Methyl [1α,2β(Z)]-(±)-3-oxo-2-(pent-2-enyl)cyclopentaneacetate

Endpoint summary

Description of key information

In chemico, OECD 442C (DPRA) - Positive

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

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:

weight of evidence

Study period:

Experimental Start Date: 19 July 2021
Experimental Completion Date: 27 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

not specified

Specific details on test material used for the study:

Jasmoneige (Methyl Jasmonate)
Chemical name Methyl 3-oxo-2-(pent-2-enyl)cyclopentaneacetate
CAS number 39924-52-2
lot number 0900175
Description: colourless to pale yellow liquid.
Received on 03 June 2021 and stored at 2-8°C, protected from light.
Expiry date was given as 25 March 2023 (2.5 years after production; production date 24 September 2020)
Purity: 99.03% (purity sum of three isomers); 6.19% trans-trans isomer, 87.80% trans-cis isomer,
5.03% cis-cis isomer

Details of test system:

THP-1 cell line [442E]

Details on the 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) x100

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

CD86/CD54 Expression Measurement
Two independent runs (experiments) were needed to drive a prediction. Each independent run was performed 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 10*6 cells/mL. The cells were then distributed into a 24-well plate (500 μL/1 x 10*6 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, resuspended 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 Experiment 1 measurements, an unusual distribution (cell population was undefined and merging with the debris and/or below the axis) was initially noted on the FACS plot for the well containing the test concentration of 269.88 μg/mL for CD86 analysis, indicating that a flow cytometer blockage had occurred. Accordingly, this sample was therefore transferred to a fresh well 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 were therefore not reported.

Vehicle / solvent control:

DMSO

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:

other: Multiple concentrations

Parameter:

EC200, CD54 [442E]

Value:

200 %

Cell viability:

>50%

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

other: multiple concentrations

Parameter:

EC150, CD86 [442E]

Value:

150 %

Cell viability:

>50%

Vehicle controls validity:

valid

Positive controls validity:

valid

Dose Finding Assay
A reduction in cell viability was observed for both runs of the additional dose finding
assay (run 3 and 4). As the viability response for both run 3 and run 4 were aligned,
the CV75 determination was based on these, and the data from run 1 and 2 are
presented for information only.
The CV75 value for the test article was determined to be 559.62 μg/mL.

Dose Finding Assay: Cell Viability

Viability (%) at Concentration (µg/mL)

Run	7.81	15.63	31.25	62.5	125	250	500	1000
1*	99.2	98.9	99.0	98.9	98.8	99.0	97.9	92.5
2*	98.8	99.0	98.7	99.0	98.9	98.7	97.5	31.7
3	98.5	98.7	98.5	98.7	98.9	98.6	94.9	0.6
4	98.4	98.6	98.2	98.6	98.5	98.2	84.4	0.7

* Included for information only, CV75 calculated from runs 3 and 4.

CD86/CD54 Expression Results
In both Experiments the RFI values for CD86 were <150% and the RFI values for CD54 were >200% at multiple concentrations (with cell viability >50%). The test article therefore gave a positive prediction in the assay.
The EC200 value for CD54 calculated by linear regression of endpoint assay data was 362.5 μg/mL. No EC150 value was calculated for CD86 as this marker was negative in both experiments.

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

Concentration (µg/mL)	RFI (CD86) Exp 1	Exp 2	RFI (CD54) Exp 1	Exp 2
187.41	107	90	169	139
224.90	95	105	167	156
269.88	105	99	168	180
323.85	87	101	175	198
388.62	98	126	217	208
466.35	105	80	174	251
559.62	79	72	193	263
671.54	82	60	333	317
Solvent/vehicle	103	124	82	106
control (DMSO)
Positive control	326	371	1253	1155
(DNCB)

 

Assay Acceptance Criteria ResultsAll 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 test article, the cell viability was more than 50% in all tested concentrations in each independent run.

