Farnesol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro: The genotoxicity potential of the test item was assessed in bacterial gene mutation, human lymphocyte chromosome aberration and mouse lymphoma cell mutation assays. Negative results were observed in all in vitro assays, therefore there is no evidence of genotoxicity of the test item.

Genetic toxicity in vivo: No positive results were observed in any of the in vitro genotoxicity studies required for the Annex VII or VIII information requirements under REACH, therefore in vivo data for genetic toxicity is not required.

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

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

Target gene:

Histidine reversion his- to his+

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

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

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

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Healthy donors not receiving medication
- Cell cycle length, doubling time or proliferation index: Mitotic activity began at about 40 hours after PHA stimulation and reached a maximum at around 3 days.
- Sex, age and number of blood donors if applicable: Female donor (46 years old) for Experiment I and a 45 year-old male and a 24 year-old male donor for Experiment II

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1) containing 10 % FCS (fetal calf serum) and supplemented with Phytohemagglutinin (PHA, final concentration 3 µg/mL), the anticoagulant heparin (25,000 U.S.P.-U/mL) and HEPES (final concentration 10 mM). All incubations were done at 37°C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).
- Periodically checked for karyotype stability: The chromosome constitution remained diploid during short-term culture.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Preliminary cytotoxicity tests were performed in duplicate in order to determine test concentrations. Test item concentrations ranged between 14.7 and the highest concentration of 2270 µg/mL (approximately 10 mM). Precipitation of the test item was observed at 79.0 µg/mL and above in the absence of S9 and at 741.2 µg/mL in the presence of S9 mix. In addition, 22 hours treatment with >45.2 µg/mL in the absence of S9 induced strong toxic effects. Therefore, top concentrations of 139.9 µg/mL and 750 µg/mL were chosen for tests in the absence and presence of S9, respectively.

- EXPERIMENT I: 14.7, 25.8, 45.2, 79.0, 138.3, 242.0, 423.6, 741.2, 1297.1 and 2270.0 µg/mL were tested
- EXPERIMENT II without S9: 0.9, 1.6, 2.8, 4.9, 8.5, 14.9, 26.1, 45.7, 80.0, 139.9 µg/mL
- EXPERIMENT II with S9: 4.9, 8.5, 14.7, 14.9, 25.8, 26.1, 45.2, 45.7, 79.0, 80.0, 138.3, 139.9, 242.0, 244.9, 423.6, 428.6, 741.2, 750.0, 1297.1, 2270.0 µg/mL (test was repeated).

Vehicle / solvent:

- Solvent (vehicle): Dimethyl sulfoxide (DMSO 99.5% purity)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

EXPOSURE
- The chromosomes were prepared for 22 hours (Experiment I) and 46 hours (Experiment II) after exposure initiation. Thus, exposure duration was 4, 22 and 46 hours.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis) was determined in addition to the number of polyploid cells in 250 metaphase cells (% polyploid metaphases) was scored.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

OTHER EXAMINATIONS:
- Analysis of metaphase cells: Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations.
- Metaphase plates (n=100) were scored for cytogenetic damage.
- Determination of chromosome aberrations: the number of induced structural chromosome aberrations

Evaluation criteria:

CLASSIFICATIONS

- Non-mutagenic: the number of induced structural chromosome aberrations is in the range of testing facilities historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
- Mutagenic: the number of induced structural chromosome aberrations is not in the range of the testing facilities historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps) or a concentration-related increase is observed
- Aneugenic: the number of induced numerical aberrations is not in the range of our historical control data (0.0 – 0.8 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p<0.05).

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Clear cytotoxicity was observed at the high concentrations in Experiment I, while in Experiment II concentrations showing clear cytotoxic effects were not scorable for cytogenetic damage. In both experiments, in the absence and presence of an S9 metabolic activation system, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of cells after treatment (0.0 – 3.8% aberrant cells) were within historical values for controls (0.0 – 4.0% aberrant cells). Slight increases in the aberrant cells were noted, however, as these fell within the laboratory’s control data, the statistical significance and dose-dependency were regarded as biologically insignificant.

