Ethyl (2E,4Z)-2,4-decadienoate

Endpoint summary

Description of key information

The provided collection of tests represents a complete Integrated Approach to Testing and Assessment (IATA). Although the individual tests on their own may be insufficient for classification, the combined weight of evidence concludes that there is no risk of sensitisation for ethyl (2E,4Z)-decadienoate.

Three OECD skin sensitisation tests demonstrate that no evidence of potential sensitising effects were observed when assessing the first key event of protein reactivity (OECD 422C, in chemico direct peptide reactivity assay), the second key event of activation of keratinocytes (OECD 422D, in vitro luciferase activity test) and the third key event of dendritic cell activation (OECD 422E, in vitro human cell line activation test). Taken together, these tests support that ethyl (2E,4Z)-decadienoate is not sensitising to the skin. 

In addition, a patch test conducted on human volunteers in 1982 found that ethyl decadienoate is not sensitising. This test did not include a positive control, however this is typical of a human "patch test"; it would not be ethical to intentionally induce an allergic reaction in human volunteers. 

Finally we note that ethyl (2E4Z)-decadienoate is intended for use solely in cosmetic products, an application for which animal testing is strictly forbidden.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

March 8-April 20, 1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

other: JID 47: 393-409, 1966

GLP compliance:

no

Type of study:

patch test

Justification for non-LLNA method:

A LLNA test was not performed as this in vivo study using humans was already available.

Species:

other: human

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: volunteers


ENVIRONMENTAL CONDITIONS
- IN-LIFE DATES: From: March 8, 1982 To: April 20, 1982

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100%

Day(s)/duration:

10 days

Adequacy of induction:

other: a pre-test showed the test substance showed no significant evidence of irritation, so test subjects were pretested with 3% SLS.

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100%

Day(s)/duration:

48 hrs

Adequacy of challenge:

not specified

No. of animals per dose:

27 (Originally 30, but 3 volunteers did not complete the study.)

Details on study design:

RANGE FINDING TESTS:

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 5 exposures for 48 hrs
- Exposure period: 10 days total
- Site: forearms
- Frequency of applications: every other day
- Duration: 48 hrs

B. CHALLENGE EXPOSURE
- No. of exposures: 27
- Day(s) of challenge: After a 10-14 day rest from the induction
- Exposure period: 48 hrs
- Control group: SLS and petrolatum were used as controls on the same volunteers
- Site: forearms
- Evaluation (hr after challenge): 48 hrs

OTHER: Prior to the induction, patch sites were pretreated with 5% SLS. For the challenge test, 5% SLS was applied 30 minutes prior to the challenge exposure on the left side, and no SLS was used on the right side.

Challenge controls:

SLS and petrolatum were used as controls on the same volunteers.

Positive control substance(s):

yes

Remarks:

SLS

Positive control results:

5% SLS produced very little reactivity.

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

27

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

100%

No. with + reactions:

1

Total no. in group:

27

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

27

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

100%

No. with + reactions:

1

Total no. in group:

27

Test Substance Challenge Results

Subject No.	With SLS at 48 hrs	With SLS at 72 hrs	Without SLS at 48 hrs	Without SLS at 72 hrs
1	N	N	N	N
2	N	N	N	N
3	N	N	N	N
4	N	N	N	N
5	N	N	N	N
6	N	N	N	N
7	--	--	--	--
8	N	N	N	N
9	N	N	N	N
10	N	N	N	N
11	N	N	N	N
12	N	N	N	N
13	N	N	N	N
14	N	N	N	N
15	--	--	--	--
16	--	--	--	--
17	N	N	N	N
18	N	N	N	N
19	N	N	N	N
20	N	N	N	N
21	N	N	N	N
22	N	N	N	N
23	N	N	N	N
24	N	B	N	N
25	N	N	N	N
26	N	N	N	N
27	N	N	N	N
28	N	N	N	N
29	N	N	N	N
30	--	--	--	--

Control Challenge Results

Subject No.	With SLS at 48 hrs	With SLS at 72 hrs	Without SLS at 48 hrs	Without SLS at 72 hrs
1	N	N	N	N
2	N	N	N	N
3	N	N	N	N
4	N	N	N	N
5	N	N	N	N
6	N	N	N	N
7	--	--	--	--
8	N	N	N	N
9	N	N	N	N
10	N	N	N	N
11	N	N	N	N
12	N	N	N	N
13	N	N	N	N
14	N	N	N	N
15	--	--	--	--
16	--	--	--	--
17	N	N	N	N
18	N	N	N	N
19	N	N	N	N
20	N	N	N	N
21	N	N	N	N
22	N	N	N	N
23	N	N	N	N
24	N	B	N	N
25	N	N	N	N
26	N	N	N	N
27	N	N	N	N
28	N	N	N	N
29	N	N	N	N
30	--	--	--	--

Interpretation of results:

study cannot be used for classification

Conclusions:

No significant irritant or allergic reactions were noted.