Expression Assay: MFI and Cell Viability Values

Experiment 1

Concentration	MFI (Geo Mean)			Corrected MFI			Viability
(µg/mL)	CD86	CD54	Isotype	CD86	CD54	IgG	CD86	CD54
187.41	1081	816	563	518	253	98.8	98.3	98.4
224.90	1025	816	566	459	250	98.6	98.3	98.3
269.88	1087	830	578	509	252	98.6	98.3	98.6
323.85	1017	858	595	422	263	98.5	98.6	98.2
388.62	1092	940	615	477	325	97.6	98.1	97.6
466.35	1139	889	628	511	261	97.9	98.0	98.0
559.62	1030	935	646	384	289	98.1	97.9	97.9
671.54	1084	1186	687	397	499	94.5	94.1	94.7
Culture medium	996	706	523	473	183	98.8	98.4	98.3
DMSO 0.2%	993	658	508	485	150	98.5	98.4	98.5
DNCB 4 µg/mL	2163	2461	581	1582	1880	84.5	83.0	83.7

Experiment 2

Concentration	MFI (Geo Mean)			Corrected MFI			Viability
(µg/mL)	CD86	CD54	Isotype	CD86	CD54	IgG	CD86	CD54
187.41	1048	793	563	485	230	98.4	98.4	98.5
224.90	1143	837	578	565	259	98.4	98.3	98.4
269.88	1144	908	609	535	299	98.4	98.1	98.3
323.85	1164	949	620	544	329	97.8	98.0	97.4
388.62	1278	946	600	678	346	98.3	97.5	98.0
466.35	1080	1067	650	430	417	97.6	97.2	97.3
559.62	1075	1125	688	387	437	96.7	96.2	95.6
671.54	1045	1251	724	321	527	93.2	92.7	92.7
Culture medium	934	658	501	433	157	98.6	98.3	98.3
DMSO 0.2%	1032	659	493	539	166	98.5	98.4	98.4
DNCB 4 µg/mL	2589	2509	592	1997	1917	86.9	85.7	86.5

 

Interpretation of results:

study cannot be used for classification

Conclusions:

The test article, Jasmoneige (Methyl Jasmonate), was considered to be positive in the human Cell Line Activation Test.

Executive summary:

The study was conducted to investigate the potential of Jasmoneige (Methyl Jasmonate) 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 dose finding, the test article was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 500 mg/mL giving a maximum test concentration of 1000 µg/mL. The viability response between the two runs performed for the initial dose finding assay was not aligned, and an additional dose finding assay was therefore performed to clarify the response. The CV75 value for the test article was determined to be

559.62 µg/mL.

 

For the expression measurements, test concentrations in a range from 187.41 to

671.54 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.

 

In both Experiments the RFI values for CD86 were <150% and the RFI values for CD54 were >200% at multiple concentrations (with cell viability at >50%). The test article therefore gave a positive prediction in the assay.

 

The EC200 value for CD54 was calculated to be 362.5 μg/mL. No EC150 value was calculated for CD86 as this marker was negative in both experiments.

 

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

 

The test article, Jasmoneige (Methyl Jasmonate), 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:

Experimental Start Date: 6th July 2021
Experimental Completion Date: 23rd July 2021

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 KeratinoSens™ test method)

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:

Methyl Jasmonate, also known as Jasmoneige
Chemical name Methyl 3-oxo-2-(pent-2-enyl)cyclopentaneacetate
CAS number 39924-52-2,
Lot number 0900175
Description: colourless to pale yellow liquid.
Received: 03 June 2021 and stored at 2-8°C, protected from light.
Expiry date: 25 March 2023 (2.5 years after production; production date 24 September 2020)
Purity: 99.03% (purity sum of three isomers); 6.19% trans-trans isomer, 87.80% trans-cis isomer,
5.03% cis-cis isomer

Details on the study design:

Test Article Formulation
Preliminary solubility data indicated that Methyl Jasmonate was soluble in dimethyl sulfoxide (DMSO) up to at least 50.89 mg/mL, which was in excess of the target concentration 200 mM (equivalent to 44.86 mg/mL).
Test formulations were prepared using DMSO. This was the first of the listed vehicles that produced a visually clear solution at a concentration of 200 mM.
Serial dilutions were made (from the 200 mM stock) using the vehicle to obtain 12 master concentrations ranging from 0.098 to 200 mM.
The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations range from 0.98 to 2000 μM.