No biologically relevant increase in the rate of polyploid metaphases were detected after treatment (0.0 - 0.2%) in Experiment I and II, relative to solvent vehicle controls (0.0%). EMS (825, 660 or 770 µg/mL) or CPA (22.5 µg/mL) positive controls were successfully identified in the assay.

Table 1. Summary of results of the chromosomal aberration study

Exp Preparation interval Test item concentration in µg/mL Polyploid cells in % Mitotic indices in % of control Incl. gaps* Aberrant cells in % excl. gaps* With exchanges Exposure period 4 hours without S-9 mix I 22-hours Solvent control^1 0.0 100.0 1.5 1.5 0.5     Positive control^2 0.0 79.0 11.0 9.0^s 1.5     25.8 0.2 105.5 2.0 1.5 0.0     45.2 0.2 67.7 1.0 1.0 0.0     79.0 0.0 47.5 0.5 0.5 0.0 Exposure period 22 hours without S-9 mix I 22-hours Solvent control^1 0.0 100.0 1.0 0.5 0.0     Positive control^3 0.4 37.2 11.5 11.5 5.5     14.7 0.0 81.1 0.5 0.5 0.0     25.8 0.2 65.6 1.0 0.5 0.0     45.2 0.2 49.9 2.5 2.5 0.0 Exposure period 46 hours without S-9 mix II 46-hours Solvent control^1 0.0 100.0 1.0 0.5 0.0     Positive control^4 0.0 109.3 10.0 11.5^s 0.0     8.5 0.0 117.2 3.5 0.5 0.0     14.9 0.0 106.2 1.0 0.5 0.0     26.1 0.0 79.7 2.5 2.0 0.0 Exposure period 4 hours with S-9 mix I 22-hours Solvent control^1 0.0 100.0 1.5 1.0 0.0     Positive control5 0.0 55.0 9.5 9.5^s 0.0     79.0 0.2 109.6 1.0 0.5 0.0     138.3 0.0 115.0 1.5 1.5 0.5     242.0** 0.2 50.8 4.0 3.8^s 0.0 II 46-hours Solvent control^1 0.0 100.0 2.0 2.0 0.0     Positive control5 0.0 75.3 11.5 11.5^s 1.5     79.0 0.2 97.9 0.5 0.5 0.5     138.3 0.0 98.6 1.5 1.5 0.5     242.0 0.2 102.5 0.0 0.0 0.0

* Inclusive cells carrying exchanges

** Evaluation of 200 metaphases per culture

S Aberration frequency statistically significant higher than corresponding control values

1 DMSO 0.5% (v/v)

2 EMS 825.0 µg/mL

3 EMS 660.0 µg/mL

4 EMS 770.0 µg/mL

5 CPA 22.5 µg/mL

Conclusions:

It can be concluded that, under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro in the absence or presence of metabolic activation.

Executive summary:

The test item dissolved in DMSO was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro, either in the presence or absence of an exogenous S9 metabolic activation system. S9 liver fraction was prepared from Wistar Hanlbm rats exposed to phenobarbital (80 mg/kg bw) and β-naphthoflavone (80 mg/kg bw). The S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final concentration of 0.75 mg/mL in the short-term cultures of human lymphocytes stimulated to replicate. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix and 22 hours without S9 mix and chromosomes were prepared for 22 hours. In Experiment II, the exposure periods were 4 hours with S9 mix and 46 hours without S9 mix, with 46 hours’ chromosome preparation. The assay adhered to acceptability criteria for ethylmethane sulfonate (EMS) and cyclophosphamide (CPA) positive controls in the absence or presence of S9 mix, respectively. In both studiess the number of induced structural chromosome aberrations of the test item were within the range of historical control data (0.0 – 4.0% aberrant cells, exclusive gaps). Thus, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. This study is reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to OECD 473 and EU B.10.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase Locus (TK+/-)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory culture
- Suitability of cells: The L5178Y cell line has successfully been used in in vitro experiments for many years. L5178Y cells are characterised by a high proliferation rate and cloning efficiencies of untreated cells of usually more than 50 % both necessary for the appropriate performance of the study. The cells have a stable karyotype with a near diploid chromosome number.
- Cell cycle length, doubling time or proliferation index: Doubling time 10 - 12 h
- Methods for maintenance in cell culture if applicable: The cells are subcultured two times prior to treatment.
- Modal number of chromosomes: 40 ± 2

