cis-hex-3-en-1-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phases of the study were performed between 22 October 2012 and 18 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

cis-hex-3-en-1-ol

EC Number:

213-192-8

EC Name:

cis-hex-3-en-1-ol

Cas Number:

928-96-1

Molecular formula:

C6H12O

IUPAC Name:

hex-3-en-1-ol

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): cis-hex-3-en-ol (LFA)
- Physical state: clear colourless liquid
- Lot/batch No.: 1Z50650
- Expiration date of the lot/batch: 06 June 2015
- Storage condition of test material: room temperature, in the dark, under nitrogen

Method

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10 % donor horse serum (giving R10 media) at 37 °C with 5 % CO2 in air
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.

Test concentrations with justification for top dose:

- Preliminary toxicity test: 3.91 to 1001.6 µg/mL
- Experiment 1 and 2: 0, 3.91, 7.83, 15.65, 31.3, 62.6, 125.2, 250.4, 500.8, 1001.6 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Sigma batch BCBH3157V at 400 μg/mL and 150 μg/mL for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation

Controls 2

Positive control substance:

cyclophosphamide

Remarks:

Acros batch A0302605 at 2 μg/mL was used as the positive control in the presence of metabolic activation for Experiments 1 and 2

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Preliminary toxicity test
- Exposure duration: 4 h with and without metabolic activation (S9); 24 h without S9
- Expression time (cells in growth medium): 24 h

Experiment 1
- Preincubation period:
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 d
- Selection time (if incubation with a selection agent): 10 to 14 d

Experiment 2
- Preincubation period:
- Exposure duration: 4 h with metabolic activation; 24 h without metabolic activation
- Expression time (cells in growth medium): 2 d
- Selection time (if incubation with a selection agent): 10 to 14 d

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
STAIN (for cytogenetic assays): 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS)

NUMBER OF REPLICATIONS: The treatments were performed in duplicate (A + B), both with and without metabolic activation at 6 dose levels of the test item, vehicle and positive controls

NUMBER OF CELLS EVALUATED: on day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL TFT in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (% V) in non-selective medium

DETERMINATION OF CYTOTOXICITY
- Method: The daily cell counts were used to obtain a Relative Suspension Growth (% RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (% V) data a Relative Total Growth (RTG) value.

OTHER
Preliminary toxicity test
- A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 h exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24 h exposure period without S9. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 10^5 cells/mL.
- The cultures were incubated at 37 °C with 5 % CO2 in air and sub-cultured after 24 h by counting and diluting to 2 x 10^5 cells/mL. After a further 24 h the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (% RSG) value.
- Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 μg/mL or 10 mM.
ii) The presence of excessive precipitate where no test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20 % survival (the maximum level of toxicity required). This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al 2002)

Experiment 1
- Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (2 % S9 final concentration) at 6 dose levels of the test item (62.6 to 1001.6 μg/mL in both the absence and presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9-mix if required, 0.2 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL.
- The treatment vessels were incubated at 37 °C for 4 h with continuous shaking using an orbital shaker within an incubated hood.
- At the end of the treatment period the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and subcultured every 24 h for the expression period of 2 d, by counting and dilution to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.

Experiment 2
- An exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL cultures in R10 medium for the 4 h treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 h therefore 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm^2 tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (1 % S9 final concentration) at 6 dose levels of the test item (62.6 to 1001.6 μg/mL in both the absence and presence of metabolic activation), vehicle and positive controls. To each culture vessel was added 2 mL of S9-mix if required, 0.2 mL of the treatment dilutions, (0.2 mL for the positive control) and sufficient R0 medium to give a final volume of 20 mL (R10 is used for the 24 h exposure group).
- The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker within an incubated hood for 24 h in the absence of metabolic activation and 4 h in the presence of metabolic activation
- At the end of the treatment period the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5 % CO2 in air and subcultured every 24 h for the expression period of 2 d, by counting and dilution to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.

Evaluation criteria:

For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. The IMF must exceed some value based on the global background MF for each method (agar or microwell). The Global Evaluation Factor (GEF) value is 126 x 10^-6 for the microwell method.

Any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10^-6 and demonstrate a positive linear trend will be considered positive.

If a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance.

When a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST: In the 4-hour exposure groups there was evidence of modest reductions in the Relative Suspension Growth (% RSG) of cells treated with the test item when compared to the concurrent vehicle controls. However, much greater reductions in % RSG were observed in the 24-hour exposure group and optimum levels of toxicity were achieved at the 10 mM limit dose level. Precipitate of the test item was not observed at any of the dose levels. Based on the % RSG values observed, the maximum dose level in the subsequent Mutagenicity Test was the 10 mM limit dose of 1001.6 μg/mL.