Treatment Plate Preparation
The cells were 80-90% confluent. On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated for 24±1 hours an incubator set to 37°C, 5% CO2.
For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.

Treatment
At the end of the 24-hour incubation period, the medium was removed and replaced with fresh culture medium (containing serum but without Geneticin) to which test article and control formulations were added.
On each plate, one well per replicate was left empty (no cells and no treatment) to assess background values.
Aliquots of 50 μL of each of the final concentrations were transferred to give three luciferase replicates on a white-walled plate and a single viability replicate on a clear-walled plate. Each plate was sealed and incubated for 48±1 hours in a humidified incubator set to 37°C, 5% CO2.
For each test article and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of two independent repetitions each containing three replicates of each concentration.
Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells. The cells came from different batches.
It may be noted that initial Experiment 2 treatments were terminated due to a technical error, and the data are not reported. These treatments were repeated in order to provide the Experiment 2 data which are presented in this report.

Cytotoxicity Assessment
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The plate was sealed and placed in an incubator set to 37°C, 5% CO2 for 4 hours. The MTT medium was removed and 200 μL SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator set to 37°C, 5% CO2, and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm.

Luciferase Activity Measurements
After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plate was then incubated for 20 minutes in an incubator set to 25°C, loaded into the luminescence plate reader and read using the following parameters: 100 μL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.

Vehicle / solvent control:

DMSO

Negative control:

other: DMSO. The negative control was diluted into culture medium containing serum so that the final concentration was 1%.

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The EC1.5 value for the positive control were 5.96 and 9.17 μM in Experiments 1 and 2, respectively. The average induction in the three replicates for the positive control at 64 μM were 1.18 and 5.86 in Experiments 1 and 2, respectively. The average induction for the positive control at 64 μM in Experiment 1 was outside the range specified in the Protocol. As however the EC1.5 was within the range seen at these laboratories and a clear positive dose response was observed for luciferase induction, these data were considered to be acceptable.

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

1.16

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC50 [442D]

Value:

2 000 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC30 [442D]

Value:

2 000 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC30 [442D]

Value:

1 208.46 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC50 [442D]

Value:

865.72 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.19

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.13

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Other effects / acceptance of results:

Calculation of Maximal Average Fold Induction and Determination of Luciferase Activity
There were no EC1.5 values for Experiments 1 or 2 as there were no statistically significant increases in induction.

Viability
The cell viability measurement was not applicable as there were no EC1.5 determining concentrations in Experiments 1 or 2.
An anomalous toxicity response which was not in line with any other treatment concentration was noted on the viability plate at 7.81 mM in Experiment 1. The unusual response was noted following treatment and incubation, and a viability of -0.06% was subsequently returned following MTT assay. The preceding three and subsequent eight test concentrations in Experiment 1, and also all test concentrations in Experiment 2 had no reduction in viability (all values >70%). It was therefore considered that a technical error likely occurred on the viability plate at 7.81 mM in
Experiment 1 which resulted in this anomalous toxicity, and accordingly, the obtained viability value was disregarded and is presented in Table 9.3 for information only.
As all viability results for Experiment 2 were above 70%, the IC50 and IC30 values for this experiment were >2000 μM and could not be calculated. The geometric mean IC50 and IC30 values of Experiments 1 and 2 could therefore not be calculated.

Assay Acceptance
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 8 to 64 μM in Experiment 1 and at concentrations of 16 to 64 μM in Experiment 2.
The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 11.33% and 12.74% in Experiments 1 and 2, respectively.