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 complete culture medium. The cell cultures are incubated at 37 ± 1.5°C in a
humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
- Periodically 'cleansed' against high spontaneous background: Yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/􀁅-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

EXPERIMENT 1:
- Without S9 mix: 1.3; 2.5; 5.0; 10.0; and 20.0 µg/mL
- With S9 mix: 4.4; 8.8; 17.5; 35.0; and 70.0 µg/mL

EXPERIMENT 2:
- Without S9 mix: 5.0; 10.0; 20.0; 27.5; and 35.0 µg/mL
- With S9 mix: 8.8; 17.5; 35.0; 52.8; and 70.0 µg/mL

Vehicle / solvent:

-Solvent Vehicle: Dimethyl sulfoxide (DMSO)
- Immediately before treatment, the test item was dissolved in DMSO (99.5%) to achieve a final DMSO concentration in culture medium of 0.5% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

In addition, a metabolic activation positive control, cyclophosphamide (CPA) was also tested.

Details on test system and experimental conditions:

Cell cultures were selectively grown ('cleansed') in RPMI 1640-HAT medium supplemented with hypoxanthine, aminopterin and thymidine, and screened for mycoplasma contamination and karyotype stability.
 
In order to test the effect of metabolism on the in vitro system. S9 liver fraction was prepared from Wistar Hanlhm rats exposed to phenobarbital (80 mg/kg bw) and β-naphthoflavone (80 mg/kg bw). The S9 supernatant was thawed and mixed with S9 cofactor solution to achieve final concentrations of 28.3 mg/mL and 31.0 mg/mL in experiment I and II, respectively.
 
Forty-eight-hour toxicity was observed at concentrations of 27.5 and 35.0 µg/mL without metabolic activation and at 105.0 µg/mL with metabolic activation (experiment I).

Method of determination of toxicity:cloning efficiency; relative total growth

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibility exceeds a threshold of 126 colonies per 10 million cells above the solvent control, in a dose-dependent manner. The mutagenic response is considered to be reproducible if it occurs in both parallel cultures. Clastogenic effects are indicated by a dose-dependent shift in the ratio of small versus large colonies.

Statistics:

A linear regression (least squares) was performed to assess a possible dose-dependent increase of mutant frequencies using SYSTAT Softare.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No substantial and reproducible dose dependent increase of the mutation frequency was observed in experiments with and without metabolic activation.

