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

Endpoint summary

Description of key information

In chemico, OECD 442C (DPRA) - Negative

In vitro, OECD 442D (ARE-Nrf2 Luciferase Test) - Positive

In vitro, OECD 442E (h-CLAT) - Positive

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:

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

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

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]

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.

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]

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.

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 March 2020 to 17 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

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
- Molecular weight: 168.24 g/mol
- Expiry/Retest date: 26 November 2021

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

- Cell Culture
The cells were the transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct supplied by Givaudan (Dubendorf, Switzerland). The cells were routinely grown and subcultured in maintenance medium at 37 ± 2 °C in a humidified atmosphere containing 5% CO2 in air. Maintenance medium was 500 mL Dulbecco’s Modified Eagles Medium containing Glutamax (DMEM) (Gibco 21885), supplemented with 50 mL foetal bovine serum (FBS) (Gibco 10500) and 5.5 mL Geneticin (Gibco 10131).

- Cell Culture from Frozen Stocks
Vials of cells, stored frozen in cryotubes at -196°C under liquid nitrogen, in DMEM containing 10% dimethyl sulphoxide (DMSO) and 20% FBS, were thawed rapidly at 37°C in a water-bath. The cells were then resuspended in 9 mL of pre-warmed maintenance medium without geneticin and pelleted by centrifugation at 125 g for 5 minutes. The cell pellet was resuspended in maintenance medium without geneticin in tissue culture flasks. The flasks were incubated until 80-90% confluent cell monolayers had been obtained. Geneticin-containing medium was used in subsequent passages. Establishing cell cultures from frozen stocks and subsequent passage was conducted prior to the start of this study.

- Cell Passage
Actively growing cell stocks were maintained and expanded by subculturing (passage). When the cells had reached 80-90% confluence, the medium from each flask was removed, the cells washed twice with Dulbecco’s phosphate buffered saline (DPBS) (Gibco 14190) and harvested using trypsin-EDTA (Ethylenediaminetetraacetic acid) solution. Cultures were incubated at 37 ± 2°C in a humidified atmosphere containing 5% CO2 in air until complete detachment and disaggregation of the cell monolayer had occurred. The cells were then resuspended in medium to neutralise the trypsin (cells from several flasks were pooled at this point). The cells were resuspended and distributed into flasks containing fresh maintenance medium. This passage procedure was repeated to provide a sufficient number of cells for a test, and were passaged at least twice before using the cells in a test. The passages of KeratinoSens™ cells were limited to 25 passages from frozen stock.

- Preparation of Test Cell Cultures
The cells from flasks of actively growing cultures were detached and disaggregated as described above. The number of viable cells in the prepared cell suspension were determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer. The cell suspension was diluted with maintenance medium without geneticin to give1 x 10E5 viable cells/mL and 100 µL volumes pipetted into all wells except one well of sterile 96-well flat-bottomed microtitre plates. On each occasion four plates were prepared in parallel: three white plates for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. One well of each plate received 100 µL maintenance medium without geneticin with no cells. The plates were incubated for 24 ± 2 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air, to allow the cells to attach.

- Test Item Solubility
Prior to commencing testing, the solubility of the test item in assay medium containing 1% DMSO at 200 mM was assessed.
The test item, was found to be soluble in DMSO at 200 mM, the highest concentration as recommended by the guideline this test follows.

- Preparation of the Test Item
A stock solution of the test item, was prepared by weighing the test item into a tared glass container and diluting to 200 mM in DMSO.

TEST PROCEDURE:
- Preparation of the 100 x solvent plate:
A 100x solvent plate was set up by adding 200 µL of the stock solution of the test item to one well in column 12. 200 µL of the 6.4 mM stock solution of cinnamic aldehyde was added to the appropriate well in column 11 as per the example plate layout below. One well was left blank. All other wells contained 100 µL of the appropriate solvent.
Serial halving dilutions of the test item were prepared by transferring 100 µL from each dilution into 100 µL of solvent. Pipette tips were discarded after each transfer and then fresh pipette tips were used to mix each concentration prior to the next transfer.
The 6.4 mM stock solution of cinnamic aldehyde was diluted from column 11 to column 7 using the same procedure of serial halving dilutions.

