(Z)-3-hexenyl salicylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 March 1994 to 13 April 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guidelines and the study was conducted under GLP conditions. The study was performed on a structurally similar substance the results of which are read-across and considered to be representative of the effects of the registered substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302, L 133, p. 61-63

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA 40 CFR; Ch. I; Part 798; Detection of gene mutation in somatic cells in culture; pp. 717-720 (7-1-86 Edition)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

25485-88-5

Cas Number:

25485-88-5

IUPAC Name:

25485-88-5

Test material form:

liquid

Details on test material:

- Name of test material: CHS, cyclohexyl salicylate
- Physical state: Clear colourless liquid
- Storage condition of test material: at room temperature
- Molecular weight (if other than submission substance): 220.3

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HRPT locus)

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) supplemented with 10 % foetal calf serum.
- Properly maintained: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes (HAT medium)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction

Test concentrations with justification for top dose:

Experiment I
Without S9 mix: 0, 0.75, 2.5, 5.0, 7.5 and 8.5 µg/mL
With S9 mix: 0, 3.0, 6.0, 10, 30 and 60 µg/mL

Experiment II
Without S9 mix: 0, 1.0, 2.5, 5.0, 7.5 and 10 µg/mL
With S9 mix: 0, 6.0, 10, 30 and 60 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:ethanol
- Justification for choice of solvent/vehicle: The vehicle was selected by the solubility of the test material in the vehicle and the non-toxic properties of the vehicle to the cell culture.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium. Plastic culture flasks were seeded with approximately 1.5 x 10⁶ (single culture) and 5 x 10² cells (in duplicate) into MEM with 10 % FCS. After 24 hours, the culture medium was replaced with serum free medium containing the appropriate test material concentration either with or without 50 µL/mL of S9 mix. After 4 hours this was replaced with complete medium after washing with saline G twice. All cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % CO₂.

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 1 week. Cultures were treated on day 2 and were transfered to selective medium on day 9
- Selection time (if incubation with a selection agent): 1 week and 10 days for experiment I and II, respectively. Flasks seeded on day 9 of the experiment were fixed and stained on day 16 (experiment I) and day 18 (experiment II)
- Fixation time (start of exposure up to fixation or harvest of cells): Day 16 and day 18 for experiments I and II, respectively.

SELECTION AGENT: 11 µg/mL thioguanine
STAIN: 10 % methylene blue in 0.01 % KOH solution.

NUMBER OF REPLICATIONS:
Mutagenicity was evaluated in 5 replicate flasks per concentration

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency.

Evaluation criteria:

The test was considered positive if either a concentration related increase of the mutant frequency or a reproducible positive response for one of the concentrations was observed.
The test was considered non-mutagenic if neither a concentration related increase in the mutant frequency was observed, or if a response observed at one test point was found not to be reproducible.
A response was considered significant if the test material produces a reproducible response at one of the concentrations with a mutant frequency three times higher than the spontaneous mutant frequency in the experiment.
The test material is classified as mutagenic if there is a reproducible concentration-related response of the mutant frequency. A concentration-related response may be considered if a threefold increase was not observed in the mutant frequency (any such result would be compared with the negative control data).

The stained colonies with more than 50 cells were counted. Were colony size was uncertain, the colony was checked using a microscope.

Statistics:

No adequate statistical evaluation was available.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Neither a reproducible relevant increase in mutations was observed not a dose dependent increase in mutations were observed

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without metabolic activation, the test material was cytotoxic in both experiments at the two highest concentrations tested.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test material was evaluated in a pre-test on toxicity. Toxicity of the test material was evidenced by a reduction in cloning efficiency when tested up to 60.00 µg/mL (the limit of solubility). Based on the results of the pre-test, the concentrations for the definitive tests were selected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Without metabolic activation, elevated toxicity was observed in the cloning efficiency of the cells with concentrations of 7.5 and 8.5 µg/mL in experiment I. The cell density at the first subcultivation was also reduced for these two concentrations (54.2 and 31.4 % of the solvent control value).

