dilutions, the medium and solvent controls, respectively were added to the cells. All dose groups were tested in 7 replicates. At the end of the incubation period of 24 ± 1 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 [%]) as compared to the solvent control is calculated using this formula:

Relative Absorbance (%) = 100 x (mean absorbance of test item - 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 Doses
Two independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable CV75. The mean of at least 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 item in the main experiment (h CLAT). The highest concentration used was 1.2 × CV75 and seven further concentrations of the test item 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
• mean viability of the solvent control is ≥ 90% in comparison to the medium control

Experimental Design and Procedures of h-CLAT
The test item was tested in three independent runs.

Treatment of the Cells
For the test item exposure the highest dose solution 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.
Each volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 1 hours.
Each concentration of the test item, medium control, positive and DMSO control was tested 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 item-treated and not test item treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 x g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, 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 procedure.
The cells were gently mixed by hand 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, 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. 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) were set for the optimal detection of DNA-bound 7 AAD fluorescence signal.

Preparation of the acquisition
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 compensated to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, such that the MFI of control cells were set in the range between 1.0 and 4.0 (Geo Mean) (FACSCalibur, Becton Dickinson).
A R1 gate was set at the middle position between the peak of the negative fraction. The negative fraction corresponds to the living cells and was kept for the subsequent analyses. The percentage of R1-gated cells should exceed 95 %.
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 in the flow line.

Acquisition
Dead cells were gated-out 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 are 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 test item treated cells) - (MFI test item treated isotype control cells) / (MFI solvent control cells) - MFI solvent isotpe 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 conrol cells / Mean cytotox of test item treated cells) x 100

Where the Mean cytotox is the mean of GeoMean(7-AAD) isotype control, GeoMean(7-AAD) CD 54 and GeoMean(7-AAD) CD 86.

Acceptance Criteria
The study is considered as valid, if the following criteria are met:
• 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 positive control (DNCB), RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
• In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
• For medium and DMSO controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
• For the test item resulting in negative outcome, the cell viability at the 1.2 x CV75 should be less than 90%. (If the cell viability at the 1.2 x CV75 is more than 90% for a positive tested test item, the data will be acceptable. If 5 mg/mL in saline, 1 mg/mL in DMSO or the highest soluble dose will be used as the maximal test concentration instead of CV75-based dose, the data for test item are accepted independent by the cell viability.)
• The cell viability of at least 4 doses in each experiment should be ≥50%.

Evaluation of Results
The test item is tested in 2 independent runs. If the RFI of CD86 is ≥ 150% or if the RFI of CD54 is ≥ 200% in both independent run data, the test item is considered to be a sensitiser. Otherwise it is considered to be a non-sensitiser. In case of different results in both runs, a third run has to be performed. If the RFI of CD86 is ≥ 150% at any dose in at least 2 of 3 independent run data, or if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data, the test item is considered to be a sensitiser. Otherwise it is considered to be a non-sensitiser.
Test chemicals 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 chemicals with a Log Pow > 3.5 could still be used to support the identification of the test chemical as a skin sensitiser.

Positive control results:

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

Run / experiment:

other: Experiment 2

Parameter:

other: CD54 induction

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: CD86 induction

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 3

Parameter:

other: CD86 induction

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the skin sensitising potential of FRET 11-0078 dissolved in culture medium when administered to THP-1 cells for 24 ± 1 hours. The dose for the main experiment (h-CLAT) of FRET 11-0078 was previously determined by two XTT tests.
Cytotoxic effects were observed following incubation with the test item starting with the concentration of 1250 µg/mL in the first XTT test and 625 µg/mL in the second XTT up to the highest tested concentration (2500 µg/mL). The mean CV75 value of both XTT tests was calculated as 576 µg/mL.
The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT):
193, 231, 278, 333, 400, 480, 576 and 691 µg/mL
The test item with a log10 Pow: 4.08 to 4.19 was tested in 3 independent runs. The RFI of CD86 was greater than 150% in at least one dose of 2 out of 3 independent run data. In addition, the RFI of CD54 was greater than 200% in three test item concentrations of the second run. Therefore the test item is considered to be a sensitiser.
In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%). The RFI values of the positive controls (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was >50%

RESULTS

Results of the Dose Finding Assay (XTT Test)

