3(or 4)-(4-methylpenten-3-yl)cyclohex-3-ene-1-methyl acetate

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:

18 October - 18 November 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted and well described study in accordance with GLP and OECD Guideline 471 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3(or 4)-(4-methylpenten-3-yl)cyclohex-3-ene-1-methyl acetate

EC Number:

276-650-6

EC Name:

3(or 4)-(4-methylpenten-3-yl)cyclohex-3-ene-1-methyl acetate

Cas Number:

72403-67-9

Molecular formula:

C15H24O2

IUPAC Name:

[3-(4-methylpent-3-en-1-yl)cyclohex-3-en-1-yl]methyl acetate; [4-(4-methylpent-3-en-1-yl)cyclohex-3-en-1-yl]methyl acetate

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Myraldyl acetate
- Physical state: Clear colourless liquid
- Analytical purity: 99.6 %
- Lot/batch No.: 9000478420
- Date of receipt: 14 August 2002
- Expiration date of the lot/batch: 15 July 2004
- Storage condition of test material: Stored at 1-10 °C in the dark

Method

Target gene:

None

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: see section: "Any other information on materials and methods incl. tables"

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Range-Finder Experiment and Mutation Experiment 1 (plate-incorporation method):
- TA1535, TA1537, TA98, TA100 and TA102: 1.6, 8, 40, 200, 1000 and 5000 µg/plate, in the absence and presence of S9-mix

Mutation Experiment 2 (preincubation method):
Without S9 mix
- TA100 and TA1535: 8.192, 20.48, 51.2, 128, 320, 800, 2000 and 5000 µg/plate
- TA98, TA1537 and TA102: 3.277, 8.192, 20.48, 51.2, 128, 320, 800 and 2000 µg/plate
With S9 mix
- TA98, TA100 and TA1535: 8.192, 20.48, 51.2, 128, 320, 800, 2000 and 5000 µg/plate
- TA1537 and TA102: 3.277, 8.192, 20.48, 51.2, 128, 320, 800 and 2000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Test substance preparation: Test article solutions were prepared by dissolving Myraldyl acetate in sterile anhydrous analytical grade DMSO, immediately prior to assay to give the maximum required treatment solution concentration. This solution was filter-sterilised (Gelman Acrodisc CR filter, 0.2 µm pore size) and further dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 6 h of the initial formulation of the test article.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see table 7.6.1/1

Remarks:

with and without metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: - All the tester strains, with the exception of strain TA102, were originally obtained from the UK NCTC. Strain TA102 was originally obtained from Glaxo Group Research Limited.

METHOD OF APPLICATION: In agar (plate incorporation); preincubation

DURATION
- Preincubation period: 1 hour at 37 ± 1 °C, with shaking
- Incubation period: Plates were inverted and incubated at 37 ± 1 °C in the dark for 3 days in both direct plate and preincubation methods

NUMBER OF REPLICATIONS:
- Negative (solvent) and positive controls were included in quintuplicate and triplicate, respectively.
- Treatment (test item) groups were included in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: The bacterial background lawn was inspected for signs of toxicity.

OTHER:
- Colony counting: Colonies were counted electronically using a Seescan Colony Counter (Seescan Plc) or manually where confounding factors such as split agar or the presence of microcolonies affected the accuracy of the automated counter.

Evaluation criteria:

Acceptance criteria
The assay was considered valid if the following criteria were met:
- the mean negative control counts fell within the normal historical ranges
- the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation
- no more than 5 % of the plates were lost through contamination or some other unforeseen event.

Evaluation criteria
The test article was considered to be mutagenic if:
- the assay was valid (see above)
- Dunnett's test gave a significant response (p ≤ 0.01) and the data set(s) showed a significant dose correlation
- the positive responses described above were reproducible

Statistics:

- The m-statistic was calculated to check that the data were Poisson-distributed (Mahon et al., 1989), and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis (Mahon et al., 1989).
- Due to limitations in the statistical package used to analyse the mutation data, where eight dose levels were treated (Experiment 2), the program was unable to assign Dunnett's test significance. For these treatments, significance levels were determined using Dunnett's test tables. In these cases, significance was only attributed at the 5 % and 1 % levels, and not at the 0.5 % level (as for the other data sets).