In experiment II, the cloning efficiency of the cells was considerably reduced in the two upper concentrations without S9 mix. The cell density at first subcultivation was reduced to 61.2 % of the solvent control. The 10.0 µg/mL treatment group could not be evaluated due to the severe reduction in the cell density (4.0 % of the solvent control) at the first subcultivation.

In the presence of metabolic activation, no toxic effects were observed up to 60.0 µg/mL (the limit of solubility) in the cloning efficiency of either experiment. No reduction in cell culture at first subcultivation was obtained in experiment I, and only a slight reduction was observed at 60.0 µg/mL in experiment II.

Any other information on results incl. tables

Table 1: Toxicity results from mutagenicity experiment 1

Treatment group	Number of cells per flask				Factor calculated*	Cells seeded	Cells survived**
	Seeded (I/II)	Found I	Found II	Mean
Without S9 mix
Negative control	501	360	334	347.0	0.69	438000	302220
Solvent control with ethanol	506	310	341	325.5	0.64	441000	282240
Positive control with 0.60 mg EMS	630	356	396	376.0	0.60	489000	293400
0.75 µg	505	344	356	350.0	0.69	421500	290835
2.50 µg	Culture not continued
5.00 µg	503	334	316	325.0	0.65	423000	274950
7.50 µg	506	319	330	324.5	0.64	427500	273600
8.50 µg	507	312	301	306.5	0.60	436500	261900
With S9 mix
Negative control	506	358	330	344.0	0.68	493000	335240
Solvent control with ethanol	504	322	306	314.0	0.62	354000	219480
Solvent control with DMSO	500	310	298	304.0	0.61	484500	295545
Positive control with 3.85 µg DMBA	506	295	337	316.0	0.62	411000	254820
3.00 µg	Culture not continued
6.00 µg	501	314	338	326..0	0.65	435000	282750
10.00 µg	500	329	321	325.0	0.65	470500	305825
30.00 µg	508	362	344	353.0	0.69	414000	285660
60.00 µg	503	302	328	315.0	0.63	427000	269010
*Factor calculated (mean/cells seeded I/II). ** Cells survived after plating in TG containing medium Only flasks containing 50 or more cells after 7 days of culturing in normal medium were scored

 

Table 2:Mutagenicity results from mutagenicity experiment 1

Treatment group	Number of mutant colonies per flask* found after plating in TG medium							Mutant colonies per 10^6cells
	I	II	III	IV	V	Mean	Standard deviation
Without S9 mix
Negative control	3	3	2	2	0	2.0	1.2	6.6
Solvent control with ethanol	2	1	3	2	1	1.8	0.8	6.4
Positive control with 0.60 mg EMS	136	143	153	129	168	145.8	15.3	496.9
0.75 µg	1	1	0	1	1	0.8	0.4	2.8
2.50 µg	Culture not continued
5.00 µg	7	5	3	7	5	5.4	1.7	19.6
7.50 µg	2	1	1	0	1	1.0	0.7	3.7
8.50 µg	1	1	2	2	1	1.4	0.5	5.3
With S9 mix
Negative control	4	2	5	2	4	3.4	1.3	10.1
Solvent control with ethanol	7	9	5	0	3	4.8	3.5	21.9
Solvent control with DMSO	4	3	2	1	2	2.4	1.1	8.1
Positive control with 3.85 µg DMBA	91	85	98	112	107	98.6	11.1	386.9
3.00 µg	Culture not continued
6.00 µg	6	5	2	2	7	4.4	2.3	15.6
10.00 µg	3	2	2	2	3	2.4	0.5	7.8
30.00 µg	2	1	3	3	2	2.2	0.8	7.7
60.00 µg	5	4	1	3	9	4.4	3.0	16.4
Only flasks containing 50 or more cells after 7 days of culturing in normal medium were scored

 