EXPERIMENT 1: There was evidence of modest dose-related toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the RTG and % RSG values. There was no evidence of any significant dose related reductions in viability (% V) in either the absence or presence of metabolic activation; therefore indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with both positive control substances.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels, including the 10 mM limit dose level, in either the absence or presence of metabolic activation. Precipitate of the test item was not observed at any of the dose levels.

EXPERIMENT 2: There was evidence of modest toxicity following exposure to the test item in the 4-hour exposure group in the presence of metabolic activation, and more marked toxicity in the 24-hour exposure group in the absence of metabolic activation, as indicated by the % RSG and RTG values. There was no evidence of any significant dose related reductions in viability (% V) in either the absence or presence of metabolic activation; therefore indicating that residual toxicity had not occurred. Based on the % RSG and RTG values observed, optimum levels of toxicity were achieved in the 24-hour exposure group in the absence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances
The 24-hour exposure without metabolic activation demonstrated that the extended time point had a marked effect on the toxicity of the test item. The lowering of the S9 concentration to 1 % in this second experiment did not result in greater levels of toxicity being observed when compared to 4-hour exposure groups in the presence of 2 % metabolic activation in the Preliminary Toxicity Test and Experiment 1.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels in the presence of metabolic activation, including the 10 mM limit dose level. A very modest but statistically significant dose related (linear-trend) increase in mutant frequency was observed in the 24-hour exposure group in the absence of metabolic activation. However, statistically significant increases in mutant frequency were not observed at any of the individual dose levels and the GEF was also not exceeded at any of the dose levels, including the 10 mM limit dose level that achieved optimum levels of toxicity. There was also no evidence of any significant increases in absolute numbers of mutant colonies and the mutant frequency values observed would have been considered acceptable for vehicle controls. The response was, therefore, considered to be artefactual and of no toxicological significance. Precipitate of the test item was not observed at any of the dose levels.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary toxicity test

The dose range of the test item used in the preliminary toxicity test was 3.91 to 1001.6 μg/mL. The results for the Relative Suspension Growth (% RSG) were as follows:

Dose (µg/mL)	% RSG (-S9) 4-Hour exposure	% RSG (+S9) 4-Hour exposure	% RSG (-S9) 24-Hour exposure
0	100	100	100
3.91	116	108	112
7.83	116	89	111
15.65	113	108	119
31.3	117	101	122
62.6	111	96	113
125.2	118	60	85
250.4	113	73	81
500.8	113	59	46
1001.6	88	79	12

Summary of Results

Experiment 1

4-Hour –S9				4-Hour +S9
Treatment (µg/mL)	% RSG	RTG	MF§	Treatment (µg/mL)	% RSG	RTG	MF§
0	100	1.00	157.56	0	100	1.00	128.80
62.6	89	1.10	108.68	62.6	93	1.00	154.20
125.2	102	1.04	131.97	125.2	91	1.09	127.08
250.5	87	1.02	132.37	250.5	83	0.96	113.87
500.8	74	0.89	116.84	500.8	82	0.96	154.48
751.2	86	0.87	131.97	751.2	88	0.93	153.47
1001.6	68	0.65	149.19	1001.6	68	0.82	148.99
Linear trend                                            NS				Linear trend                                     NS
EMS 400	76	0.58	1108.37	CP 2	63	0.42	1222.64

  

Experiment 2

4-Hour –S9				4-Hour +S9
Treatment (µg/mL)	% RSG	RTG	MF§	Treatment (µg/mL)	% RSG	RTG	MF§
0	100	1.00	120.30	0	100	1.00	122.50
62.6	88	0.87	103.16	62.6	89	1.03	108.50
125.2	84	0.89	129.14	125.2	86	0.90	103.50
250.5	71	1.02	118.93	250.5	80	0.72	130.32
500.8	31	0.45	132.83	500.8	83	0.96	120.77
751.2	14	0.26	130.88	751.2	83	0.85	123.40
1001.6	5	0.13	159.99	1001.6	75	0.74	144.28
Linear trend                                             *				Linear trend                                    NS
EMS 150	49	0.37	1388.93	CP 2	46	0.27	1028.72

% RSG = Relative Suspension Growth

RTG = Relative Total Growth

MF§= 5- TFT resistant mutants/ 10⁶ viable cells 2 days after treatment

NS= Not significant

* = p < 0.05

EMS = Ethylmethanesulphonate

CP = Cyclophosphamide

Applicant's summary and conclusion

Conclusions:

The potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line was assessed according to OECD guideline 476. The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line according to OECD guideline 476.

Methods:

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (DMSO) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9 final concentration). In Experiment 2, the cells were treated with the test item at six dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9 final concentration) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test and for Experiments 1 and 2 was 62.6 to 1001.6 μg/ml in both the absence and presence of metabolic activation.

Results:

The maximum dose levels used in the Mutagenicity Test was the 10 mM limit dose of 1001.6 μg/mL. Precipitate of test item was not observed at any of the dose levels during the course of the study. The vehicle (DMSO) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at teh TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.