- Preparation of the Dilution Plate:
The 100x solvent plate was replicated into the bottom of the same 96 well plate (rows G and H as directed by the authorized method) by adding 240 µL of assay medium to each well and then 10 µL solution per well from the 100x solvent wells was added to equivalent wells at the bottom of the same plate. Assay medium was
495 mL DMEM (Gibco 21885), supplemented with 5.0 mL FBS.

- Treatment of Cultured Plates:
Approximately 24 hours after the test cell culture plates were established, the medium was removed from the wells by careful inversion of the plates and blotting onto a paper towel. 150 µL of assay medium was added to every well of the 96 well plates. 50 µL from each well of the dilution plate was transferred to equivalent wells in the 96 well plates. Three white plates were dosed for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay.
The plates were then covered with a plate seal and placed in the incubator at 37 ± 2oC, in a humidified atmosphere of 5% CO2 in air for 48 ± 2 hours.

- Cell Viability Measurement:
After incubation, the transparent plate was removed from the incubator and the plate seal discarded. The cell culture medium was removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 100 µL fresh assay medium was added to each well. 10 µL of MTT solution (5 mg/mL in PBS) was added to each well of the 96-well plate. The plate was incubated at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air for 4 hours ± 10 minutes. The medium was then removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 50 µL of DMSO was added to each well. The plate was then placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2in air, protected from light, for at least 10 minutes. The absorbance value of each well was read using a plate reader with a 540 nm filter.

- Luciferase Measurement:
Luciferase was measured using the Steady Glo®Luciferase Assay system kit supplied by Promega (E2550). Steady-Glo®luciferase reagent was prepared by transferring the contents of one bottle of Steady-Glo®buffer to one bottle of Steady-Glo®substrate. The reagent was mixed by inversion until the substrate had dissolved. The reconstituted reagent was used on the same day it was prepared for test 1. Frozen reconstituted reagent was used for test 2 and was thawed to room temperature before use.

After incubation the medium was removed from the wells of the triplicate white plates by careful inversion of the plates and blotting on sterile absorbent paper. 100 µL of fresh assay medium was added to each well before 100 µL of Steady-Glo®luciferase reagent was added to each well of the plate. The plates were shaken on a plate shaker for at least 5 minutes until the cells had lysed. Luminescence (emitted light) was measured using a SpectraMax L luminometer. Each plate was read fortotal photon countwith an integration time of 1 second. The plates were dark adapted for 1 minute prior to measurement.

- Number of Tests Required:
Two independent tests each containing three replicates (i.e. n=6) were required to make a conclusion. Each independent test was performed on a different day with fresh stock solutions of the chemicals and independently harvested cells.

Vehicle / solvent control:

DMSO

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The results for the positive control, cinnamic aldehyde, are shown in tables 3 and 4.

The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 μM) in both tests.
The EC1.5 values of the positive control, cinnamic aldehyde, were 20.53 μM and 26.81 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory. The average induction in the three replicates for cinnamic aldehyde at 64 µM were 2.45 and 2.24 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.73

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: unit not applicable for Imax

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

2.04

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: unit not applicable for Imax

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

198.39 µM

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

151.81 µM

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC30 [442D]

Remarks:

(µM)

Value:

364.66 µM

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC30 [442D]

Value:

338.97 µM

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC50 [442D]

Value:

409.36 µM

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC50 [442D]

Value:

385.49 µM

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

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

The I_max for Jasminlactone was 1.73 in test 1 and 2.04 in test 2. The I_max for both tests was >1.5 fold and statistically significant compared to the DMSO control. 