Table 3: Toxicity results from mutagenicity experiment 2

Treatment group	Number of cells per flask				Factor calculated*	Cells seeded	Cells survived**
	Seeded (I/II)	Found I	Found II	Mean
Without S9 mix
Negative control	503	301	318	309.5	0.62	372000	230640
Solvent control with ethanol	507	363	318	340.5	0.67	375000	251250
Positive control with 0.60 mg EMS	505	295	302	298.5	0.59	393000	231870
1.00 µg	504	292	324	308.0	0.61	369000	225090
2.50 µg	509	291	273	282.0	0.55	348000	191400
5.00 µg	505	275	277	276.0	0.55	378000	207900
7.50 µg	510	259	255	257.0	0.50	411000	205500
10.00 µg	Not possible to evaluate, toxic to cultures
With S9 mix
Negative control	510	390	367	378.5	0.74	375000	277500
Solvent control with ethanol	507	302	340	321.0	0.63	363000	228690
Solvent control with DMSO	505	341	287	314.0	0.62	366000	226920
Positive control with 3.85 µg DMBA	510	301	310	305.5	0.60	360000	216000
6.00 µg	504	340	362	351.0	0.70	378000	264600
10.00 µg	515	388	360	374.0	0.73	366000	267180
30.00 µg	511	323	337	330.0	0.65	369000	239850
60.00 µg	511	265	270	267.5	0.52	390000	202800
*Factor calculated (mean/cells seeded I/II). ** Cells survived after plating in TG containing medium Only flasks containing 50 or more cells after 7 days of culturing in normal medium were scored

 

Table 4: Mutagenicity results from mutagenicity experiment 2

Treatment group	Number of mutant colonies per flask* found after plating in TG medium							Mutant colonies per 10^6cells
	I	II	III	IV	V	Mean	Standard deviation
Without S9 mix
Negative control	0	1	1	1	1	0.8	0.4	3.5
Solvent control with ethanol	3	4	1	4	3	3.0	1.2	11.9
Positive control with 0.60 mg EMS	93	81	51	61	69	71.0	16.5	306.2
1.00 µg	1	0	0	0	2	0.6	0.9	2.7
2.50 µg	1	2	1	1	0	1.0	0.7	5.2
5.00 µg	1	2	4	0	0	1.4	1.7	6.7
7.50 µg	0	5	5	1	1	2.4	2.4	11.7
10.00 µg	Not possible to evaluate, toxic to cultures
With S9 mix
Negative control	1	5	3	3	5	3.4	1.7	12.3
Solvent control with ethanol	7	3	1	1	3	3.0	2.4	13.1
Solvent control with DMSO	2	1	0	4	1	1.6	1.5	7.1
Positive control with 3.85 µg DMBA	90	103	96	86	95	94.0	6.4	435.2
6.00 µg	1	3	1	3	3	2.2	1.1	8.3
10.00 µg	1	0	0	1	1	0.6	0.5	2.2
30.00 µg	5	4	3	1	3	3.2	1.5	13.3
60.00 µg	3	1	2	3	0	1.8	1.3	8.9
Only flasks containing 50 or more cells after 7 days of culturing in normal medium were scored

 

Applicant's summary and conclusion

Conclusions:

negative with and without metabolic activation

Under the conditions of the test, the test material did not induce gene mutations at the HRPT locus in V79 cells.

Executive summary:

The ability of the test material to induce mutations at the HPRT locus in Chinese Hamster V79 cells was investigated. In two independent experiments, both performed with and without metabolic activation. The test material was dissolved in ethanol and tested in the following concentrations:

Experiment I:

Without S9 mix: 0.75, 2.5, 5.0 7.5 and 8.5 µg/mL

With S9 mix: 3.0, 6.0 10, 30 and 60 µg/mL

Experiment II:

Without S9 mix: 1.0, 2.5, 5.0, 7.5 and 10.0 µg/mL

With S9 mix: 6.0, 10, 30 and 60 µg/mL

Doses were selected based on a pre-test for toxicity. Without S9 mix, the highest concentration demonstrated strong toxicity. With S9 mix, no toxicity was observed up to the limit of solubility (60 µg/mL).

No relevant increase in mutant colony numbers were obtained in either experiment with or without metabolic activation. The positive negative, and vehicle controls were all demonstrated to be valid under the conditions of the test.

Under the conditions of the test, the test material did not induce gene mutations at the HRPT locus in V79 cells.