Results of the first XTT test for Test Item FRET 11-007

Test Group	Concentration [µg/mL]	Cytotoxicity	Mean Absorbance*	Standard Deviation	Chem. Blank	Mean Absorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	1.019	0.223	0.237	0.782	96.66
Solvent Control	-	no	1.050	0.116	0.241	0.809	100.00
Test Item	19.6	no	0.876	0.145	0.234	0.642	79.41
	29.1	no	0.887	0.069	0.216	0.671	82.96
	78.1	no	0.975	0.062	0.235	0.740	91.49
	156.3	no	0.998	0.207	0.238	0.760	93.96
	312.5	no	0.948	0.045	0.240	0.708	87.47
	625	no	0.851	0.068	0.243	0.609	75.26
	1250	yes	0.373	0.032	0.239	0.134	16.57
	2500	yes	0.280	0.016	0.232	0.049	6.05

 

*       mean absorbance (absolute) of 7 wells

**       relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 103.5%.

The CV75 value of the first XTT test: 627.8 µg/mL

Results of the second XTT test for Test Item FRET 11-0078

Test Group	Concentration [µg/mL]	Cytotoxicity	Mean Absorbance*	Standard Deviation	Chem. Blank	Mean Absorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.757	0.030	0.234	0.523	100.69
Solvent Control	-	no	0.750	0.057	0.230	0.520	100.00
Test Item	19.6	no	0739	0.022	0.231	0.508	97.77
	39.1	no	0.734	0026	0.229	0.505	97.20
	78.1	no	0.751	0.026	0.239	0.512	98.41
	156.3	no	0.714	0.030	0.233	0.481	92.60
	312.5	no	0.687	0.020	0.227	0.460	88.49
	625	no	0.589	0.041	0.233	0.356	68.57
	1250	yes	0.410	0.041	0.235	0.176	33.83
	2500	yes	0.309	0.065	0.230	0.078	15.07

*       mean absorbance (absolute) of 7 wells

**       relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 99.4%.

The CV75 value of the second XTT test: 524.1 µg/mL

The mean CV75 value of both XTT tests: 576 µg/mL

Results of the h-CLAT Test

Results of the first h-CLAT run for the Test Item FRET 11-0078

	Concentration (µg/mL)	Antibody	RFI (%)	Cell Viability (%)
Medium Control	-	CD54	100.0	100.0
	-	CD86	100.0	100.0
DMSO Control	-	CD54	100.0	100.0
	-	CD86	100.0	100.0
Positive Control (DNCB)	2.0	CD54	308.8*	66.2
		CD86	837.1*
	3.0	CD54	528.6*	69.6
		CD86	950.5*
Test Item	193	CD54	122.1	96.3
		CD86	118.8
	231	CD54	111.6	94.5
		CD86	112.5
	278	CD54	118.6	92.0
		CD86	113.3
	333	CD54	125.6	86.0
		CD86	120.3
	400	CD54	127.9	83.9
		CD86	146.9
	480	CD54	130.2	83.2
		CD86	139.1
	576	CD54	120.9	90.6
		CD86	99.2
	691	CD54	124.4	89.7
		CD86	109.4

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

Results of the second h-CLAT run for the Test Item FRET 11-0078

	Concentration (µg/mL)	Antibody	RFI (%)	Cell Viability (%)
Medium Control	-	CD54	100.0	100.0
	-	CD86	100.0	100.0
DMSO Control	-	CD54	100.0	100.0
	-	CD86	100.0	100.0
Positive Control (DNCB)	2.0	CD54	397.6*	74.6
		CD86	694.3*
	3.0	CD54	561.4*	72.2
		CD86	766.5*
Test Item	193	CD54	150.0	89.1
		CD86	134.2
	231	CD54	153.6	85.1
		CD86	148.0
	278	CD54	150.0	82.7
		CD86	190.8*
	333	CD54	251.8*	68.1
		CD86	312.5*
	400	CD54	180.4	86.7
		CD86	228.3*
	480	CD54	207.1	83.0
		CD86	228.3*
	576	CD54	146.4	97.8
		CD86	173.7*
	691	CD54	223.2*	68.5
		CD86	267.1*

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

Results of the third h-CLAT run for the Test Item FRET 11-0078

	Concentration (µg/mL)	Antibody	RFI (%)	Cell Viability (%)
Medium Control	-	CD54	100.0	100.0
	-	CD86	100.0	100.0
DMSO Control	-	CD54	100.0	100.0
	-	CD86	100.0	100.0
Positive Control (DNCB)	2.0	CD54	200.8*	83.2
		CD86	383.9*
	3.0	CD54	553.7*	78.1
		CD86	849.6*
Test Item	193	CD54	121.3	102.9
		CD86	124.3
	231	CD54	107.4	102.7
		CD86	121.4
	278	CD54	123.1	101.5
		CD86	159.2*
	333	CD54	102.8	96.2
		CD86	178.6*
	400	CD54	160.2	94.2
		CD86	196.1*
	480	CD54	154.6	91.2
		CD86	284.8*
	576	CD54	153.7	83.7
		CD86	338.8*
	691	CD54	156.5	82.6
		CD86	346.6*

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

Interpretation of results:

other:

Remarks:

this h-CLAT test indicates a skin sensitizing potential of the test item, being one indicator of the testing battery for the assessment of the skin sensitisation potential.

