4-methylpent-1-ene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Test start: 16 August 2004. Test completed: 14 December 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to GLP and guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-methylpent-1-ene

EC Number:

211-720-1

EC Name:

4-methylpent-1-ene

Cas Number:

691-37-2

Molecular formula:

C6H12

IUPAC Name:

4-methylpent-1-ene

Details on test material:

- Name of test material (as cited in study report): 4-methyl-1-pentene
- Analytical purity: 98.36%
- Lot/batch No.: 3B24A

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose finding test: 3000, 1000, 333, 111, 37.0, 12.3 and 4.12 μg/plate
Main test: 3000, 1500, 750, 375, 188, 93.8 and 46.9 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide

Controls

Controls 1

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide

Remarks:

Used with S. typhimurium TA 100 , TA98 and E.coli WP2 for the direct method

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Used with S. typhimurium TA1535 for the direct method.

Controls 3

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Used with S. typhimurium TA1537 for the direct method

Controls 4

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Used for all strains for the metabolic activation method

Controls 5

Untreated negative controls:

yes

Remarks:

Dimethyl sulfoxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: The test substance and control substance preparations were treated by the pre-incubation method

Preincubation of bacterial strains:
Twelve mL of pre-incubation medium (nutrient broth) was put into an L-shaped tube of approximately 40 mL, 12 μL of thawed bacteria was seeded, after the L-shaped tube was ice-cooled, the culture was incubated with reciprocal shaking for 10 hours in a shaking thermostatic chamber (Personal-11 EX, Taitec Co., Ltd.) set at 37°C, oscillation of 40 mm and shaking speed of 100 times/minute. At the completion of incubation, the OD660 nm of the bacterial culture was measured with a colorimeter (Mini Photo 518, Taitec Co., Ltd.), and the viable bacteria count for each strain was calculated from the viable bacteria count-OD660 nm equation. Cultures confirmed to have sufficient growth with viable bacteria counts exceeding 1 x 109 cells/mL were used in the test

Treatment of test substance and control substance preparations :
The test substance and control substance preparations were treated by the pre-incubation method.

0.1 mL of the test substance preparation or control substance preparation was mixed with 0.5 mL of 0.1 mol/L Na-phosphate buffer (pH 7.4) in the direct method, or with 0.5 mL of S9 mix in the metabolic activation method. Then 0.1 mL of bacterial culture was added to the mixture and it was pre-incubated for 20 minutes in a shaking thermostatic chamber (Personal-11 EX, Taitec Co., Ltd.) set at 37°C, oscillation of 40 mm and shaking speed of 100 times/minute. After the completion of pre-incubation, 2 mL of overlay medium containing 0.05 mmol/L L-histidine and 0.05 mmol/L D-biotin for S. typhimurium and 0.05 mmol/L L-tryptophan for E. coli were added and mixed, then overlaid on the minimum glucose agar. The overlay medium was allowed to harden in a level place, then plates for each bacterial strain at each dose were placed in pouches (Mitsubishi Gas Chemical Company, Inc.) and static incubation was conducted for 48 hours in an incubator (MIR-262: Sanyo Electric Co., Ltd.) set at 37°C. In each test, the presence or absence of contaminating bacteria was confirmed in the highest concentration of test substance preparation and the S9 mix


NUMBER OF REPLICATIONS: One plate for each dose was used in the dose-finding test and 3 plates for each dose were used in the main test.

Observation of plates:
After each plate was observed visually for precipitation of the test substance and for growth inhibition under a stereoscopic microscope (SZ6045TR, Olympus Optical Co., Ltd.), the number of revertant colonies was counted. A colony counter (BMS-400, Toyo Sokki Co., Ltd.) was used for counting in the positive control group. In the negative control group and test substance treatment groups, S. typhimurium TA100 and E. coli WP2uvrA were counted using the colony counter. S. typhimurium TA98, TA1535 and TA1537, whose revertant colonies were small, were counted visually using a digital colony counter (DC-3, Fuji Medical Co.) to avoid missing the count.

The judgment of growth inhibition in the bacterial strains was done according to the following criteria based on standard operating procedure. A score of 1 or higher was considered growth inhibition.

0: Growth inhibition is not observed.
Microscopic background colonies (visible at magnification of about 50) are observed over the surface of the medium, with no difference from the background colonies of the control group observed.

1: Slight growth inhibition is observed.
Background colonies are decreased compared to the control group, and individual colonies are larger.

2: Moderate growth inhibition is observed.
Large, raised revertant colonies and small, flat background colonies are coexisting.

3: Strong growth inhibition is observed.
Background colonies have grown to the same extent as revertant colonies, and the two are difficult to distinguish.

4: No viable bacteria are observed.

Mean ± standard deviation was calculated for the numbers of revertant colonies in the test groups

Evaluation criteria:

If the mean number of revertant colonies in a test substance treatment group was twice or more that in the negative control group, and the number of colonies increased with the dose level, and those results were reproducible, the result was judged positive

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results of the main test were negative, with the mean number of revertant colonies in the test substance treatment groups was less than twice that of the negative control group in each bacterial strain by both the direct method and with metabolic activation, and no increase in revertant colonies with increasing doses. Precipitation of the test substance was observed at all doses, and growth inhibition at high doses was observed in each strain. These results were reproduced in the second test.

The mean number of revertant colonies in the negative control group was within the range of the control values based the background data of the test facility. The mean numbers of revertant colonies in each positive control group showed clear increase and was at least twice the value in the negative control group. These results confirm that the bacterial strains have appropriate sensitivity to mutagens.

Any other information on results incl. tables

Tables of results and dose response curves are included in the attached background information.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It was judged from these results that 4-methyl-1-pentene does not induce genetic mutations in the test bacteria strains under the conditions of this study.