Table 1. Summary of results

Conc. µg/mL S-9 mix Relative cloning efficiency 1 Relative total growth Mutant colonies/ 10^6cells Threshold Relative cloning efficiency 1 Relative total growth Mutant colonies / 10^6cells threshold Experiment I / 4-hour treatment Culture I Culture II Solvent control with DMSO   - 100.00 100.00 122 248 100.0 100.0 146 272 Positive control with MMS 19.5 - 78.0 38.4 405 248 200.0 54.6 285 272 Test item 1.3 - 139.9 143.1 90 248 109.2 205.6 64 272 Test item 2.5 - 108.1 129.8 89 248 100.0 135.5 85 272 Test item 5.0 - 97.0 95.7 164 248 129.6 224.4 78 272 Test item 10.0 - 97.0 85.3 134 248 123.5 217.2 72 272 Test item 20.0 - 13.2 14.5 147 248 2.5 33.4 149 272 Test item 27.5 - 0.7 Culture was not continued* 0.0 Culture was not continued* Test item 35.0 - 0.0 Culture was not continued* 0.0 Culture was not continued* Solvent control with DMSO   + 100.0 100.0 120.0 246 100.0 100.0 119 245 Positive control with CPA 3.0 + 94.3 53.9 281 246 54.6 53.3 206 245 Positive control with CPA 4.5 + 39.4 20.7 531 246 33.1 20.3 357 245 Test item 4.4 + 68.9 119.7 78 246 80.8 82.5 131 245 Test item 8.8 + 120.4 122.0 96 246 71.6 73.1 131 245 Test item 17.5 + 109.5 180.0 58 246 77.2 99.9 87 245 Test item 35.0 + 41.4 65.2 91 246 54.6 60.3 132 245 Test item 70.0 (p) + 0.4 1.9 315 246 22.7 15.3 226 245 Test item 105.0 (p) + 0.0 Culture was not continued*   Culture was not continued* Experiment II / 24-hour treatment Culture I Culture II Solvent control with DMSO   - 100.00 100.00 123 249 100.0 100.0 147 273 Positive control with CPA 13.0 - 17.8 10.4 598 249 14.9 8.6 725 273 Test item 1.3 - 96.7 Culture was not continued** 101.6 Culture was not continued** Test item 2.5 - 117.8 Culture was not continued** 98.4 Culture was not continued** Test item 5.0 - 127.8 118.7 65 249 91.1 65.1 116 273 Test item 10.0 - 89.2 79.5 151 249 101.6 55.4 122 273 Test item 20.0 - 58.3 61.4 79 249 66.2 44.1 156 273 Test item 27.5 - 19.5 16.1 144 249 10.8 11.0 112 273 Test item 35.0 - 0.3 0.0 153 249 1.3 0.0 147 273 Experiment II/ 4-hour treatment Culture I Culture II Solvent control with DMSO   + 100.0 100.0 119 245 100.0 100.0 106 232 Positive control with CPA 3.0 + 59.1 27.3 274 245 58.9 42.3 359 232 Positive control with CPA 4.5 + 27.2 15.7 531 245 32.1 12.4 597 232 Test item 4.4 + 95.1 103.3 108 245 79.5 98.4 135 232 Test item 8.8 + 96.7 81.5 127 245 79.5 100.6 132 232 Test item 17.5 + 90.6 92.0 118 245 101.5 73.0 218 232 Test item 35.0 + 85.1 59.4 100 245 64.2 55.4 153 232 Test item 52.5 + 13.1 6.7 167 245 2.5 1.3 241 232 Test item 70.0 + 12.4 Culture was not continued* 0.8 Culture was not continued*

p = precipitation

Threshold = number of mutant colonies per 10⁶cells of each solvent control plus 126

* Culture was not continued due to exceedingly severe toxic effects

** Culture was not continued since a minimum of four concentrations is required by the guidelines

The values printed in bold italics are judged as invalid, since the acceptance criteria are not met

Conclusions:

The test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the L5178Y cell line, in the absence or presence of S9 liver fraction metabolic acitvation.

Executive summary:

The study was performed to investigate the potential of the test item to induce mutation at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. This in vitro test is an assay for the detection of forward gene mutations at the autosomal thymidine kinase (TK) locus of heterozygous L5178Y/TK+/- cells to TK-/- mutants. In addition, evidence has been obtained that small TK-/- colonies may result from chromosomal damage to the TK locus and adjacent genes. Gene and chromosome mutations are considered as an initial step in the carcinogenic process. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation. Following a 48-hour phenotypic expression time, treated cell populations were monitored for the loss of the functional TK enzyme. TK catalyses the conversion of trifluorothymidine (TFT) to cytostatic and cytotoxic trifluorothymidine-monophosphate derivative. Therefore, cells deficient in TK due to a forward mutation are resistant to TFT. No substantial and reproducible dose dependent increase of the mutation frequency was observed in experiments with and without metabolic activation. It can be stated that under the experimental conditions defined, the test item did not induce mutation in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. This study is considered to be reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to OECD 476 and EU B.17.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study conducted according to GLP, however no guideline reported. One strain (TA102 or E.coli) has been omitted based on current guideline recommendations and the plate incorporation method was used for both experiments; no analytical data on purity of the test substance was included.