Luminescence Readings for Experiment 1

		Concentration (µM)
Substance		0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
	Plate 1	402359	482958	388531	463364	535506	447711	545741	467357	467398	412005	431378	563452
	Plate 2	459264	441075	486799	541909	453109	507595	486831	505190	479749	464852	496811	502881
Methyl Jasmonate	Plate 3	488889	465327	470398	477052	460814	475381	464764	459837	452389	486306	450089	607077
	Mean fold
	Induction	0.92	0.94	0.91	1.01	0.99	0.97	1.02	0.97	0.95	0.92	0.94	1.13*

Substance	Individual Values
Negative control	Plate 1	418088	463819	504379	515926	530726	457400
	Plate 2	386501	453527	455478	607793	469996	474925
	Plate 3	480423	474502	501564	510936	531838	603037

 

Concentration (µM)
Substance		4	8	16	32	64
	Plate 1	718252	840496	1057104	2074031	5435230
Positive control	Plate 2	637787	751442	1090331	1986846	5763708
	Plate 3	683427	788623	1097392	1968326	5194634
	Mean fold Induction	1.39	1.62#	2.21#	4.11#	11.18#

*              Imax

#              Luciferase activity induction statistically significant above the threshold of 1.5

 

Luminescence Readings for Experiment 2

Concentration (µM)
Substance		0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
	Plate 1	606307	482318	473109	523432	492419	435818	524813	431267	613633	531213	439794	491825
	Plate 2	491285	534258	497339	541145	541341	558171	550899	499604	662367	557156	497099	535143
Methyl Jasmonate	Plate 3	466586	429721	581515	520107	522527	627890	510305	480316	596500	490784	453397	465583
	Mean fold
	Induction	0.99	0.92	0.98	1.00	0.98	1.02	1.00	0.89	1.19*	1.00	0.88	0.94

Substance	Individual Values
Negative control	Plate 1	604784	463981	502707	502184	498945	479730
	Plate 2	495566	552454	480448	525347	594084	598658
	Plate 3	515906	419065	458233	501998	593701	692315

	Concentration (µM)
Substance		4	8	16	32	64
Positive control	Plate 1	729083	702254	961233	1575379	2960198
	Plate 2	851784	802070	1139438	1593390	3528909
	Plate 3	752865	729654	1065849	1742544	2761048
	Mean fold Induction	1.48	1.41	2.00#	3.11#	5.86#

*              Imax

#              Luciferase activity induction statistically significant above the threshold of 1.

MTT-Absorbance Readings

	concentration	(µM)
Substance	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Methyl Jasmonate                                      Experiment 1	0.474	0.471	0.453	0.000	0.459	0.509	0.483	0.498	0.555	0.640	0.649	0.556
Experiment 2	0.668	0.599	0.578	0.644	0.608	0.614	0.568	0.709	0.766	0.856	0.897	0.686
Viability (%)                                      Experiment 1	99.08	98.41	94.57	-0.06*	95.91	106.27	100.81	103.99	115.90	133.63	135.51	116.19
Experiment 2	101.80	91.40	88.18	98.15	92.76	93.67	86.59	108.17	116.75	130.52	136.86	104.59

*Data considered anomalous and disregarded

The Imax values were calculated as follows:

Parameter	Experiment 1	Experiment 2	Mean
Imax	1.13	1.19	1.16

There were no EC1.5 values for Experiments 1 or 2 as there were no statistically significant increases in induction.

Interpretation of results:

study cannot be used for classification

Conclusions:

The Imax values were less than 1.5 in both experiments, therefore Methyl Jasmonate was considered to be negative in the ARE-Nrf2 Luciferase Test.