Executive summary:

The skin sensitization of ethyl decadienoate was tested in a maximization test using 27 human volunteers. No volunteers showed significant reactions during the challenge phase. The test had significant methodoligical deficiencies, most notably, the test substance concentration and scoring system was not reported.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January 25-March 1, 2018

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))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

An in chemico method was chosen to see if the skin sensitization potential of the test substance could be determined without the use of vertebrate animals.

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
Preparation: A stock solution of 100 mM was prepared.
Reference controls: Yes, reference control A was used to verify the accuracy of the calibration curve for peptide quantification. Reference control B was used to verify the stability of the peptides over the analysis time. Reference control C was used to verifty that the solvent does not impact the percent peptide depletion (PPD). Each of the reference control solutions contained acetonitrile.
Solvent: acetonitrile
Proteins used: cysteine (amino acid sequence Ac-RFAACAA) 0.667 mM and lysine (amino acid sequence Ac-RFAAKAA) 0.667 mM, prior to use the peptides were stored at -80 degrees C and protected from light
Raio test item/protein: 1:10 cysteine, 1:50 lysine
Co-elution control: Prepared similarly to the test substance sample preparations but without peptide solution to determine if the test substance absorbs at 220 nm.
Postitive control: cinnamic aldehyde dissolved in acetonitrile
Incubation time: 24 hrs
Temperature: 25 degrees C
Detection method: HPLC, 220 nm for quantitation, 258 nm for co-elution; 100 mm x 2.1 mm, 3.5 um
Injection volume: 10 uL
Column temperature: 30 degrees C
Run Time: 20 minutes
Mobile phase A: 0.1% v/v trifluoroacetic acid in water
Mobile phase B: 0.085% v/v trifluoroacetic acid in acetonitrile
Replicates: 3

Acceptance criteria:
1) standard calibration curve with r2 > 0.99
2) mean PPD for postive control for cysteine is between 60.8-100%, and the maximum standard deviation is < 14.9%
3) mean PPD for positive control for lysine is between 40.2-69.0%, and the maximum standard deviation is < 11.6%
4) the mean peptide concentration for reference control A is 0.50 +/- 0.05 mM
5) coefficient of variation of the peptide peak areas for reference control B and C in solvent is < 15.0%

Run / experiment:

other: Mean of three replicates

Parameter:

other: Percent peptide depletion

Value:

0

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Due to phase separation, the skin sensitization potential could not be predicted.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: Yes, the reactivity of the postitive control to the peptides was 70.65%.
- Acceptance criteria met for variability between replicate measurements: yes

No turbidity or precipitation was observed in the test solutions. Phase separation was noted after incubation. Minimal reactivity of the test item to the peptides was seen. The co-elution controls were valid. The mean depletion of both cysteine and lysine was 0.0%. Since a phase separation was observed, the concentration of 100 mM of test item, and full contact between the peptides and test item could not be guaranteed. No prediction of skin sensitization could therefore be made.

Interpretation of results:

study cannot be used for classification

Conclusions:

Prediction of skin sensitivity could not be made due to the observed phase separation.

Executive summary:

The skin sensitization potential of the test substance was determine in a peptide reactivity assay. The test evaluates the reactivity of the test sustance to peptides containing lysine and cysteine. Although the control show the test to be valid, phase seperation of the test substance means a prediction of sensitivity cannot be made.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 12-May 4, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422E (In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation)

Version / remarks:

Adopted 9 October 2017

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

An in vitro method was chosen to see if the skin sensitization potential of the test substance could be determined without the use of vertebrate animals.

Details on the study design:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.
In the present study ETHYL 2,4-DECADIENOATE was dissolved in DMSO. Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:
1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL
Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

Dose Groups
1. Medium Control: cell culture medium
2. Solvent Control: 0.2% DMSO (v/v) in cell culture medium
3. Positive Control: 4 µg/mL DNCB
4. Test Item: 8 concentrations of the test item
(dose finding assay/ main experiment)
dose finding assay 1:
1000, 500, 250, 125, 62.50, 31.25, 15.63, 7.81 µg/mL
main experiment 1 and 2:
1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL

Positive control results:

The positive controls 2,4-dinitrochlorobenzene and NiSO4 led to upregulation of the cell surface markers CD54 and CD86. The negative control lactic acid did not induce an upregulation of CD54 and CD86.
The cell batch was accepted for further testing.

