Reaction mass of citronellyl propionate and geranyl propionate and neryl 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:

26 June 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:

Geranyl propionate

EC Number:

203-344-1

EC Name:

Geranyl propionate

Cas Number:

105-90-8

Molecular formula:

C13H22-O2

IUPAC Name:

(2E)‐3,7‐dimethylocta‐2,6‐dien‐1‐yl propanoate

Test material form:

liquid

Method

Target gene:

Salmonella typhimurium strains: Histidine
Escherichia coli WP2uvrA: Tryptophan

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Each tester strain contained additional mutations

Remarks:

rfa : deep rough (defective lipopolysaccharide cellcoat) gal : mutation in the galactose metabolism chl : mutation in nitrate reductase bio : defective biotin synthesis uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: 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 of S9: 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.
The highest concentration of the test item used in the subsequent mutation assays was 5000 μg/plate or the level at which the test item inhibited bacterial growth.

First experiment Direct Plate Assay: 52, 164, 512, 1600 and 5000 μg/plate.
Second experiment Pre-incubation assay: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (TA1537, TA98, TA100 and WP2uvrA); 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (TA1535; test item was tested up to concentrations of 1600 μg/plate and 5000 μg/plate in respectively the absence and presence of S9-mix)
Third experiment Pre-incubation assay: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (TA100; concentrations of 1600 μg/plate and 5000 μg/plate in respectively the absence and presence of S9-mix)

Vehicle / solvent:

- Vehicle used: DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

With metabolic activation

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Remarks:

Without metabolic acivation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 3

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

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

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: bacterial background lawn

Evaluation criteria:

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

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no cytotoxicity nor precipitates, but tested up to and including recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

FIRST EXPERIMENT: DIRECT PLATE ASSAY (dose-range finding study with two tester strains is reported as part of the direct plate assay)
- Precipitate: Precipitation of the test item on the plates was not observed in 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 the absence of S9-mix in TA98 and in the presence of S9-mix in TA1535, TA1537 and TA98.
- 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.

SECOND EXPERIMENT: PRE-INCUBATION ASSAY
- Precipitate: 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, except in WP2uvrA in the absence 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.

THIRD EXPERIMENT: PRE-INCUBATION ASSAY
- Precipitate: Precipitation of the test item on the plates was not observed.
- 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 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.

Any other information on results incl. tables

Table 1. Dose-Range Finding Test: Mutagenic Response of Geranyl 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	812	±	91		1254	±	59
Solvent control	101	±	4		22	±	2
1.7	75	±	2		18	±	2
5.4	81	±	14		18	±	3
17	88	±	11		14	±	8
52	76	±	7		22	±	3
164	82	±	5		19	±	4
512	77	±	9		21	±	2
1600	81	±	6		19	±	8
5000	96	±	24	^n NP	17	±	11	^n NP

With S9-mix

Positive control	1248	±	165		254	±	25
Solvent control	75	±	6		17	±	9
1.7	73	±	15		21	±	4
5.4	75	±	8		20	±	1
17	66	±	2		18	±	4
52	72	±	5		22	±	10
164	82	±	15		28	±	10
512	70	±	4		28	±	9
1600	80	±	7		27	±	5
5000	89	±	11	^n NP	22	±	3	^n NP

 

NP	No precipitate
n	Normal bacterial background lawn

 

Table 2. Experiment 1: Mutagenic Response of Geranyl 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	987	±	114		1403	±	151		1777	±	181
Solvent control	5	±	2		3	±	2		16	±	5
52	10	±	2		4	±	3		7	±	5
164	11	±	4		3	±	2		9	±	5
512	8	±	0		1	±	2		6	±	3
1600	8	±	2		3	±	4		12	±	8
5000	8	±	2	^n NP	6	±	1	^n NP	6	±	3	^n NP a

 

With S9-mix

 

Positive control	277	±	68		300	±	76		923	±	786
Solvent control	5	±	2		5	±	2		15	±	5
52	11	±	7		8	±	2		19	±	5
164	9	±	4		3	±	2		12	±	2
512	6	±	2		7	±	2		9	±	2
1600	5	±	5	^n	4	±	1	^n	13	±	4	^n
5000	4	±	1	^NP s				^NP e MC				^NP e MC

