Citronellyl formate

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:

10 May 2017 - 10 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial forward mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Citronellyl formate

EC Number:

203-338-9

EC Name:

Citronellyl formate

Cas Number:

105-85-1

Molecular formula:

C11H20O2

IUPAC Name:

3,7-dimethyloct-6-en-1-yl formate

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II:
Strains TA 1535 and WP2 uvrA: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
The remaining strains: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Justification for top dose: according to OECD guideline 471

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine: TA 1537, TA 98 without metabolic activation, 2-aminoanthracene: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: experiment I: in agar (plate incorporation); experiment II: preincubation

DURATION
- Preincubation period: 60 minutes.
- Exposure duration: ≥ 48 hours

NUMBER OF REPLICATIONS: 3 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants and inspection of the bacterial background lawn

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

no statistical analysis

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations ≥ 333 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations ≥ 100 µg/plate

Vehicle controls validity:

valid

Untreated negative 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:

at concentrations ≥ 333 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations ≥ 100 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at concentrations ≥ 333 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose.

Any other information on results incl. tables

Table 1: Summary of Experiment I

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		10 ± 4	12 ± 3	22 ± 5	162 ± 12	36 ± 6
	Untreated		11 ± 4	9 ± 3	25 ± 2	175 ± 24	37 ± 3
	Test item	3 µg	12 ± 2	12 ± 2	26 ± 9	151 ± 4	34 ± 7
		10 µg	13 ± 3	13 ± 3	25 ± 5	142 ± 13	35 ± 10
		33 µg	10 ± 1	11 ± 4	26 ± 5	137 ± 7	40 ± 2
		100 µg	9 ± 5	7 ± 4	16 ± 5	110 ± 15	40 ± 9
		333 µg	10 ± 4	8 ± 3^R	20 ± 6^R	54 ± 2^R	41 ± 1
		1000 µg	7 ± 3	10 ± 2^R	17 ± 5^R	51 ± 4^R	37 ± 6
		2500 µg	8 ± 3	8 ± 5^R	16 ± 3^R	57 ± 10^R	40 ± 4
		5000 µg	8 ± 2	10 ± 2^R	13 ± 1^R	35 ± 3^M R	41 ± 8
	NaN3	10 µg	1284 ± 13			2059 ± 79
	4-NOPD	10 µg			346 ± 17
	4-NOPD	50 µg		62 ± 5
	MMS	2.0 µL					934 ± 30
With Activation	DMSO		14 ± 3	18 ± 6	26 ± 7	158 ± 7	47 ± 2
	Untreated		14 ± 6	16 ± 5	33 ± 4	166 ± 8	54 ± 8
	Test item	3 µg	12 ± 3	16 ± 3	32 ± 5	130 ± 26	47 ± 5
		10 µg	13 ± 3	18 ± 8	31 ± 4	134 ± 8	39 ± 9
		33 µg	13 ± 4	12 ± 1	28 ± 9	143 ± 16	51 ± 8
		100 µg	12 ± 5	15 ± 2	32 ± 8	148 ± 9	53 ± 11
		333 µg	9 ± 3	10 ± 4^R	31 ± 4^R	94 ± 24^R	40 ± 11
		1000 µg	11 ± 3^R	4 ± 1^M R	16 ± 2^R M	25 ± 2^R	29 ± 8
		2500 µg	6 ± 2^R M	3 ± 1^M R	1 ± 1^M R	1 ± 1^R	21 ± 6
		5000 µg	7 ± 3^M R	3 ± 1^M R	0 ± 0^R	0 ± 0^R	25 ± 2
	2-AA	2.5 µg	533 ± 60	154 ± 14	3890 ± 433	4101 ± 294
	2-AA	10.0 µg					444 ± 24

NaN3: sodium azide, 2-AA: 2-aminoanthracene, 4-NOPD: 4-nitro-o-phenylene-diamine, MMS:methyl methane sulfonate, R: Reduced background growth, M: Manual count