Conclusions:

The test item FRET 11-0078 with a log10 Pow: 4.08 to 4.19 was found to be positive under the test conditions of this study. Therefore, this h-CLAT test indicates a skin sensitizing potential of the test item, being one indicator of the testing battery for the assessment of the skin sensitisation potential.

Executive summary:

An alternative method has been developed for the evaluation of the skin sensitisation potential by measuring phenotypic changes, such as CD86 and CD54 expression on dendritic cells. The human leukemia cell line THP-1 is used as surrogate for human myeloid dendritic cells, since these cells show also enhanced CD86 and/or CD54 expression when treated with sensitisers.

The purpose of the Human Cell Line Activation Test (h-CLAT) will be to assess the skin sensitising potential of FRET 11-0078 in an appropriate solvent (DMSO, saline or culture medium) when administered to THP-1 cells for 24 hours. The dose for the main experiment (h-CLAT) of FRET 11-0078 will be determined by a XTT test.

This human cell line activation test can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the skin sensitising potential of FRET 11-0078 dissolved in culture medium when administered to THP-1 cells for 24 ± 1 hours. The dose for the main experiment (h-CLAT) of FRET 11-0078 was previously determined by two XTT tests.

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 1250 µg/mL in the first XTT test and 625 µg/mL in the second XTT up to the highest tested concentration (2500 µg/mL). The mean CV75 value of both XTT tests was calculated as 576 µg/mL.

The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT):

193, 231, 278, 333, 400, 480, 576 and 691 µg/mL

The test item with a log10 Pow: 4.08 to 4.19 was tested in 3 independent runs. The RFI of CD86 was greater than 150% in at least one dose of 2 out of 3 independent run data. In addition, the RFI of CD54 was greater than 200% in three test item concentrations of the second run. Therefore the test item is considered to be a sensitiser.

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

In conclusion, the test item FRET 11-0078 with a log10 Pow: 4.08 to 4.19 was found to be positive under the test conditions of this study. Therefore, this h-CLAT test indicates a skin sensitizing potential of the test item, being one indicator of the testing battery for the assessment of the skin sensitisation potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Migrated from Short description of key information:
Respiratory sensitisation can be assessed using human data such as indicated in R7.3.5.2 of the ECHA guidance (2015) that indicate respiratory reactions e. g. from consumer experience or occupational exposure. In case no such data are available the respiratory sensitisation can be assessed using the integrated evaluation strategy for respiratory sensitisation data in the ECHA guidance (R7A, Fig. 7.3-2, 2015), which says that if the substance is a non skin sensitiser, than it is unlikely to be a respiratory sensitiser.

Justification for classification or non-classification

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the skin sensitising potential of FRET 11-0078 dissolved in culture medium when administered to THP-1 cells for 24 ± 1 hours.

The study was performed in accordance with OECD Guidelines for the Testing of Chemicals: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), July 2016.

The dose for the main experiment (h-CLAT) of FRET 11-0078 was previously determined by two XTT tests.

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 1250 µg/mL in the first XTT test and 625 µg/mL in the second XTT up to the highest tested concentration (2500 µg/mL). The mean CV75 value of both XTT tests was calculated as 576 µg/mL.

The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT):

193, 231, 278, 333, 400, 480, 576 and 691 µg/mL

The test item with a log10 Pow: 4.08 to 4.19 was tested in 3 independent runs. The RFI of CD86 was greater than 150% in at least one dose of 2 out of 3 independent run data. In addition, the RFI of CD54 was greater than 200% in three test item concentrations of the second run. Therefore the test item is considered to be a sensitiser.

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

In conclusion, the test item FRET 11-0078 with a log10 Pow: 4.08 to 4.19 was found to be positive under the test conditions of this study. Therefore, this h-CLAT test indicates a skin sensitizing potential of the test item, being one indicator of the testing battery for the assessment of the skin sensitisation potential.