(Z)-tetrahydro-6-(2-pentenyl)-2H-pyran-2-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Start Date: 16 November 2020
Experimental Completion Date: 08 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Test material

Test material information

Constituent 1

Reference substance name:

(Z)-tetrahydro-6-(2-pentenyl)-2H-pyran-2-one

EC Number:

247-074-2

EC Name:

(Z)-tetrahydro-6-(2-pentenyl)-2H-pyran-2-one

Cas Number:

25524-95-2

Molecular formula:

C10H16O2

IUPAC Name:

(Z)-tetrahydro-6-(2-pentenyl)-2H-pyran-2-one

Test material form:

liquid

Details on test material:

Identification: JASMINLACTONE
Chemical name: (Z)-tetrahydro-6-(2-pentenyl)-2H-pyran-2-one
CAS number: 25524-95-2
Appearance/Physical state: Clear, colorless liquid
Lot: 8500119
Purity: 99.63%
Expiry date: 26 November 2021
Storage conditions: Approximately 4 °C, in the dark, under Nitrogen

Specific details on test material used for the study:

TEST MATERIAL
- Test item identity (including alternative names): (Z)-tetrahydro-6-(2-pentenyl)-2H-pyran-2-one
- Lot number: 8500119
- Storage conditions: Refrigerated (2-8oC) under nitrogen
- Purity: 99.63%
- Appearance: Colorless to pale yellow liquid
- Expiry/Retest date: 26 November 2021

In vitro test system

Details of test system:

THP-1 cell line [442E]

Details on the study design:

Test Article Formulation
Dose Finding Assay
A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control.

The test article was formulated at 500 mg/mL in DMSO then eight stock solutions were prepared by 2-fold serial dilutions using the corresponding solvent. The stock solutions were then further diluted 250-fold in culture medium (working solutions). The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate to obtain a final range of concentrations in the plate of 7.8 1000 µg/mL.

The test article was prepared shortly before testing. Preparation was conducted under subdued lighting with the aid of vortex mixing.

CD86/CD54 Expression Measurement
Eight stock solutions of each test article were prepared by 1.2-fold serial dilutions using the corresponding solvent, and then further diluted 250-fold into the culture medium to give eight working solutions ranging from 0.335 x CV75 to 1.2 x CV75. The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate to obtain a final range of concentrations in the plate of 265.50-951.35 µg/mL.

The test article was prepared shortly before testing. Preparation was conducted under subdued lighting with the aid of vortex mixing.

Cell Culture Maintenance
THP-1 cells were cultured in a humidified incubator set to 37ºC, 5% CO2, in RPMI 1640 medium supplemented with 10% heat inactivated (HI) foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin and 100 µg/mL streptomycin.

The cells were passaged every 2-3 days at a density of 0.1 to 0.2 x 10E6 cells/mL and maintained at a density from 0.1 x 10E6 to 0.8 x 10E6 cells/mL. Cell density did not exceed 1 x 10E6 cells/mL, and cells did not exceed 30 passages.

Reactivity Check
This was performed using DNCB (CAS no. 97-00-7, ≥99% purity), nickel sulphate (CAS no. 10101-97-0, ≥99% purity) and lactic acid (CAS no. 50-21-5, ≥85% purity) two weeks after thawing.
DNCB and nickel sulphate should produce a positive response of both CD86 and CD54 and lactic acid should produce a negative response of both CD86 and CD54. Only cells which passed the reactivity check were used for the assay.

Plate Preparation
THP-1 cells were pre-cultured in culture flasks either at a density of 0.2 x 10E6 cells/mL for 48 hours or at a density of 0.1 x 10E6 cells/mL for 72 hours. On the days of testing, cells were harvested from the flasks and were resuspended with fresh culture medium at 2 x 10E6 cells/mL. The cells were then distributed into a 24 well flat-bottom plate (500 µL/well, expression assay) or a 96-well flat-bottomed plate (80 µL/1.6 x 10E5 cells per well, DRF assay).

Study Design
Dose Finding Assay
The test article working solutions or solvent controls were mixed 1:1 (v/v) with the cell suspensions in the 96-well plates. The plates were sealed and then incubated for 24±0.5 hours (incubator set to 37ºC, 5% CO2).

After the 24-hour incubation period, all cells from the 96-well flat-bottomed plate were transferred into a 96-well round-bottomed plate. The cells were washed at least twice in 200 µL of phosphate buffered saline containing 0.1% bovine serum albumin (FACS buffer) and re-suspended in 190 µL of FACS buffer. 10 µL of propidium iodide solution (PI) was added just before FACS analysis (final concentration of PI = 0.625 µg/mL).

PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of 10,000 viable cells were acquired.

Cell viability was calculated using the following equation:

Cell Viability = (number of living cells / total number of acquired cells) x 100

The CV75 value, i.e. a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), was calculated by log-linear interpolation using the following equation:

Log CV75 = (75 - c) x Log (b) - (75-a) x Log (d) / a - c

Where:
a was the minimum value of cell viability over 75% in testing groups
c was the maximum value of cell viability below 75% in testing groups
b and d were the concentrations showing the value of cell viability a and c respectively.

CD86/CD54 Expression Measurement
Three independent runs (experiments) were needed to drive a prediction. Each independent run was performed on a different day or on the same day provided that for each run:

a) Independent fresh stock solutions and working solutions of the test article and antibody solutions were prepared and

b) Independently harvested cells were used (i.e. cells were collected from different culture flasks).

The cells may have come from the same passage.

On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2 x 10E6 cells/mL. The cells were then distributed into a 24-well plate (500 µL/1 x 106 cells per well).

