Citronellyl butyrate

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:

11 May - 1 Jun 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Citronellyl butyrate

EC Number:

205-463-4

EC Name:

Citronellyl butyrate

Cas Number:

141-16-2

Molecular formula:

C14H26O2

IUPAC Name:

citronellyl butyrate

Method

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

The pre-experiment is reported as Experiment 1.

Experiment 2:
3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA100
33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for remaining strains

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA), methylmethanesulfonate (MMS)

Remarks:

+S9: 2-AA (2.5 µg/plate, TA1535, TA1537, TA98, TA100; 10 µg/plate, WP2 uvrA); -S9: sodium azide (10 µg/plate, TA1535, TA100); 4-NOPD (10 µg/plate, TA98; 50 µg/plate, TA1537); MMS (2 µL/plate, WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (Experiment 1); preincubation (Experiment 2)

DURATION
- Preincubation period: 1 h
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

A test substance is considered as a mutagenic 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:

Mean values and standard deviations were calculated.

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:

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

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:

exp. 1 and 2: +S9 mix: 2500 - 5000 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:

exp. 2: +S9 mix: at 5000 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:

exp. 1 and 2: +S9 mix: from 333 to 5000 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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the overlay agar in the test tubes from 2500 to 5000 µg/plate in Experiment 1 and at 5000 µg/plate Experiment 2. No precipitation of the test substance in the overlay agar on the incubated agar plates was observed.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I (all strains were tested in the pre-experiment).

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred with metabolic activation in TA100 from 333 to 5000 µg/plate in both experiments and in TA 1537 and TA 98 at 5000 µg/plate in Experiment 2. Reduced background growth was observed with metabolic activation in TA 1537 and TA 100 from 2500 to 5000 µg/plate in both experiments.

Any other information on results incl. tables

Table 1. Test results of Experiment 1 (plate incorporation).

With or without S9-Mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA	TA1537	TA98
-	0 (DMSO)	13 ± 3	158 ± 6	46 ± 3	16 ± 4	25 ± 6
-	0	11 ± 5	174 ± 20	48 ± 5	13 ± 7	27 ± 4
-	3	14 ± 2	169 ± 19	43 ± 6	19 ± 4	25 ± 7
-	10	12 ± 6	145 ± 12	45 ± 11	18 ± 2	27 ± 4
-	33	15 ± 6	155 ± 9	43 ± 2	19 ± 4	27 ± 6
-	100	11 ± 6	157 ± 8	47 ± 4	14 ± 2	29 ± 4
-	333	15 ± 4	155 ± 21	45 ± 11	20 ± 8	24 ± 3
-	1000	13 ± 2	155 ±13	45 ± 7	20 ± 3	28 ± 6
-	2500	9 ± 3	143 ± 6	48 ± 5	17 ± 3	27 ± 7
-	5000	13 ± 2	135 ± 11	51 ± 2	13 ± 6	31 ± 5
Positive controls, –S9-Mix	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentration [μg/plate]	10	10	2.0 µL	50	10
	Mean No. of colonies/plate (average of 3 plates ± SD)	1127 ± 36	1994 ± 51	877 ± 46	77 ± 7	408 ± 29
+	0 (DMSO)	13 ± 2	142 ± 11	52 ± 6	23 ± 4	44 ± 4
+	0	12 ± 3	193 ± 18	53 ± 15	22 ± 3	42 ± 3
+	3	13 ± 5	164 ± 19	57 ± 4	23 ± 5	43 ± 7
+	10	17 ± 1	157 ± 10	45 ± 6	23 ± 3	39 ± 3
+	33	13 ± 3	167 ± 9	61 ± 2	25 ± 4	38 ± 6
+	100	16 ± 4	165 ± 21	61 ± 3	23 ± 3	44 ± 3
+	333	14 ± 2	47 ± 8	62 ± 8	29 ± 4	35 ± 3
+	1000	14 ± 3	55 ± 8	51 ± 12	28 ± 3	29 ± 5
+	2500	12 ± 2	32 ± 3^R	41 ± 10	17 ± 4^R	28 ± 8
+	5000	14 ± 6	7 ± 2^M, R	27 ± 5	18 ± 4^R	29 ± 11
Positive controls, +S9-Mix	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentration [μg/plate]	2.5	2.5	10	2.5	2.5
	Mean No. of colonies/plate (average of 3 plates ± SD)	348 ± 36	4596 ± 182	474 ± 37	239 ± 13	4760 ± 275

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

R: reduced background growth

 

Table 2. Test results of Experiment 2 (preincubation).

With or without S9-Mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± SD)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA	TA1537	TA98
-	0 (DMSO)	16 ± 1	157 ± 18	41 ± 4	8 ± 3	31 ± 8
-	0	13 ± 3	194 ± 9	45 ± 4	7 ± 3	35 ± 5
-	3	-	175 ± 18	-	-	-
-	10	-	143 ± 7	-	-	-
-	33	11 ± 1	146 ± 6	39 ± 6	8 ± 2	29 ± 11
-	100	12 ± 1	131 ± 14	49 ± 4	9 ± 2	26 ± 6
-	333	14 ± 2	124 ± 24	39 ± 8	8 ± 2	32 ± 10
-	1000	12 ± 2	123 ± 14	38 ± 2	10 ± 2	35 ± 6
-	2500	12 ± 2	122 ± 6	41 ± 6	8 ± 2	37 ± 3
-	5000	9 ± 2	109 ± 2	48 ± 12	8 ± 3	30 ± 6
Positive controls, –S9-Mix	Name	NaN3	NaN3	MMS	4-NOPD	4-NOPD
	Concentration [μg/plate]	10	10	2.0 µL	50	10
	Mean No. of colonies/plate (average of 3 plates ± SD)	1190 ± 38	2127 ± 88	757 ± 66	90 ± 16	334 ± 27
+	0 (DMSO)	13 ± 3	176 ± 19	54 ± 7	13 ± 3	50 ± 10
+	0	12 ± 6	195 ± 13	53 ± 3	13 ± 3	38 ± 5
+	3	-	173 ± 10	-	-	-
+	10	-	145 ± 3	-	-	-
+	33	14 ± 5	164 ± 9	49 ± 4	13 ± 2	48 ± 9
+	100	14 ± 4	170 ± 29	50 ± 6	12 ± 3	39 ± 4
+	333	11 ± 1	49 ± 4	45 ± 5	15 ± 3	41 ± 8
+	1000	11 ± 1	50 ± 5	41 ± 15	14 ± 5	43 ± 8
+	2500	10 ± 1	41 ± 7^R	38 ± 3	13 ± 4^M, R	36 ± 4
+	5000	8 ± 2	3 ± 1^M, R	25 ± 3	4 ± 1^M, R	17 ± 2
Positive controls, +S9-Mix	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentration [μg/plate]	2.5	2.5	10	2.5	2.5
	Mean No. of colonies/plate (average of 3 plates ± SD)	374 ± 9	5014 ± 176	449 ± 51	149 ± 24	5531 ± 111

NaN3: sodium azide

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methylmethanesulfonate

2-AA: 2-aminoanthracene

M: manual count

R: reduced background growth

Applicant's summary and conclusion

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation up to 5000 µg/plate.

Executive summary:

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with CITRONELLYL BUTYRATE 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. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.