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

Skin sensitisation

Administrative data

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

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 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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

243-497-1

EC Name:

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

Cas Number:

20073-13-6

Molecular formula:

C13H20O3

IUPAC Name:

methyl 2-[(1S,2S)-3-oxo-2-[(2Z)-pent-2-en-1-yl]cyclopentyl]acetate

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

In vitro test system

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]

Results and discussion

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.

In vitro / in chemico

Results

Results 1

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

1.16

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

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

Results 3

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

Results 4

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

Results 5

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

Results 6

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

Results 7

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.