The test article working solution or solvent control was mixed 1:1 (v/v) with the cell suspensions in the 24-well plates. The plates were sealed and then incubated for 24±0.5 hours (incubator set to 37ºC, 5% CO2).

After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation (approximately 250 g, 5 minutes) and washed twice with 1 mL of FACS buffer. After washing, the cells were blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) on ice for 15 minutes.

After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate and centrifuged (approximately 250 g, 3 minutes).

After centrifugation, the cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes.

The stained cells were washed three times with an excess of FACS buffer, re-suspended in FACS buffer and 12.5 µg/mL PI solution was added (to give a final PI concentration of 0.625 µg/mL).

The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.
It may be noted that for Main Experiment run 2 measurements an unusual distribution (cell population was undefined and merging with the debris and/or below the axis) was initially noted on the FACS plots for the wells containing test concentrations of 792.79 to 951.35 μg/mL, indicating that a flow cytometer blockage had occurred. Accordingly, these samples were therefore transferred to fresh wells and the FACS analysis re-run. Sample distribution was confirmed to be normal for the re-run analysis and the data from the initial run are therefore not reported.

Data Evaluation
Analysis of Results
Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 for the positive control cells and test article-treated cells were calculated according to the following equation:

RFI = ((MFI of chemical-treated cells – MFI of chemical-treated isotype control cells / (MFI of solvent/vehicle-treated cells – MFI of solvent/vehicle-treated isotype control cells) x 100

The cell viability from the isotype control cells (stained with mouse IgG1 antibodies) was
calculated using the following equation:

Cell Viability = (number of living cells / total number of acquired cells) x 100

Prediction Model
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, otherwise the h-CLAT prediction is considered NEGATIVE:
• 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%).

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Results and discussion

Positive control results:

For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54, and cell viability was >50% in each independent run.

In vitro / in chemico

Results

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC200, CD54 [442E]

Value:

435.33 µg/mL

Cell viability:

As discordant results were obtained in the two endpoint assays, a third experiment was performed. In Experiment 3, the RFI values for CD86 were <150% at all concentrations and the RFI values for CD54 were >200% at multiple concentrations (with cell viability >50%).

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Any other information on results incl. tables

Dose Finding Assay

The CV75 value for the test article was 792.79 µg/mL.

CD86/CD54 Expression Results

The relative fluorescence intensity (RFI) values for the test article were calculated as follows:

Concentration (µg/mL)	RFI (CD86)			RFI (CD54)
	Exp 1	Exp 2	Exp 3	Exp 1	Exp 2	Exp 3
265.50	93	104	75	82	122	159
318.60	86	92	85	87	88	226
382.33	71	80	93	146	81	347
458.79	146	96	93	224	169	324
550.55	104	100	88	235	169	347
660.66	148	173	107	220	177	297
792.79	112	68	108	211	35	328
951.35	207	130	149	163	181	566
Solvent/vehicle control (DMSO)	121	93	131	100	101	85
Positive control (DNCB)	355	382	298	310	375	1881

In Experiment 1, the RFI values for CD86 were <150%, except at a concentration of 951.35 μg/mL where the RFI was 207% with cell viability <50%. The RFI values for CD54 in Experiment 1 were >200% at multiple concentrations (with cell viability >50%). This experiment therefore gave a positive prediction.

In Experiment 2, the RFI values for CD86 were <150%, except at a concentration of 660.66 μg/mL where the RFI was 173% with cell viability <50%. The RFI values for CD54 were <200% at all concentrations. This experiment therefore gave a negative prediction.

As discordant results were obtained in the two endpoint assays, a third experiment was performed. In Experiment 3, the RFI values for CD86 were <150% at all concentrations and the RFI values for CD54 were >200% at multiple concentrations (with cell viability >50%). This experiment therefore gave a positive prediction.

Therefore, based on the majority result of the three independent runs, the test article gave a positive prediction in the assay.

The EC200 value for CD54 calculated by linear regression of endpoint assay data was 435.33 μg/mL. No EC150 value was calculated for CD86 as this marker was negative in all experiments.

Assay Acceptance Criteria Results

All assay acceptance criteria were met.

The cell viabilities of medium and solvent/vehicle control were higher than 90% in each independent run.

In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).

For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.

For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54, and cell viability was >50% in each independent run.

For the test article, the cell viability was more than 50% in at least four tested concentrations in each independent run.

Applicant's summary and conclusion

Interpretation of results:

other: The substance is likely to be a skin sensitiser

Conclusions:

The test article, Jasminlactone, was considered to be positive in the human Cell Line Activation Test.

Executive summary:

The study was conducted to investigate the potential of Jasminlactone to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The human Cell Line Activation Test (h-CLAT) was performed according to OECD TG 442E.

For the dose finding assay, the test article was dissolved in dimethyl sulphoxide (DMSO) at a concentration of 500 mg/mL giving a maximum test concentration of 1000 µg/mL. A reduction in viability was noted at the highest concentration resulting in mean CV75 of 792.79 µg/mL.

For the expression measurements, test concentrations in a range from 265.50 to 951.35 μg/mL (after dilution in medium) were used.

Aliquots of 500 µL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 10⁶ cells per well.

After blocking, the cells were stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes.

The stained cells were washed, re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

There were no RFI values for CD86 which were >150% and had >50% cell viability in any experiment. In Experiment 2, the RFI values for CD54 were <200% at all concentrations, however in Experiments 1 and 3, the RFI values for CD54 were >200% with >50% cell viability at multiple concentrations. Therefore, based on the majority result of the three independent runs, the test article gave a positive prediction in the assay.

The EC200 value for CD54 was calculated to be 435.33 μg/mL.

All acceptance criteria of the h-CLAT assay parameters were met in each experiment.

The test article, Jasminlactone, was considered to be positive in the human Cell Line Activation Test.