Key result

Run / experiment:

other: Relative Fluorescence Intensity of CD86

Parameter:

other: RFI

Value:

90

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Relative Fluorescence Intensity of CD54

Parameter:

other: RFI

Value:

71

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. In theory, the test item is considered to be no skin sensitiser. However, since the log KOW is higher than 3.5, the results must be considered as inconclusive.

Table4:  CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	95.2	93.3	95.1	3235	1599	831	2404	768	105	149	389	192
Solvent Control	0.20%	94.8	94.1	95.1	3266	1483	969	2297	514	100	100	337	153
DNCB	4.00	84.3	84.9	84.3	8562	2099	875	7687	1224	335	238	979	240
ETHYL 2,4-DECADIENOATE	1000	94.1	94.1	94.4	3871	1500	1079	2792	421	122	82	359	139
	833.33	94.1	93.9	93.6	4078	1636	1036	3042	600	132	117	394	158
	694.44	93.7	93.7	93.8	4052	1611	1036	3016	575	131	112	391	156
	578.70	93.4	94.0	92.7	3880	1546	1000	2880	546	125	106	388	155
	482.25	94.3	94.2	94.1	4062	1580	1003	3059	577	133	112	405	158
	401.88	94.1	94.5	94.1	4222	1693	1101	3121	592	136	115	383	154
	334.90	94.4	94.7	94.8	4077	1515	1005	3072	510	134	99	406	151
	279.08	95.2	95.1	94.7	3738	1526	991	2747	535	120	104	377	154

 

 

Table5: CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	93.3	91.8	92.8	5237	1393	701	4536	692	106	111	747	199
Solvent Control	0.20%	93.1	92.6	92.4	4963	1303	679	4284	624	100	100	731	192
DNCB	4.0	84.3	83.3	83.1	10948	2051	747	10201	1304	238	209	1466	275
ETHYL 2,4-DECADIENOATE	1000.00	79.1	79.0	78.5	5511	1479	807	4704	672	110	108	683	183
	833.33	83.3	84.2	83.6	5025	1428	823	4202	605	98	97	611	174
	694.44	85.9	85.6	85.8	4889	1723	803	4086	920	95	147	609	215
	578.70	90.8	91.1	90.9	4749	1433	870	3879	563	91	90	546	165
	482.25	92.7	91.5	92.2	4438	1371	813	3625	558	85	89	546	169
	401.88	90.8	91.2	90.8	4520	1306	866	3654	440	85	71	522	151
	334.90	90.8	91.0	90.6	4666	1334	770	3896	564	91	90	606	173
	279.08	90.0	90.3	90.4	4627	1311	790	3837	521	90	83	586	166

 

Interpretation of results:

study cannot be used for classification

Executive summary:

Thein vitrohuman cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the presentstudy ETHYL 2,4-DECADIENOATE was dissolved in DMSO.Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:

1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 94.1% (CD86), 94.1% (CD54) and 94.4% (isotype IgG1 control) in the first experiment and to 79.1% (CD86), 79.0% (CD54) and 78.5% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

In theory, the test item is considered to be no skin sensitiser. However, since the log KOW is higher than 3.5, the results must be considered as inconclusive.

The controls confirmed the validity of the study for all experiments.

In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. However, since the log KOW is higher than 3.5, the results must be considered as inconclusive.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January 23-March 16, 2018

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)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

An in vitro method was chosen to see if the skin sensitization potential of the test substance could be determined without the use of vertebrate animals.

Details on the study design:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and
non-sensitisers.
In the present study ETHYL 2,4-DECADIENOATE was dissolved in DMSO.
Based on a molecular weight of 196.28 g/mol a stock solution of 200 mM was prepared.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:
2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM
Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

Positive control results:

Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%; Alfa Aesar; Lot No.: 10176010) was used as positive control. CA was dissolved in DMSO (AppliChem; Lot No.: 0001055932) at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

Fold induction of positive control at 64 uM was > 1.5 in all experiments.

Key result

Parameter:

other: Luciferase activity (fold induction)

Value:

1.5

Vehicle controls validity:

valid

Positive controls validity:

valid

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered asnonsensitiser.