Qualifier:

no guideline followed

Principles of method if other than guideline:

No guidelines reported, however experimental design was similar to OECD 471 and EU B.13/14. The study deviates from the OECD guideline method in that one strain (TA 102 or E. coli WP2) has been omitted and the plate incorporation method was used for both experiments.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat liver (S9)

Test concentrations with justification for top dose:

Range-finding study: 19.5, 39, 78, 156, 312, 625, 1250, 2500, 5000 and 10000 µg/plate; a mild bacteriotoxic effect was observed at the two highest concentrations.
Definitive study: 8, 40, 200, 1000 and 5000 µg/plate

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

other: 9-aminoacridinehydrochloride, 4-nitro-1,2-phenylene diamine, 2-aminoanthracene

Remarks:

Positive controls sodium azide, 9-aminoacridinehydrochloride and 4-nitro-1,2-phenylene diamine were used only without S 9 mix, and the positive control 2-aminoanthracene only with S 9 mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 16 hours
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 4 plates per strain and dose, both with and without S 9 mix

NUMBER OF CELLS EVALUATED: 0.1 mL of bacterial suspension at 10E6 cells/mL

Evaluation criteria:

The following criteria were used for the acceptance of an assay:
- The negative controls have to be within the expected range as defined by literature data (Maron and Ames 1983).
- The positive controls have to show sufficient effects as defined by the laboratories' experience.
- The titer determination must yield a sufficient bacterial density in the suspension.

A reproducible and dose related increase of mutant counts for at least one strain is considered positive. For TA 100, TA 98 andn TA 1535 a twofold increase of revertants compared to the negative controls should be reached, whereas for TA 1537 a threefold increase should be reached. Otherwise the results are estimated as negative.

Statistics:

Not mandatory under OECD test guidleine 471. The mean values and standard deviations were calculated for the number of revertants formed per plate.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A mild bacteriotoxic effect was observed at the two highest concentrations.

Salmonella typhimurium TA 100 Dose µg/plate S-9 mix Revertants/plate x SD Quotient In the absence of an S-9 metabolising system 0 - 200/168/180/* 182.67 16.16 1.00 8 - 164/128/165/156 153.25 17.3 0.84 40 - 164/150/128/136 144.5 15.86 0.79 200 - 94/119/123/126 115.5 14.61 0.63 1000 - 33/21/38/34 31.5 7.32 0.17 5000 - 15/10/9/* 11.33 3.21 0.06 10µg Na azid - 4000/4500/3800/4600 4225 386.0 23.0 Presence of an S-9 metabolising system 0 + 130/115/120/* 121.66 7.63 1.00 8 + 104/115/110/134 115.75 12.97 0.95 40 + 171/189/180/149 172.25 17.15 1.42 200 + 100/114/134/121 117.25 14.17 0.96 1000 + 74/67/54/60 63.75 8.65 0.52 5000 + 32/30/19/25 26.50 5.80 0.21 2µg 2AA * unsterile + 4600/4800/5000/4500 4725 221.0 39.0 *

Salmonella typhimurium TA 1535
Dose µg/plate	S-9 mix	Revertants/plate	x	SD	Quotient
In the absence of an S-9 metabolising system
0	-	13/21/14/20	17.00	4.10	1.00
8	-	17/14/8/13	13.00	3.74	0.76
40	-	11/6/8/11	9.00	2.45	0.52
200	-	8/6/11/9	8.50	2.08	0.50
1000	-	10/12/4/8	8.50	3.41	0.50
5000	-	3/15/7/9	8.50	5.00	0.50
10µg Na azid	-	2240/2500/2300/2200	2310	133	136
Presence of an S-9 metabolising system
0	+	20/17/10/20	16.75	4.71	1.00
8	+	19/21/19/15	18.50	2.51	1.05
40	+	18/30/18/20	21.50	5.74	1.23
200	+	26/15/16/25	20.50	5.80	1.17
1000	+	9/18/12/*	13.00	4.58	0.76
5000	+	18/11/13/*	14.00	3.60	0.82
2µg 2AA * unsterile	+	160/152/198/*	170	24.57	10.0

