ETHYL 2-METHYL-4-PENTANOATEETH OXANOATE-369 CRUDE

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 November 2020 - 17 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Substance information is used to read across from

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 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

June 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(Z)‐ethyl 2‐methylpent‐3‐enoate

EC Number:

854-058-4

Cas Number:

58625-89-1

Molecular formula:

C8H14O2

IUPAC Name:

(Z)‐ethyl 2‐methylpent‐3‐enoate

Test material form:

liquid

Method

Target gene:

Salmonella typhimurium: histidine locus
Escherichia coli: tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Rat liver microsomal enzymes (S9 homogenate) were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The above solution was filter (0.22 μm)-sterilized.
- concentration S9 in the S9-mix: 5% (v/v) S9-fraction

Test concentrations with justification for top dose:

Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
First experiment: 52, 164, 512, 1600 and 5000 μg/plate.
Second experiment: 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (without S9-mix); 52, 164, 512, 1600 and 5000 μg/plate (with S9-mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10^9 cells/mL
- Test substance added in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period (experiment 2): 30 ± 2 minutes by 70 rpm at 37 ± 1°C
- Exposure duration/duration of treatment: 48 ± 4 h at 37.0 ± 1.0°C (experiment 1); 48 ± 4 h at 37.0 ± 1.0°C (experiment 2, after preincubation)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: No cytotoxicity Experiment 2: Cytotoxicity in the absence and presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: No cytotoxicity Experiment 2: Cytotoxicity in the absence and presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: No cytotoxicity Experiment 2: Cytotoxicity in the absence and presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: No cytotoxicity Experiment 2: Cytotoxicity in the absence and presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: No cytotoxicity Experiment 2: Cytotoxicity in the absence and presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Experiment 1:
- Precipitate: Precipitation of the test item on the plates was not observed in any tester strain.
- Toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the
number of revertants were observed.
- Mutagenicity: In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Experiment 2:
- Precipitation of the test item on the plates was not observed any tester strain.
- Toxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
- Mutagenicity: In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Acceptability
All bacterial strains showed negative responses over the entire dose-range, i.e. no dose-related increase in the number of revertants in two independently repeated experiments.
The negative control values were within the laboratory historical control data ranges.
The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA98 in the presence of S9-mix in the first experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was above the historical control data range, this deviation in the mean plate count of the positive control had no effect on the results of the study.

Any other information on results incl. tables

Table 1. Dose-Range Finding Test:  Mutagenic Response of Ethyl 2-methyl-3- pentenoate in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Direct Plate Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
	TA100	WP2uvrA

 

Without S9-mix

 

Positive control	799	±	113		1746	±	122
Solvent control	92	±	5		16	±	4
1.7	102	±	7		18	±	3
5.4	119	±	6		19	±	4
17	97	±	6		16	±	2
52	91	±	11		15	±	1
164	94	±	19		19	±	5
512	80	±	6		15	±	8
1600	87	±	7		20	±	2
5000	80	±	7	^n NP	12	±	3	^n NP

 

With S9-mix

 

Positive control	1367	±	44		383	±	7
Solvent control	85	±	12		14	±	4
1.7	90	±	6		16	±	7
5.4	96	±	7		13	±	5
17	95	±	24		12	±	7
52	83	±	10		16	±	2
164	84	±	2		15	±	11
512	86	±	10		12	±	6
1600	70	±	3		15	±	3
5000	80	±	12	^n NP	21	±	11	^n NP

 

NP	No precipitate
n	Normal bacterial background lawn

 

Table 2. Experiment 1:  Mutagenic Response of Ethyl 2-methyl-3-pentenoate in the Salmonella typhimurium Reverse Mutation Assay 

Direct Plate Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium.
	TA1535	TA1537	TA98

 

Without S9-mix

 

Positive control	972	±	39		1135	±	85		1795	±	78
Solvent control	8	±	7		3	±	1		15	±	2
52	10	±	2		4	±	3		11	±	4
164	8	±	4		6	±	4		16	±	3
512	8	±	3		4	±	4		15	±	5
1600	6	±	4		3	±	2		15	±	7
5000	11	±	3	^n NP	2	±	2	^n NP	12	±	3	^n NP

 

With S9-mix

 

Positive control	306	±	8		243	±	12		2334	±	77
Solvent control	8	±	3		5	±	2		17	±	3
52	12	±	3		5	±	5		18	±	5
164	9	±	4		3	±	2		17	±	3
512	7	±	4		3	±	0		16	±	6
1600	15	±	5		2	±	2		13	±	1
5000	7	±	1	^n NP	4	±	3	^n NP	14	±	7	^n NP

 

NP	No precipitate
n	Normal bacterial background lawn

Table 3. Experiment 2:  Mutagenic Response of Ethyl 2-methyl-3-pentenoate in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Pre-incubation Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.
	TA1535	TA1537	TA98	TA100	WP2uvrA

 

Without S9-mix

 

Positive control	1139	±	89		164	±	15		1896	±	106		838	±	59		1501	±	71
Solvent control	8	±	3		3	±	2		6	±	4		102	±	17		17	±	6
Positive control ^1	1023	±	45		118	±	11		1988	±	44		627	±	23		1251	±	236
DMSO ^1	11	±	5		2	±	1		11	±	4		81	±	15		16	±	5
5.4 ^1	10	±	3		3	±	1		9	±	4		76	±	12		12	±	2
17 ^1	6	±	1		3	±	2		9	±	4		86	±	10		14	±	7
52	6	±	5		1	±	0		10	±	2		106	±	12		22	±	6
164	9	±	1	^n	3	±	1	^n	11	±	7	^n	109	±	12	^n	17	±	3
512	6	±	3	^s	4	±	3	^s	9	±	4	^s	99	±	11	^s	26	±	4	^n
1600				^e MC				^e MC				^e MC				^e MC				^e MC
5000				^e NP  MC				^e NP  MC				^e NP  MC				^e NP  MC				^e NP  MC

 

 

With S9-mix

 

Positive control	250	±	24		153	±	29		661	±	27		2185	±	48		425	±	74
Solvent control	9	±	3		3	±	1		14	±	6		95	±	8		34	±	4
52	5	±	0		3	±	2		16	±	4		96	±	4		22	±	7
164	8	±	4		3	±	2		13	±	3		106	±	13		35	±	4
512	7	±	5	^n	4	±	1	^n	15	±	5	^n	83	±	1	^n	32	±	2
1600	5	±	2	^m	1	±	2	^m	7	±	8	^m	70	±	8	^m	25	±	3	^n
5000				^e NP  MC				^e NP  MC				^e NP  MC				^e NP  MC				^e NP  MC

 

 

MC	Microcolonies
NP	No precipitate
e	Bacterial background lawn extremely reduced
m	Bacterial background lawn moderately reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced
^1	Data from an additional experiment

 

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and the Escherichia coli Reverse Mutation Assay (Plate Incorporation and Pre-Incubation Methods) performed according to OECD 471.

Executive summary:

The substance is tested in the Ames test according to OECD TG 471 and following GLP. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. The following concentrations were tested: 52, 164, 512, 1600 and 5000 μg/plate in experiment 1; 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (without S9-mix); 52, 164, 512, 1600 and 5000 μg/plate (with S9-mix) in experiment 2. In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The test item did not induce a dose-related increase in the number of revertant colonies in each of the five tester strains (TA1535, TA1537, TA98 and TA100 WP2uvrA) both in the absence and presence of S9-metabolic activation. Based on this the substance is not mutagenic in this test.