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Cytotoxicity

Table1: Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100	100	100	0.0
Positive Control	4.00	94.3	96.5	95.4	1.6
	8.00	97.1	99.2	98.1	1.5
	16.00	96.6	96.2	96.4	0.3
	32.00	94.3	101.4	97.8	5.0
	64.00	93.7	88.3	91.0	3.8
Test Item	0.98	97.7	90.1	93.9	5.3
	1.95	102.4	93.1	97.8	6.6
	3.91	100.8	94.7	97.8	4.3
	7.81	97.4	98.3	97.8	0.7
	15.63	94.8	93.2	94.0	1.1
	31.25	81.3	83.5	82.4	1.6
	62.50	6.1	7.6	6.9	1.1
	125.00	0.7	1.6	1.1	0.7
	250.00	0.7	0.1	0.4	0.4
	500.00	0.5	0.1	0.3	0.3
	1000.00	3.7	2.8	3.3	0.6
	2000.00	15.4	4.7	10.0	7.6

 

Luciferase Activity

Table2: Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.16	1.21	1.05	1.14	0.08
	8.00	1.19	1.25	1.13	1.19	0.06
	16.00	1.68	1.45	1.38	1.50	0.16	*
	32.00	1.80	1.81	2.03	1.88	0.13	*
	64.00	5.22	4.72	4.96	4.97	0.25	*
Test Item	0.98	1.23	1.17	0.95	1.12	0.15
	1.95	1.12	1.11	0.85	1.03	0.15
	3.91	1.00	0.95	0.96	0.97	0.03
	7.81	1.06	1.09	1.01	1.05	0.04
	15.63	0.92	1.06	0.91	0.96	0.09
	31.25	1.17	1.35	0.95	1.16	0.20
	62.50	0.09	0.06	0.38	0.18	0.18
	125.00	0.00	0.00	0.03	0.01	0.01
	250.00	0.00	0.02	0.02	0.01	0.01
	500.00	0.00	0.04	0.05	0.03	0.03
	1000.00	0.11	0.06	0.45	0.21	0.21
	2000.00	0.43	0.16	0.07	0.22	0.19

* = significant induction according to Student’s t-test, p<0.05

Table3: Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.04	1.11	0.99	1.05	0.06
	8.00	1.17	1.33	1.02	1.18	0.15
	16.00	1.24	1.29	1.22	1.25	0.03
	32.00	1.70	1.71	1.65	1.69	0.03	*
	64.00	3.08	2.66	3.11	2.95	0.25	*
Test Item	0.98	1.13	1.16	1.10	1.13	0.03
	1.95	0.82	0.96	1.08	0.96	0.13
	3.91	0.95	0.95	0.91	0.94	0.02
	7.81	0.90	1.00	0.98	0.96	0.05
	15.63	1.17	1.10	0.92	1.06	0.13
	31.25	0.93	0.94	0.92	0.93	0.01
	62.50	0.21	0.27	0.59	0.36	0.21
	125.00	0.02	0.00	0.38	0.13	0.21
	250.00	0.00	0.00	0.29	0.10	0.17
	500.00	0.00	0.00	0.29	0.10	0.17
	1000.00	0.28	0.46	0.52	0.42	0.12
	2000.00	0.38	0.00	0.55	0.31	0.28

* = significant induction according to Student’s t-test, p<0.05

 

Table4: Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.14	1.05	1.09	0.06
	8.00	1.19	1.18	1.18	0.01
	16.00	1.50	1.25	1.38	0.18
	32.00	1.88	1.69	1.78	0.14	*
	64.00	4.97	2.95	3.96	1.43
Test Item	0.98	1.12	1.13	1.12	0.01
	1.95	1.03	0.96	0.99	0.05
	3.91	0.97	0.94	0.95	0.03
	7.81	1.05	0.96	1.01	0.06
	15.63	0.96	1.06	1.01	0.07
	31.25	1.16	0.93	1.04	0.16
	62.50	0.18	0.36	0.27	0.13
	125.00	0.01	0.13	0.07	0.09
	250.00	0.01	0.10	0.05	0.06
	500.00	0.03	0.10	0.06	0.05
	1000.00	0.21	0.42	0.31	0.15
	2000.00	0.22	0.31	0.27	0.06

* = significant induction according to Student’s t-test, p<0.05

 

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

Thein vitroKeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and
non-sensitisers.

In the present study ETHYL 2,4-DECADIENOATE was dissolved in DMSO.

Based on a molecular weight of 196.28 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, nosignificant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5value could be calculated.

In the second experiment,nosignificant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5value could be calculated.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non sensitiser.

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered asnonsensitiser.

The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

OECD 422 tests confirm that ethyl (2E,4Z)-decadienoate does not display the potential for activation of the first, second, and third key events for sensitisation. A human patch test confirms that ethyl (2E,4Z)-decadienoate does not cause an allergic reaction.