Executive summary:

The study was conducted to investigate the potential of Methyl Jasmonate to induce genes that are regulated by the antioxidant response element (ARE). 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 test article was dissolved in dimethyl sulfoxide (DMSO) to the final stock concentration (200 mM). Serial dilutions were then made using DMSO to obtain 12 master concentrations of the test article in the range 0.098 to 200 mM.
The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations ranged from 0.98 to 2000 μM.
Aliquots of 50 μL of each of the final concentrations were transferred to give three luciferase replicates on a white-walled plate and a single viability replicate on a clear-walled plate. Each plate was sealed using a plate sealer and then incubated for 48±1 hours in a humidified incubator set to 37°C, 5% CO2.
After the 48-hour exposure period, the medium in the viability plate was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The plate was sealed and incubated for 4 hours in an incubator set to 37°C, 5% CO2. The MTT medium was then removed and 200 μL SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator set to 37°C, 5% CO2, and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm.
After the 48-hour exposure period, the cells in the luciferase plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plate was then incubated for 20 minutes in an incubator set to 25°C, loaded into the luminescence plate reader and read.
The Imax value in both experiments was less than 1.5.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Experimental Start Date: 19 July 2021
Experimental Completion Date: 08 December 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Deviations:

yes

Remarks:

Please see details of study design

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Jasmoneige (Methyl Jasmonate)
Chemical name Methyl 3-oxo-2-(pent-2-enyl) cyclopentaneacetate
CAS number 39924-52-2
Lot number 0900175
Molecular weight 224.3 g/mol,
Appearance: colourless to pale yellow liquid.
Received on 03 June 2021 and stored at 2-8°C, protected from light.
Expiry date was given as 25 March 2023 (2.5 years after production; production date 24 September 2020)
Purity was stated as 99.03% (purity sum of three isomers); 6.19% trans-trans isomer, 87.80% trans-cis isomer, 5.03% cis-cis isomer

Details on the study design:

Test Article Formulation
The test article was dissolved in acetonitrile. The acetonitrile (batch numbers 2049762, expiry 13 August 2021; 2034317, expiry 13 August 2023 and 2043930, expiry 19 May 2024) was supplied by Fisher. This was the first of the listed vehicles that produced a visually clear solution at a concentration of 100 mM.
The positive control was dissolved in acetonitrile at a concentration of 100 mM.
A stock solution containing cysteine at approximately 0.667 mM was prepared in 100 mM Phosphate Buffer pH 7.5 and a stock solution containing lysine at approximately 0.667 mM was prepared in 100 mM ammonium acetate buffer pH 10.2.
Formulations were prepared shortly before testing

Test Article Incubation
Each test solution was prepared at ratios of 1:10 and 1:50 (based on molecular weight) with the cysteine and lysine stock solutions, respectively. The preparations were placed in an incubator set at 25˚C for 24±2 hours. Prior to, and at the end of the incubation period the samples were visually inspected for precipitate formation. No precipitation was observed in the cysteine samples. Precipitation was observed pre incubation in all lysine test article and co-elution samples and post-incubation in all lysine test article, co-elution and positive control incubations. Precipitate was removed from all affected post-incubation samples by centrifugation at 400 g for 5 minutes; and the remaining sample aspirated to a clean autosampler vial.

Analytical Method
The following HPLC conditions were applied:
Column: Agilent Zorbax SB-C18 2.1 mm x 100 mm, 3.5 µm
Wavelength: 220 nm
Guard column: Phenomenex Security Guard C18 4 mm x 2 mm
Flow rate: 0.35 mL/min
Oven temperature: 30°C
Sample temperature: 25°C
Injection volume: 5 µL

Mobile Phase:
Phase A: 0.1% (v/v) of trifluoroacetic acid in MilliQ water
Phase B: 0.085% (v/v) of trifluoroacetic acid in acetonitrile

Gradient:
Time (min): 0; Phase A: 90; Phase B: 10
Time (min): 10; Phase A: 75; Phase B: 25
Time (min): 11; Phase A: 10; Phase B: 90
Time (min): 13; Phase A: 10; Phase B: 90
Time (min): 13.5; Phase A: 90; Phase B: 10
Time (min): 20; Phase A: 90; Phase B: 10