Salmonella typhimurium TA 98 Dose µg/plate S-9 mix Revertants/plate x SD Quotient In the absence of an S-9 metabolising system 0 - 20/30/17/19 21.50 5.80 1.00 8 - 18/18/18/17 17.75 0.50 0.83 40 - 22/24/23/15 21.00 4.08 0.98 200 - 10/18/15/17 15.00 3.56 0.69 1000 - 14/13/12/16 13.50 1.29 0.63 5000 - 10/14/24/15 15.75 5.91 0.73 10µg Na azid - 161/166/146/150 155.75 9.23 7.25 Presence of an S-9 metabolising system 0 + 22/24/29/25 25.00 2.94 1.00 8 + 23/26/19/17 21.25 4.03 0.85 40 + 16/10/24/* 16.67 7.02 0.67 200 + 20/14/21/22 19.25 3.59 0.77 1000 + 28/14/27/22 22.75 6.40 0.91 5000 + 14/15/21/13 15.75 3.59 0.63 2µg 2AA * unsterile + 1850/1760/1830/1600 1760 13.43 70.4

Salmonella typhimurium TA 1537 test no. 1 Dose µg/plate S-9 mix Revertants/plate x SD Quotient In the absence of an S-9 metabolising system 0 - 5/3/3/10 5.25 3.30 1.00 8 - 6/2/2/8 4.50 3.00 0.85 40 - 3/4/3/4 3.50 0.57 0.66 200 - 4/2/4/* 3.30 1.15 0.62 1000 - 2/0/1/* 1.00 1.00 0.19 5000 - 1/0/0/* 0.33 0.58 0.06 50µg 9-AN - 22/20/45/29 29.0 11.34 5.52 Presence of an S-9 metabolising system 0 + 11/10/13/* 11.33 1.52 1.00 8 + 23/25/20/* 22.66 2.50 2.00 40 + 8/7/15/11 10.25 3.59 0.90 200 + 11/11/7/* 9.67 2.30 0.85 1000 + 7/3/5/5 5.00 1.63 0.44 5000 + 5/4/8/10 6.75 2.75 0.59 2µg 2AA * unsterile + 388/400/368/381 384.25 13.37 33.89

Salmonella typhimurium TA 100 test no. 2 Dose µg/plate S-9 mix Revertants/plate x SD Quotient In the absence of an S-9 metabolising system 0 - 189/174/180/# 181.00 07.55 1.00 8 - 172/158/136/176 160.50 18.06 0.88 40 - 173/179/201/157 177.50 18.21 0.98 200 - 162/126/147/118 138.25 20.01 0.76 1000 - 110/142/146/104 125.50 21.56 0.69 5000 - 112/107/138/160 129.25 24.59 0.72 10µg Na azid - 1040/950/1000/1050 1010 45.46 5.58 Presence of an S-9 metabolising system 0 + 184/198/174/156 178.00 17.66 1.00 8 + 198/216/156/180 187.50 25.63 1.05 40 + 199/190/207/202 199.50 7.14 1.12 200 + 210/174/169/129 170.50 33.15 0.96 1000 + 141/128/152/159 145.00 13.54 0.81 5000 + 199/151/170/168 172.00 19.92 0.97 2µg 2AA * unsterile + 1070/1100/1200/1050 1105 66.58 6.21

Salmonella typhimurium TA 98 test no. 2 Dose µg/plate S-9 mix Revertants/plate x SD Quotient In the absence of an S-9 metabolising system 0 - 30/30/33/43 34.00 6.16 1.00 8 - 21/17/24/17 19.75 3.40 0.58 40 - 19/26/13/27 21.25 6.55 0.63 200 - 29/23/20/18 22.50 4.79 0.66 1000 - 27/21/23/16 23.67 3.06 0.70 5000 - 21/23/20/30 23.50 4.51 0.69 10µg NPDA - 260/240/250/230 245.0 19.91 7.21 Presence of an S-9 metabolising system 0 + 38/19/31/40 32.00 9.49 1.00 8 + 30/24/23/32 27.25 4.43 0.85 40 + 62/41/63/* 55.33 12.42 1.73 200 + 39/36/32/35 35.50 2.89 1.11 1000 + 32/25/35/25 29.25 5.06 0.91 5000 + 31/25/34/38 32.00 5.48 1.00 2µg 2AA * unsterile + 1200/1300/1450/1350 1325 104.1 41.41

Conclusions:

None of the four tester strains used showed any dose related and relevant increase in mutant counts over those of the negative controls at any concentration level, neither in the tests with nor without S9 mix. There was no indication of a bacteriotoxic effect of the test item in all of the employed doses and inhibition of bacterial growth was not noted.

