Farnesol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted according to OECD 471 and GLP. The test included five strains of Salmonella typhimurium, with TA102 in place of an Escherichia coli strain.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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:

2000

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:

Farnesol

EC Number:

225-004-1

EC Name:

Farnesol

Cas Number:

4602-84-0

Molecular formula:

C15H26O

IUPAC Name:

farnesol

Method

Target gene:

Histidine reversion his- to his+

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

nitroreductase deficient

Remarks:

All strains: rfa- mutation; strains TA 1535, TA 1537, TA 98 and TA 100: uvrB- mutation; strains TA 98, TA 100 and TA102: R-factor mutation; strain TA 102: hisG428 mutation

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10, 33, 100. 333. 1000. 2500 and 5000 µg/plate for pre-experiment (main experiment I) and main experiment II

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; purity >99%
- Justification for choice of solvent/vehicle: solubility properties and relative non-toxicity to the bacteria

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-ortho-phenylene -diamine, 2-aminoanthracene

Details on test system and experimental conditions:

EXPERIMENT I and EXPERIMENT 1A (repeat) -
In Experiment I strain TA 1537 without S9 mix showed a reduction in the number of revertants below the indication factor of 0.5 - indication of toxicity at nearly all concentrations, this part was repeated under identical conditions and reported as EXPERIMENT 1A

METHOD OF APPLICATION: In agar (plate incorporation);

DURATION
Exposure duration: - Exposure duration: at least 48 hours at 37C in the dark

NUMBER OF REPLICATIONS:Three

EXPERIMENT II
METHOD OF APPLICATION: ; pre incubation test
DURATION
- Preincubation period: 60min at 37C in the dark
- Exposure duration: at least 48 hours at 37C in the dark
-
NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY determined in Experiment I

---------
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 hours
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth

Evaluation criteria:

Test system is considered mutagenic if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA 102) or thrice (TA 1535 and TA1537) 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 that one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

Statistics:

Not mandatory under OECD test guidleine 471

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No substantial increase in revertant colony numbers in any of the 5 tester strains was observed following treatment with the test item at any concentration level, neither in the presence or absence of metabolic activation (S9 mix) . There was also no tendency for higher mutation rates with increasing concentration below the range normally considered to be biologically significant.


TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Observed in test tubes from 1000 to 5000 mcg/plate in both experiments - and on the incubated agar paltes from 2500 to 5000 mcg/plate
-
RANGE-FINDING/SCREENING STUDIES: As all plates were evaluable in the range finding study, this study was reported as Experiment I.


COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effects were observed at the following concentrations (mcg/plate)
TA1535 : Experiment I plate test 2500-5000 +/- S9 ) ; Experiment II pre-incubation ; 2500-5000 +/- S9
TA1537 : Experiment I plate test several doses upto 5000 - S9 ;2500 +S9 ; (Experiment Ia plate test repeat) 333-5000mcg/plate -S9; Experiment II pre-incubation ; 1000 -5000 - S9 ; 333 -5000 +S9
TA98 : Experiment I plate test 5000 - S9 ;2500 +S9 ; Experiment II pre-incubation ; 100, 2500-5000 -S9 ;2500 -5000 +S9
TA100 : Experiment I plate test 333-5000 - S9 ;1000-5000 +S9 ; Experiment II pre-incubation ; 1000-5000 +/- S9
TA102 : Experiment I plate test 2500-5000+/ - S9 Experiment II pre-incubation 2500-5000+/ - S9