Reference and Co-Elution Controls
Reference controls were prepared for each peptide.
Reference Control A and B for each peptide were prepared by adding 750 µL of peptide stock solution to 250 µL of acetonitrile.
Reference Control C for each peptide were prepared by adding 750 µL of peptide stock solution to 250 µL of acetonitrile (vehicle).
Reference Control A (in triplicate) was used to verify the HPLC system suitability prior to the analysis. Reference Control B (six replicates) was used to verify the stability of the reference controls over time and Reference Control C (in triplicate) was used to verify that the vehicle did not impact the percent peptide depletion.
Co-elution controls were prepared to detect possible co-elution of the test article with the peptides. A mixture of 750 µL of 100 mM Phosphate Buffer pH 7.5, 200 µL of acetonitrile and 50 µL of test article solution was used to detect possible co-elution of the test article with cysteine. A mixture of 750 µL of 100 mM ammonium acetate buffer pH 10.2 and 250 µL of test article solution was used to detect possible co elution of the test article with lysine.

Calibration Curves for Peptides
Calibration curves were prepared for each peptide using a range of concentrations from approximately 0.534 mM to 0.0167 mM (Standards 1 to 6).
Standard 1 was prepared at approximatively 0.534 mM by dilution of 1600 µL of the peptide stock solution (0.667 mM) with 400 µL of acetonitrile.
Standards 2 to 6 for cysteine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM Phosphate Buffer pH 7.5).
Standards 2 to 6 for lysine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM ammonium acetate buffer pH 10.2).
Samples of dilution buffer alone were also prepared.

4.5 Sample Analysis Sequence
The analysis sequence for each peptide was as follows:
System suitability Standard 1
Blank
Calibration standards and reference controls Standard 1
Standard 2
Standard 3
Standard 4
Standard 5
Standard 6
Dilution Buffer
Reference Control A, rep 1
Reference Control A, rep 2
Reference Control A, rep 3
Co-elution controls Co-elution control for test article
Reference controls Reference Control B, rep 1
Reference Control B, rep 2
Reference Control B, rep 3
First set of replicates
Reference Control C, rep 1
Positive Control, rep 1
Test sample, rep 1
Second set of replicates
Reference Control C, rep 2
Positive Control, rep 2
Test sample, rep 2
Third set of replicates
Reference Control C, rep 3
Positive Control, rep 3
Test sample, rep 3
Reference controls
Reference Control B, rep 4
Reference Control B, rep 5
Reference Control B, rep 6

Protocol Deviation
For Experiment 1 the cysteine peptide stock solution was formulated at 0.518 mg/mL (0.690 mM) rather than the protocol required 0.501 mg/mL (0.667 mM). This resulted in in a final mix ratio of test article:peptide of 9.7:1. The final mix ratio was considered sufficiently close to ratio 10:1, furthermore, all acceptance criteria were met, thereby confirming correct assay functioning
The study deviation neither affected the overall interpretation of study findings nor compromised the integrity of the study.

Run / experiment:

run/experiment 1

Parameter:

mean cystein depletion

Value:

9.35 %

Run / experiment:

run/experiment 1

Parameter:

mean lysine depletion

Value:

0 %

Run / experiment:

run/experiment 2

Parameter:

mean cystein depletion

Value:

27.35 %

Run / experiment:

run/experiment 2

Parameter:

mean lysine depletion

Value:

0.53 %

Run / experiment:

run/experiment 3

Parameter:

mean cystein depletion

Value:

41.17 %

Run / experiment:

run/experiment 3

Parameter:

mean lysine depletion

Value:

1.71 %

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

All acceptance criteria were met in all experiments.

Precipitate was observed immediately upon addition of the test article solution to the lysine peptide solution in all experiments, there was therefore uncertainty as to how much test article initially remained in solution to react with the peptide. In addition, precipitation was also observed post-incubation which may result in an underestimation of peptide depletion.