The EC_1.5 was 198.39 µM and 151.81 µM for tests 1 and 2, respectively. 

The IC₃₀ value was 364.66 µM in test 1 and 338.97 µM in test 2 and the IC₅₀ values were 409.36 µM and 385.49 µM in tests 1 and 2, respectively and showed an overall dose-response for luciferase induction.

 

The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 12.4% and 11.0% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.

 

Table 1: Results for Jasminlactone – Test 1

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	0.78	0.84	0.91	0.87	1.25	0.98	1.08	1.18	1.73	0.01	0.00	0.00
Statistically significant	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	Yes	N/A	N/A	N/A
Viability (%)	99.80	105.47	112.61	102.21	115.87	118.88	110.54	119.66	121.29	9.46	4.47	3.95
Imax	1.73
EC1.5(µM)	198.39
IC30(µM)	364.66
IC50(µM)	409.36

 Determination criteria for the skin sensitisation potential of the test item: Result

Is the Imax >1.5 fold and statistically significant Yes

Is the cellular viability >70% at the EC1.5 determining concentration: Yes

Is the EC1.5 value < 1000 µM: Yes

Is there an apparent overall dose-response for luciferase induction: Yes

KeratinoSens™ prediction: Positive

 

 

Table 2: Results for Jasminlactone– Test 2

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	0.88	1.03	0.92	1.03	1.06	1.09	1.17	1.35	2.04	0.00	0.00	0.00
Statistically significant	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	Yes	N/A	N/A	N/A
Viability (%)	103.14	101.40	103.62	106.22	117.99	106.90	118.09	118.77	108.25	0.77	1.25	0.29
Imax	2.04
EC1.5(µM)	151.81
IC30(µM)	338.97
IC50(µM)	385.49

Determination criteria for the skin sensitisation potential of the test item: Result

Is the Imax >1.5 fold and statistically significant: Yes

Is the cellular viability >70% at the EC1.5 determining concentration: Yes

Is the EC1.5 value < 1000 µM: Yes

Is there an apparent overall dose-response for luciferase induction: Yes

KeratinoSens™ prediction: Positive

 

 

Table 3: Results for Cinnamic Aldehyde – Test 1

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.18	1.22	1.36	1.86	2.45
Statistically significant	N/A	N/A	N/A	Yes	Yes
Viability (%)	104.79	109.08	110.54	107.28	116.39
Imax	2.45
EC1.5(µM)	20.53
IC30(µM)	N/A
IC50(µM)	N/A

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2-8	Yes (2.45)	Pass
EC1.5of positive control within two standard deviations of the historical mean (‑2.36 to 28.67)	Yes (20.53)	Pass
CV% of blank values < 20%	Yes (12.4%)	Pass

 

 

Table 4: Results for Cinnamic Aldehyde – Test 2

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.07	1.19	1.32	1.58	2.24
Statistically significant	N/A	N/A	N/A	Yes	Yes
Viability (%)	105.16	99.57	112.49	102.27	93.97
Imax	2.24
EC1.5(µM)	26.81
IC30(µM)	N/A
IC50(µM)	N/A

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2-8	Yes (2.04)	Pass
EC1.5of positive control within two standard deviations of the historical mean (‑2.36 to 28.67)	Yes (26.81)	Pass
CV% of blank values < 20%	Yes (11.0%)	Pass

Interpretation of results:

other: the test item is likely to be a skin sensitizer

Conclusions:

It was concluded that the test item, Jasminlactone, gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is likely to be a skin sensitizer.

Executive summary:

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, Jasminlactone, is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The I_max for Jasminlactone was 1.73 in Test 1 and 2.04 in Test 2.  The I_max for both tests was >1.5 fold and statistically significant compared to the DMSO control.  The EC_1.5 was 198.39 µM and 151.81 µM for Tests 1 and 2, respectively.  The IC₃₀ value was 364.66 µM in Test 1 and 338.97 µM in Test 2 and the IC₅₀ values were
409.36 µM and 385.49 µM in Tests 1 and 2, respectively.