Any other information on results incl. tables

Table 1. Summary results of pre-experiment and Experiment I

Metabolic activation	Test Group	Dose Level (µg/plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without activation	DMSO		13 ± 3	17 ± 3^BM	31 ± 5	104 ± 8^BM	526 ± 53
	Untreated		12 ± 2	14 ± 4^BM	32 ± 6	129 ± 7^BM	467 ± 24
	Farnesol	3	13 ± 0	15 ± 3^BM	29 ± 6	106 ± 10^BM	449 ± 21
		10	15 ± 3	12 ± 1^BM	37 ± 12	12 ± 1^BM	471 ± 86
		33	10 ± 6	6 ± 2^BM	27 ± 2	6 ± 2^BM	465 ± 36
		100	16 ± 5^MR	6 ± 2^BM	27 ± 3	6 ± 2^BM	402 ± 25
		333	10 ± 4^RM	7 ± 4^BM	26 ± 2	7 ± 4^BM	357 ± 11
		1000	7 ± 1^MR	9 ± 5^BM	19 ± 0	9 ± 5^BMR	352 ± 21
		2500	4 ± 2^PMR	6 ± 3^PMB	17 ± 4^PM	6 ± 3^PMBR	186 ± 13^PM
		5000	4 ± 3^PMR	5 ± 2^PMB	13 ± 2^PM	5 ± 2^PMB	172 ± 16^PM
	NaN3	10	2176 ± 87			2024 ± 85
	4-NOPD	10			432 ± 7^BM
	4-NOPD	50		117 ± 8^BM
	MMS	3.0					4424 ± 108
With activation	DMSO		24 ± 3	21 ± 6^BM	41 ± 6	109 ± 10^BM	551 ± 27
	Untreated		20 ± 6	17 ± 3^BM	35 ± 1	111 ± 10^BM	554 ± 21
	Farnesol	3	23 ± 12	17 ± 6^BM	36 ± 8	111 ± 10^BM	562 ± 74
		10	24 ± 5	21 ± 4^BM	33 ± 10	103 ± 6^BM	532 ± 22
		33	20 ± 6	16 ± 6^BM	49 ± 2	107 ± 12^BM	544 ± 23
		100	20 ± 7	15 ± 6^BM	43 ± 4	63 ± 6^BM	526 ± 13
		333	13 ± 2	17 ± 3^BM	28 ± 5	56 ± 7^BM	398 ± 39
		1000	13 ± 1	13 ± 6^BM	30 ± 6	28 ± 2^BMR	379 ± 16
		2500	5 ± 2^PM	7 ± 3^PMB	18 ± 1^P	21 ± 2^PMBR	215 ± 10^PM
		5000	7 ± 1^PM	14 ± 1^PMB	19 ± 6^P	18 ± 2^PMB	223 ± 11^PM
	2-AA	2.5	308 ± 14	301 ± 11^BM	1686 ± 122	2383 ± 187
	2-AA	10.0					2214 ± 287

NaN₃= Sodium azide

2-AA = 2-aminoanthracene

MMS = Methyl methane sulfonate

4-NOPD = 4-Nitro-o-phenylene-diamine

B = Extensive bacterial growth

M = Manual count

R = Reduced background growth

P = Precipitate

Table 2. Summary of Results Pre-Experiment and Experiment Ia

Metabolic activation	Test Group	Dose Level (µg/plate)	Revertant Colony Counts (Mean ± SD)
			TA 1537
Without activation	DMSO		15 ± 2
	Untreated		19 ± 2
	Farnesol	3	16 ± 4
		10	13 ± 5
		33	10 ± 2
		100	8 ± 2
		333	6 ± 3
		1000	6 ± 2^MR
		2500	3 ± 2^MR
		5000	1 ± 1^MR
	4-NOPD	50	143 ± 17