In Experiment 1, the cysteine depletion value was 9.35% and the lysine depletion value was 0%. However, due to precipitation observed in lysine samples, a conclusion could not be drawn with sufficient confidence for this peptide. As no precipitation was observed for cysteine treatments, the cysteine 1:10 prediction model was used. The cysteine depletion value (9.35%), which provided a negative prediction with no or minimal reactivity, was however within the range 9% to 17% of the cysteine 1:10 prediction model. Accordingly, as per test guideline, a confirmatory experiment (Experiment 2) was conducted.

In Experiment 2, the cysteine depletion value was 27.35%, the lysine depletion value was 0.53% and the mean of the cysteine and lysine depletion values was 13.94% which provided a positive prediction with low reactivity. However, due to precipitation observed in lysine samples, a conclusion could not be drawn with sufficient confidence for this peptide. As no precipitation was observed for cysteine treatments, the cysteine 1:10 prediction model was used, which provided a positive prediction with moderate reactivity as the cysteine depletion value of 27.35% was in the range of 23.09% – 98.24%.

As discordant predictions were obtained between Experiments 1 and 2, a third run (Experiment 3) was conducted.

In Experiment 3, the cysteine depletion value was 41.17%, the lysine depletion value was 1.71% and the mean of the cysteine and lysine depletion values was 21.44% which provided a positive prediction with low reactivity. Precipitation was again observed in lysine samples, and a conclusion could not be drawn with sufficient confidence for this peptide. As no precipitation was observed for cysteine treatments, the cysteine 1:10 prediction model was used, which provided a positive prediction with moderate reactivity as the cysteine depletion value of 41.17% was in the range of 23.09% – 98.24%.

Precipitation of the test article occurred only with the lysine peptide, therefore the cysteine 1:10 prediction model was used in each experiment. Cysteine depletion was 9.35%, 27.35% and 41.17% in Experiments 1, 2 and 3, respectively. The results indicated a positive DPRA prediction with moderate reactivity as the cysteine PPD value in two of the three experiments was between 23.09% and 98.24% for the cysteine 1:10 prediction model.

Therefore, based on the majority result of the three independent runs, the test article, Jasmoneige, was considered to be positive, with moderate reactivity in the Direct Peptide Reactivity assay

Interpretation of results:

study cannot be used for classification

Conclusions:

The test article, Jasmoneige (Methyl Jasmonate), was considered to be positive, with moderate reactivity, in the Direct Peptide Reactivity Assay.

Executive summary:

The study was conducted to quantify the reactivity of Jasmoneige (Methyl Jasmonate) towards model synthetic peptides containing either lysine or cysteine. 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 test article was dissolved in acetonitrile at a concentration of 100 mM.

The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24±2 hours in glass autosampler vials, protected from light, in an incubator set to 25°C.

The remaining concentration of cysteine- or lysine-containing peptides following the 24-hour incubation period was measured by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

Precipitation of the test article occurred only with the lysine peptide, therefore the cysteine 1:10 prediction model was used in each experiment. Cysteine depletion was 9.35%, 27.35% and 41.17% in Experiments 1, 2 and 3, respectively. The results indicated a positive DPRA prediction with moderate reactivity as the cysteine PPD value in two of the three experiments was between 23.09% and 98.24% for the cysteine 1:10 prediction model.

The test article, Jasmoneige (Methyl Jasmonate), was considered to be positive, with moderate reactivity, in the Direct Peptide Reactivity Assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

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 Jasmoneige. 

Solutions of Jasmoneige were successfully analysed by the validated DPRA analytical method in Cysteine containing synthetic peptides. Precipitation of the test article occurred with the lysine peptide, therefore only the cysteine 1:10 prediction model was used. Cysteine depletion was 9.35%, 27.35% and 41.17% in the presence of Jasmoneige, the test item is therefore classified by DPRA as positive. 

 

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 Jasmoneige is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The Imax for Jasmoneige was less than 1.5 in both experiments. 

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

It was concluded that the test item, Jasmoneige gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is not a skin sensitizer.

 

In 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, Jasmoneige, 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 442C 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 ma