Reaction mass of 3,7-dimethyl-1-octyl propionate and citronellyl propionate

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:

22 October 2020 - 19 November 2020

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 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Reaction mass of 3,7-dimethyloct-6-en-1-yl propanoate and 3,7-dimethyloctyl propanoate

EC Number:

907-873-5

Molecular formula:

C13H24O2 C13H26O2

IUPAC Name:

Reaction mass of 3,7-dimethyloct-6-en-1-yl propanoate and 3,7-dimethyloctyl propanoate

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: Trinova Biochem GmbH, Giessen, Germany
- method of preparation of S9 mix: 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). S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized.
- concentration of S9: To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.

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: 17, 52, 164, 512, 1600 and 5000 μg/plate
Second experiment:
- TA1537, TA98, TA100 and WP2uvrA: 17, 52, 164, 512, 1600 and 5000 μg/plate (without S9-mix); 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (with S9-mix)
- TA1535: 17, 52, 164, 512, 1600 and 5000 μg/plate (without S9-mix); 1.7, 5.4, 17, 52, 164, 512, 1600 μg/plate (with S9-mix)

Vehicle / solvent:

- Vehicle used: DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

With metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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: triplicates
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in agar (plate incorporation) and preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 30 ± 2 minutes by 70 rpm at 37 ± 1°C in the dark
- Exposure duration/duration of treatment: 48 ± 4 h at 37.0 ± 1.0°C in the dark
- Harvest time after the end of treatment (sampling/recovery times): Not reported

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

Evaluation criteria:

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

First experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Second experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the absence and presence of S9-mix at 164 and 512 μg/plate, respectively.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

First experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Second experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the absence and presence of S9-mix at 1600 and 164 μg/plate, respectively.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

First experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at a dose level of 5000 μg/plate in the presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Second experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the absence and presence of S9-mix at 164 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

First mutation experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Second experiment

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the absence and presence of S9-mix at 512 μg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose-range finding test/First mutation experiment:
- Precipitation of the test item on the plates was not observed in any tester strain.
- Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacter ial background lawn, was observed in tester strain TA100 and TA1537 at a dose level of 5000 μg/plate, in respectively the presence and absence of S9-mix. In strain TA1537 (presence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by an incidental fluctuation in the number of revertant colonies.
- In the direct plate test, no increase in the number of revertants was observed upon treatment
with the test item under all conditions tested.

Second experiment:
- Precipitation of the test item on the plates was not observed any tester strain.
- 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, except in WP2uvrA.
- In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Any other information on results incl. tables

Table 1. Dose-Range Finding Test:  Mutagenic Response of Citronellyl Propionate 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	781	±	51		1092	±	28
Solvent control	85	±	1		15	±	5
1.7	77	±	26		12	±	3
5.4	79	±	15		15	±	7
17	86	±	11		22	±	14
52	87	±	4		18	±	2
164	90	±	13		12	±	2
512	87	±	7		19	±	6
1600	73	±	5		18	±	7
5000	64	±	16	^n NP	19	±	1	^n NP

 

With S9-mix

 

Positive control	1345	±	82		260	±	10
Solvent control	75	±	19		23	±	3
1.7	77	±	5		18	±	8
5.4	81	±	4		25	±	3
17	75	±	13		21	±	1
52	84	±	12		18	±	2
164	88	±	8		20	±	3
512	78	±	12		16	±	3
1600	65	±	2	^n	17	±	6
5000	3	±	2	^m NP	19	±	7	^n NP

 

NP	No precipitate
m	Bacterial background lawn moderately reduced
n	Normal bacterial background lawn

 

Table 2. Experiment 1:  Mutagenic Response of Citronellyl Propionate 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	834	±	55		1015	±	144		1042	±	70
Solvent control	8	±	3		2	±	2		19	±	3
17	7	±	3		5	±	1		9	±	5
52	13	±	2		5	±	4		12	±	2
164	7	±	3		4	±	2		7	±	4
512	8	±	0		5	±	2		7	±	3
1600	10	±	5		4	±	1		9	±	5
5000	7	±	4	^n NP	1	±	1	^n NP	14	±	5	^n NP