Executive summary:

The potential of the test item to induce gene mutations was assessed in a plate incorporation test using Salmonella typhimurium strains TA1535, TA100, TA1537 and TA98. A range-finding study at ten concentrations ranging from 19.5 to 10000 µg/plate found bacteriotoxic effects at 5000 and 10000 µg/plate, the two highest concentrations tested. The doses for the definitive test were set at 8, 40, 200, 1000 and 5000 µg/plate. The test systems were evaluated with and without rat liver microsomal activation (S9) mix after 48 hours of exposure. None of the tester strains used showed any dose related and relevant increase in mutant counts over those of the negative controls at any concentration level, neither in the tests with or without S9 mix. This study is reliable with restrictions (Klimisch 2) as the study was conducted according to GLP, however no guideline reported. One strain (TA102 or E.coli) has been omitted based on current OECD 471 recommendations and the plate incorporation method was used for both experiments; no analytical data on purity of the test substance was included.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicology assesses the interaction of agents with DNA to produce gene mutations or chromosome alterations. Whilst mutagenicity refers to the production of transmissible genetic alteration, genotoxicity covers a broader spectrum of endpoints, including unscheduled DNA synthesis (UDS), sister chromatid exchanges (SCEs) and DNA strand breaks as measures of genotoxicity.

Genetic toxicity in vitro: A total of four Annex VII and Annex VIII in vitro genetic toxicity studies have been conducted with the test item. In the key chromosome aberration test using human lymphocytes, and at a range of test item dose concentrations including cytotoxic doses, the number of induced structural chromosome aberrations was within the historical control data when tested in the presence and absence of an exogenous S9 metabolic activation system (2008). Thus no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. This study is reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to OECD 473 and EU B.10.

An in vitro mammalian cell gene mutation assay conducted according to OECD 476 showed no substantial or reproducible dose dependent increases in the mutation frequency in the absence and presence of metabolic activation (2008). Under the experimental conditions, the test item did not induce mutations at the mouse lymphoma thymidine kinase locus in the L5178Y cell line. This study is considered to be reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to OECD 476 and EU B.17.

A Bacterial Reverse Mutation Assay (Ames test) was conducted to evaluate the potential of the test item to induce gene mutations Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA 98 (2008). There was no indication of a bacteriotoxic effects in any of the employed doses, and inhibition of bacterial growth was not noted. Under the experimental conditions the test substance 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 in the presence or absence of a metabolising system. This study is reliable without restrictions (Klimisch 1) as it is GLP-compliant and was conducted according to OECD 471 and EU B.13/14. This conclusion is supported by another Ames assay conducted with Salmonella typhimurium strains TA1535, TA100, TA1537 and TA98 in a plate incorporation test (1989). None of the strains tested showed any dose related and relevant increase in mutant counts over those of the negative controls at any concentration level, neither in the tests with or without S9 mix. This study is reliable with restrictions (Klimisch 2) as the study was conducted according to GLP, however no guideline reported and there were minor restrictions in experimental design and reporting.

Genetic toxicity in vivo: No positive results were observed in any of the genotoxicity studies required for the Annex VII or VIII information requirements under REACH, therefore in vivo data for genetic toxicity is not required.

Justification for classification or non-classification

Since the results from all in vitro experimental studies were negative, it can be concluded that there is no evidence of relevant intrinsic genotoxic properties that would initiate classification. Consequently, no further testing to include in vivo studies was justified and the substance was not classifiable as mutagenic according to CLP Regulation (EC) No. 1272/2008.