Table 2: Summary of Experiment II

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		11 ± 4	8 ± 3	18 ± 4	182 ± 12	41 ± 9
	Untreated		10 ± 2	10 ± 3	22 ± 1	213 ± 3	44 ± 7
	Test item	3 µg		9 ± 3	21 ± 5	180 ± 3
		10 µg	11 ± 5	8 ± 4	18 ± 8	172 ± 4	44 ± 5
		33 µg	9 ± 2	10 ± 3	18 ± 3	153 ± 18	46 ± 9
		100 µg	10 ± 2	6 ± 1^R	19 ± 6	71 ± 25^R	35 ± 3
		333 µg	9 ± 3	9 ± 3^R	18 ± 5^R	47 ± 9^R	36 ± 8
		1000 µg	8 ± 2	8 ± 1^R	19 ± 3^R	46 ± 6^R	31 ± 7
		2500 µg	11 ± 3^R	10 ± 4^R	7 ± 3^R M	26 ± 5^M R	41 ± 9
		5000 µg	10 ± 5^R	2 ± 1^M R	6 ± 1^M R	8 ± 2^M R	40 ± 7
	NaN3	10 µg	1218 ± 30			1468 ± 71
	4-NOPD	10 µg			290 ± 7
	4-NOPD	50 µg		81 ± 6
	MMS	2.0 µL					537 ± 47
With Activation	DMSO		11 ± 2	10 ± 1	34 ± 6	181 ± 13	52 ± 9
	Untreated		12 ± 1	12 ± 4	38 ± 3	201 ± 3	71 ± 14
	Test item	3 µg		13 ± 4	30 ± 8	166 ± 14
		10 µg	14 ± 2	12 ± 4	38 ± 7	171 ± 21	58 ± 11
		33 µg	10 ± 2	10 ± 4	38 ± 3	158 ± 5	57 ± 8
		100 µg	13 ± 2	11 ± 3	36 ± 9	133 ± 18	62 ± 9
		333 µg	7 ± 2^M R	3 ± 1^R M	6 ± 1^R M	71 ± 7^M R	36 ± 8^R
		1000 µg	5 ± 1^M R	2 ± 1^M R	1 ± 1^R	28 ± 9^M R	27 ± 4^R
		2500 µg	3 ± 1^M R	0 ± 0^R	0 ± 0^R	7 ± 2^R M	9 ± 2^M R
		5000 µg	2 ± 1^M R	0 ± 0^R	0 ± 0^R	0 ± 0^R	11 ± 1^M R
	2-AA	2.5 µg	376 ± 1	119 ± 20	3852 ± 420	4094 ± 290
	2-AA	10.0 µg					492 ± 60

NaN3: sodium azide, 2-AA: 2-aminoanthracene, 4-NOPD: 4-nitro-o-phenylene-diamine, MMS:methyl methane sulfonate, R: Reduced background growth, M: Manual count

Table 2: Historicla data

Strain		without S9 mix				with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent control Untreated control Positive control	12 12 1130	2.5 3.1 143.1	6 6 334	25 28 1816	12 12 388	2.5 2.9 58.2	7 7 176	26 26 668
TA 1537	Solvent control Untreated control Positive control	10 11 82	2.2 2.7 12.7	6 5 43	19 21 157	13 14 191	3.5 4.0 60.8	7 7 83	30 31 434
TA 98	Solvent control Untreated control Positive control	25 27 378	4.4 4.9 73.7	13 12 211	43 43 627	34 37 3949	6.2 6.5 771.8	15 11 360	58 57 6586
TA 100	Solvent control Untreated control Positive control	156 176 1966	26.0 23.6 293.2	78 79 498	209 217 2767	148 172 3798	32.3 25.4 830.4	73 85 536	208 218 6076
WP2uvrA	Solvent control Untreated control Positive control	41 42 798	5.6 5.8 362.7	27 30 319	63 63 4732	50 52 378	6.8 6.8 112.6	28 36 167	72 88 1265

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that the test item is non-mutagenic in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA in the absence and presence of S9-mix.

Executive summary:

The genetic toxicity of the test item was assessed using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA, in accordance with OECD 471 guideline and GLP principles in two independent experiments (direct plate assay and a preincubation assay). No precipitation of the test item occurred up to the highest investigated dose. The negative and strain-specific positive control values were within the laboratory background historical control data ranges.

The plates incubated with the test item showed reduced background growth up in all strains with metabolic activation and in all strains, except of strain WP2uvrA without metabolic activation.

Toxic effects, evident as a reduction in the number of revertants, were observed in all strains with metabolic activation and in strains TA 1537, TA 98, and TA 100 without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Based on the results of this study it is concluded that the test item is non-mutagenic in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA in the absence and presence of S9-mix.