 

With S9-mix

 

Positive control	259	±	35		343	±	27		1409	±	176
Solvent control	9	±	1		3	±	2		8	±	2
17	16	±	3		6	±	1		17	±	6
52	12	±	5		3	±	2		19	±	5
164	4	±	1		1	±	2		17	±	3
512	5	±	2		0	±	1		17	±	6
1600	8	±	4		2	±	2		21	±	2
5000	9	±	6	^n NP	3	±	3	^n NP	11	±	5	^n NP

 

NP	No precipitate
n	Normal bacterial background lawn

 

Table 3. Experiment 2:  Mutagenic Response of Citronellyl Propionate 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	513	±	368		170	±	4		1522	±	184		703	±	63		1296	±	40
Solvent control	9	±	2		4	±	3		14	±	3		103	±	8		17	±	8	^i
17	8	±	4		1	±	2		10	±	2		55	±	28	^s	9	±	2
52	12	±	4		2	±	2	^n	9	±	1		77	±	6	^s	11	±	6
164	6	±	5		2	±	2	^s	9	±	4	^n	55	±	14	^m	12	±	5
512	10	±	3	^n	9	±	6	^m	9	±	1	^s	72	±	18	^m	17	±	4
1600	5	±	1	^s	3	±	2	^m	4	±	1	^s	52	±	18	^m	14	±	3
5000	7	±	3	^s NP	6	±	9	^m NP	4	±	4	^s NP	52	±	10	^m NP	15	±	3	^n NP

 

 

With S9-mix

 

Positive control	211	±	13		135	±	18		587	±	43		2120	±	104		481	±	23
Solvent control	9	±	2		4	±	1		18	±	3		84	±	9		12	±	6
Positive control		-			201	±	30	*	648	±	18	*	1326	±	60	*		-
Solvent control		-			2	±	2	*	15	±	4	*	87	±	8	*		-
1.7	6	±	4		4	±	2	*	13	±	3	*	74	±	5	*		-
5.4	7	±	4		3	±	2	*	19	±	5	*	83	±	16	*		-
17	7	±	3		3	±	3		20	±	2		85	±	5		18	±	4
52	8	±	6	^n	8	±	4		20	±	2		91	±	10	^n	23	±	6
164	6	±	2	^s	4	±	3	^n	13	±	2	^n	51	±	12	^m	16	±	2
512		±		^e MC	0	±	0	^a	0	±	0	^a	0	±	0	^a	15	±	0
1600	0	±	0	^a NP	0	±	0	^a	0	±	0	^a	0	±	0	^a	12	±	6
5000		-			0	±	0	^a NP	0	±	0	^a NP	0	±	0	^a NP	22	±	4	^n NP

 

 

MC	Microcolonies
MP	Moderate Precipitate
NP	No precipitate
a	Bacterial background lawn absent
e	Bacterial background lawn extremely reduced
i	One Plate infected: mean of two plates
m	Bacterial background lawn moderately reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced
*	Data from an additional experiment
-	Not tested

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay performed according to OECD 471 (2020).

Executive summary:

The substance is tested in the Ames test (OECD TG 471) following GLP. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. In the first mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the strains TA100, TA1535, TA1537, TA98, and WP2uvrA. The test item did not precipitate on the plates at this dose level. Cytotoxicity was observed in tester strain TA100 at a dose level of 5000 μg/plate in the presence of S9-mix, and in tester strain TA1537 at a dose level of 5000 μg/plate in the absence of S9-mix. In the second mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the tester strains TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. Tester strain TA1535 was exposed to concentrations up to 5000 μg/plate in the absence of S9-mix and 1600 μg/plate in the presence of S9-mix. The test item did not precipitate on the plates at this dose level. Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix, except in WP2uvrA. The negative and 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. In  conclusion, based on the results of this study it is concluded that Citronellyl Propionate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.