Data showed an overall dose-response for luciferase induction.

All acceptance criteria for the positive control, cinnamic aldehyde, were met.

It was concluded that the test item, Jasminlactone, gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is likely to be a skin sensitizer.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 March 2020 to 20 March 2020

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:

The DPRA is a chemistry-based assay (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) exploiting the fact that most chemical allergens have electrophilic properties and are therefore able to react with the nucleophilic sidechains of amino acids to form covalent bonds. The underlying rationale of the assay is that if a chemical is capable of reacting with proteins then it has the potential to act as a sensitizer. The endpoint measured in the assay is the percentage depletion over time of two synthetic peptides (containing respectively a cysteine and a lysine amino acid) from the peptide mixtures following an approximate 24 hour (22-26 hours) incubation with the test item. The percentage of peptide depletion is calculated by High Performance Liquid Chromatography using ultra-violet detection.

The DPRA test allows quantification of a chemical’s reactivity and is used to categorize a substance in one of four classes of reactivity to allow discrimination between skin sensitizing and non-sensitizing chemicals and thus assesses their sensitization potential.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Purity/correction factor: 99.63% (GC)
- Appearance: Colourless to pale yellow liquid
- Expiry/retest date: 26 November 2021
- Storage conditions: Refrigerated (2 to 8°C) protected from light, under nitrogen.

Details of test system:

other: synthetic peptides (containing respectively a cysteine and a lysine amino acid)

Details on the study design:

Skin sensitisation (In chemico test system)
- Details on study design:
Peptide and Positive Control
Synthetic peptide containing Cysteine:
Alternative name: Ac-RFAACAA-OH
Batch number: 1857724
Stated purity: 96.2% (by HPLC)
Molecular Weight: 751 g/mol
Supplier: AnaSpec
Storage conditions: Frozen (-10°C to -30°C)

Synthetic peptide containing Lysine:
Alternative name: Ac-RFAAKAA-OH
Batch number: 1857068
Stated purity: 90.4% (by HPLC)
Molecular Weight: 777 g/mol
Supplier: AnaSpec
Storage conditions: Frozen (-10°C to -30°C)

Assessment of Test Item Solubility
The solubility of Jasminelactone was assessed in acetonitrile.

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).

Incubation
The appearance of the Jasminelactone and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to injection of the samples as part of analytical run. Before initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of Jasminelactone and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.

Positive control:

cinnamic aldehyde

Positive control results:

The positive control resulted in mean depletion results that result in positive DPRA prediction.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

1.2 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

1.5 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

Solubility Assessment
The solubility of Jasminlactone in acetonitrile at a nominal concentration of 100 mM was achieved.

ACCEPTANCE OF RESULTS:
All analytical acceptance criteria for each peptide run were met (see any other information on results).
- Acceptance criteria met for reference control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Reactivity Assessment

All analytical acceptance criteria for each peptide run were met:

 

		Peptide		Standard Linearity		Positive control depletion (%)		Reference controls		Test item
Acceptance criteria		Cysteine		r^2>0.99		60.8-100 (SD <14.9%)		0.45-0.55 mM (CV <15%)		SD <14.9%
		Lysine		r^2>0.99		40.2-69.0 (SD <11.6%)		0.45-0.55 mM (CV <15%)		SD <11.6%
Achieved results		Cysteine		r^2>0.999		70.4 (SD 0.51%, CV 1.21%, n=3)		A: 0.507 mM (CV 0.11%, n=3) B: 0.508 mM (CV 0.83%, n=6)		SD 0.41% CV 0.41% (n=3)
		Lysine		r^2>0.999		57.7 (SD 0.51%, CV 1.21%, n=3)		A: 0.497 mM (CV 2.60%, n=3) B: 0.509 mM (CV 0.48%, n=6)		SD 0.13% CV 0.13% (n=3)