 

MC	Microcolonies
NP	No precipitate
a	Bacterial background lawn absent
e	Bacterial background lawn extremely reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced

 

Table 3. Experiment 2: Mutagenic Response of Geranyl 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	1022	±	49		134	±	45		1415	±	175		1438	±	95		1224	±	152
Solvent control	11	±	1		5	±	2		12	±	9	^i	75	±	9		14	±	4	^i
Positive control		-			177	±	14	*		-				-				-
Solvent control		-			5	±	3	*		-				-				-
1.7	10	±	3		4	±	3	*		-				-				-
5.4	8	±	6	^n	4	±	2	*		-				-				-
17	6	±	2	^s	3	±	2		13	±	8	^n	78	±	15	^i	18	±	3
52	7	±	2	^s	5	±	2		8	±	4	^s	75	±	6	^i	24	±	6
164	6	±	1	^s	3	±	2	^n	11	±	2	^s	61	±	15	^n	22	±	6
512	7	±	3	^s				^e MC	7	±	2	^s				^e MC	14	±	2
1600	4	±	1	^m NP	0	±	0	^a	7	±	6	^s				^e MC	17	±	6
5000		-			0	±	0	^a NP	8	±	2	^NP s	0	±	0	^a NP	19	±	4	^NP n

 

 

With S9-mix

 

Positive control	213	±	3		157	±	16		473	±	4		690	±	38		373	±	23
Solvent control	10	±	6		5	±	2		15	±	3		73	±	5		16	±	4
Positive control		-				-			674	±	34	*		-				-
Solvent control		-				-			14	±	6	*		-				-
1.7		-				-			16	±	4	*		-				-
5.4		-				-			19	±	6	*		-				-
17	8	±	5		2	±	2	^s	23	±	4	^i	68			^s ii	19	±	9
52	8	±	4		1	±	2	^s	12	±	6		30	±	12	^s	17	±	2
164	8	±	2	^n	2	±	3	^s	17	±	8	^n	58			^s ii	22	±	3	^n
512	0	±	0	^a	5	±	6	^s				^e MC	27	±	13	^m	10	±	4	^s
1600	0	±	0	^a	2	±	2	^m i	0	±	0	^a	36	±	15	^m				^e MC
5000	0	±	0	^a NP	1	±	1	^m NP i	0	±	0	^a NP	39	±	10	^m NP				^e NP MC

 

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

 

Table 4. Experiment 3: Mutagenic Response of Geranyl Propionate in the Salmonella typhimurium Reverse Mutation Assay

Pre-incubation Assay

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

 

Without S9-mix

 

	Positive control	811		±	17
	Solvent control	108		±	6
	1.7	107		±	16
	5.4	87		±	21	^n
	17	59		±	14	^s
	52	61		±	8	^m
	164	27		±	14	^m
	512					^e MC
	1600					^e MC NP

 

With S9-mix

 

Positive control	1939	±	96
Solvent control	95	±	21
17	86	±	8
52	81	±	3	^n
164	77	±	5	^s
512				^a
1600				^a
5000				^a NP

 

MC	Microcolonies
NP	No precipitate
a	Bacterial background lawn absent
e	Bacterial background lawn extremely reduced
m	Bacterial background lawn moderately reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced

 

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 performed according to OECD 471 (2020).

Executive summary:

Substance is tested in the Ames test (OECD TG 471). The following concentrations were tested in 3 independent experiments, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate. In the first mutation experiment, cytotoxicity was seen as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, in the absence of S9-mix in TA98 and in the presence of S9-mix in TA1535, TA1537 and TA98. In the second mutation experiment, cytotoxicity was seen, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, in all tester strains in the absence and presence of S9-mix, except in WP2uvrA in the absence of S9-mix. Because of plate infections and excessive toxicity in tester strain TA100 in the second experiment, an additional third mutation experiment was performed. Cytotoxicity was seen, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, in the absence and presence of S9-mix. There were no positive results in all strains. Based on this the substance is not mutagenic in this test.