4-NOPD = 4-Nitro-o-phenylene-diamine

M = Manual count

R = Reduced background death

Table 3. Summary of Results Experiment II

Metabolic activation	Test Group	Dose Level (µg/plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without activation	DMSO		14 ± 1	11 ± 3	42 ± 4	118 ± 15	413 ± 9
	Untreated		13 ± 2	13 ± 8	24 ± 2	130 ± 10	485 ± 9
	Farnesol	3	13 ± 2	15 ± 4	25 ± 4	119 ± 5	437 ± 14
		10	10 ± 2	13 ± 7	24 ± 3	99 ± 11	442 ± 23
		33	15 ± 4	6 ± 6	19 ± 4	64 ± 7	406 ± 43
		100	14 ± 2	13 ± 1	17 ± 5	71 ± 6	273 ± 13
		333	3 ± 1^MR	6 ± 1^MR	25 ± 3^MR	59 ± 24^MR	379 ± 13^MR
		1000	3 ± 2^MR	2 ± 3^MR	21 ± 3^MR	39 ± 8^MR	356 ± 41^MR
		2500	0 ± 0^MR	0 ± 0^MR	11 ± 2^MR	22 ± 6^MR	123 ± 18^MR
		5000	0 ± 1^MR	0 ± 0^MR	4 ± 2^MR	14 ± 1^MR	64 ± 8^MR
	NaN3	10	2184 ± 27			2117 ± 79
	4-NOPD	10			514 ± 78
	4-NOPD	50		137 ± 27
	MMS	3.0 µL					3301 ± 380
With activation	DMSO		13 ± 1	16 ± 1	30 ± 5	143 ± 17	607 ± 38
	Untreated		12 ± 1	20 ± 8	36 ± 1	148 ± 12	678 ± 42
	Farnesol	3	9 ± 4	18 ± 3	40 ± 9	148 ± 14	604 ± 19
		10	10 ± 2	17 ± 8	43 ± 3	154 ± 11	568 ± 47
		33	9 ± 2	16 ± 3	34 ± 3	151 ± 7	648 ± 23
		100	8 ± 2	10 ± 4	30 ± 7	121 ± 13	588 ± 47
		333	2 ± 1^MR	6 ±2^MR	34 ± 6^MR	88 ± 10^MR	523 ± 56^MR
		1000	3 ± 3^MR	3 ± 1^MR	34 ± 1^MR	32 ± 4^MR	432 ± 67^MR
		2500	2 ± 2^MR	1 ± 2^MR	13 ± 6^MR	22 ± 4^MR	174 ± 13^MR
		5000	1 ± 1^MR	3 ± 3^MR	6 ± 1^MR	19 ± 2^MR	118 ± 18^MR
	2-AA	2.5	891 ± 30	266 ± 35	1858 ± 1207	2576 ± 61
	2-AA	10.0					2805 ± 70

NaN₃= Sodium azide

2-AA = 2-aminoanthracene

MMS = Methyl methane sulfonate

4-NOPD = 4-Nitro-o-phenylene-diamine

B = Extensive bacterial growth

M = Manual count

R = Reduced background growth

P = Precipitate

Applicant's summary and conclusion

Conclusions:

No substantail increase in revertant colony numbers in any of the 5 tester strains was observed following treatment with the test item at any concentration level, neither in the presence or absence of metabolic activation (S9 mix) . There was also no tendency for higher mutation rates with increasing concentration below the range normally considered to be biologically significant. Under the experimental conditions, the test item did not induce gene mutations by base pair or frameshift in the genome of the species tested. The test item is not considered to be mutagenic in the Salmonella tyhimurium reverse transcription assay.

Executive summary:

The study was performed to assess the potential of the test item to induce gene mutations according to the plate incorporation test (Experiment I) or the pre-incubation test (Experiment II) using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102. The assay was performed in two independent experiments with or without liver microsomal activation (S9 mix). Since results for TA1537 without metabolic activation showed a reduction of revertants (below the indication level of 0.5) at nearly all concentrations, this part of Experiment I was repeated under identical conditions as Experiment IA. In all experiments, test concentrations ranged from 3 to 5000 µg/plate, and toxic effects were observed at the higher concentrations tested. No substantial increase in revertant colony numbers in any of the 5 tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix) . There was also no tendency for higher mutation rates with increasing concentration below the range normally considered to be biologically significant. Appropriate mutagens were used as positive controls and showed a distinct increase in revertant colonies. Under the experimental conditions, the test item did not induce gene mutations by base pair or frameshift in the genome of the species tested. The test item is not considered to be mutagenic in the Salmonella typhimurium reverse transcription assay. This study is considered to be reliable without restrictions (Klimisch 1) as it was GLP-compliant and was performed according to OECD 471.