CV         Coefficient of Variation

SD         Standard deviation

 

The depletion of peptide in the presence of Jasminelactone was:

	Mean peak area of reference control(µV.sec)	Mean peak area of peptide with test item(µV.sec)	Mean peptide depletion by Jasminelactone (%)
Cysteine	Control B: 881040 (n=6)	870580 (n=3)	1.2
Lysine	Control B: 754010 (n=6)	742400 (n=3)	1.5

 

Applying the following depletion model (below), reactivity is classed as “No or Minimal” hence the DPRA prediction is negative and Jasminelactone is therefore predicted to not be a sensitizer. 

 

Mean of Cysteine and Lysine% depletion	Reactivity Class	DPRA Prediction
0%≤ mean% depletion ≤6.38%	No or minimal reactivity	Negative
6.38%< mean% depletion ≤22.62%	Low reactivity	Positive
22.62%< mean% depletion ≤42.47%	Moderate reactivity
42.47%< mean% depletion ≤100%	High reactivity

There were no co-elution peaks in the Cysteine and Lysine assays.

Interpretation of results:

GHS criteria not met

Conclusions:

Solutions of Jasminelactone were successfully analysed by the validated DPRA analytical method in both Cysteine and Lysine containing synthetic peptides. With no or minimal mean depletion of both peptides (1.4%) in the presence of the test item, Jasminelactone is therefore predicted by DPRA as negative and to not be a potential skin sensitizer based on this assay.

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of Jasminelactone. 

Solutions of Jasminelactone were successfully analysed by the validated DPRA analytical method in both the Cysteine and Lysine containing synthetic peptides.  With minimal to no depletion of both peptides in the presence of Jasminelactone, the test item is therefore classified by DPRA as negative and hence to not be a potential skin sensitizer. 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The prediction of the skin sensitising potential of jasminlactone was performed with BIOVIA Discovery Studio (TOPKAT) 4 .5, VEGA NIC 1.1.4 (CAESAR), OECD QSAR Toolbox 4.2, Toxtree 3.1.0 and DEREK Nexus 6.0.1.

Although the majority of models predicted jasminlactone not to be sensitising, there is uncertainty due to other lactones reported to be sensitising. Confirmatory testing was therefore performed, as below:

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

The purpose of this study was to assess the reactivity and sensitizing potential of Jasminelactone. 

Solutions of Jasminelactone were successfully analysed by the validated DPRA analytical method in both the Cysteine and Lysine containing synthetic peptides.  With minimal to no depletion of both peptides in the presence of Jasminelactone, the test item is therefore classified by DPRA as negative and hence to not be a potential skin sensitizer. 

In vitro ARE-Nrf2 Luciferase Test (KeratinoSens™) (OECD 442D):

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether Jasminlactone is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The Imax for Jasminlactone was 1.73 in Test 1 and 2.04 in Test 2. The Imax for both tests was >1.5 fold and statistically significant compared to the DMSO control. The EC1.5 was 198.39 µM and 151.81 µM for Tests 1 and 2, respectively. The IC30 value was 364.66 µM in Test 1 and 338.97 µM in Test 2 and the IC50 values were 409.36 µM and 385.49 µM in Tests 1 and 2, respectively. Data showed an overall dose-response for luciferase induction.

All acceptance criteria for the positive control, cinnamic aldehyde, were met.

 It was concluded that the test item, Jasminlactone, gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is likely to be a skin sensitizer.

I n Vitro Skin Sensitisation (human Cell Line Activation Test) (OECD 442E):

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

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of available in chemico and in vitro skin sensitisation studies, the substance is considered to have skin sensitisation potential due to positive results in two in-vitro studies (OECD 442D and 442E) and therefore the substance will be classified for skin sensitisation (Cat 1). The in-vitro studies are not suitable to assess to potency of the substance for skin sensitisation and a sub-categorisation (1A or 1B) is therefore not made.