Results and discussion

Test results

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test article precipitate was noted on all plates which had been treated at 5000 µg/plate in all experiments.

RANGE-FINDING/SCREENING STUDIES:
- TA 100 strain: Evidence of toxicity in the form of a diminution of the background bacterial lawn was observed following the top dose treatment in both the absence and presence of S-9. In addition, no revertant colonies were observed following the top dose treatment in the presence of S-9.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

EXPERIMENT 1:
- Evidence of toxicity in the form of a diminution of the background bacterial lawn was observed following the highest one or two dose treatments in all strains (TA98, TA1535, TA1537 and TA102) both in the absence and presence of S-9. No revertant colonies were observed following the top dose treatment of strains TA98, TA1537 and TA102 in the presence of S-9.

EXPERIMENT 2:
- Evidence of toxicity in the form of slight thinning of the background bacterial lawn was observed following the top one or two dose treatments of all strains in the absence of S-9, except strain TA102 where no toxicity was observed. For TA102 treatments in the absence of S-9, it was considered that treatments had been performed up to doses where evidence of toxicity would have been predicted. No toxicity was observed following any TA1535 treatments in the presence of S-9.
- Evidence of toxicity ranging from a slight thinning of the background lawn to complete killing of the test bacteria was observed following the top two dose treatments of strain TA98, the top three dose treatments of strain TA102 and the top four dose treatments of strains TA100 and TA1537 in the presence of S-9.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical negative (solvent) and positive control data (12 October 2001 to 21 November 2001).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

See the attached document for information on tables of results

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test conditions, Myraldyl acetate is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98, TA100 and TA102) strains.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102) were exposed to Myraldyl acetate at the following concentrations:

Range-Finder Experiment and Mutation Experiment 1 (plate-incorporation method):

- TA1535, TA1537, TA98, TA100 and TA102: 1.6, 8, 40, 200, 1000 and 5000 µg/plate, in the absence and presence of S9-mix

Mutation Experiment 2 (preincubation method):

Without S9 mix

- TA100 and TA1535: 8.192, 20.48, 51.2, 128, 320, 800, 2000 and 5000 µg/plate

- TA98, TA1537 and TA102: 3.277, 8.192, 20.48, 51.2, 128, 320, 800 and 2000 µg/plate

With S9 mix

- TA98, TA100 and TA1535: 8.192, 20.48, 51.2, 128, 320, 800, 2000 and 5000 µg/plate

- TA1537 and TA102: 3.277, 8.192, 20.48, 51.2, 128, 320, 800 and 2000 µg/plate

Metabolic activation system used in this test 10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254. Vehicle and positive control groups were also included in mutagenicity tests.

 

In Range-Finder Experiment with TA100, evidence of toxicity in the form of a diminution of the background bacterial lawn was observed following the top dose treatment in both the absence and presence of S-9. In addition, no revertant colonies were observed following the top dose treatment in the presence of S-9. Test article precipitate was noted on all plates which had been treated at 5000 µg/plate in all experiments. In Experiment 1, evidence of toxicity in the form of a diminution of the background bacterial lawn was observed following the highest one or two dose treatments in all strains (TA98, TA1535, TA1537 and TA102) both in the absence and presence of S-9. No revertant colonies were observed following the top dose treatment of strains TA98, TA1537 and TA102 in the presence of S-9. In Experiment 2, evidence of toxicity in the form of slight thinning of the background bacterial lawn was observed following the top one or two dose treatments of all strains in the absence of S-9, except strain TA102 where no toxicity was observed. For TA102 treatments in the absence of S-9, it was considered that treatments had been performed up to doses where evidence of toxicity would have been predicted. No toxicity was observed following any TA1535 treatments in the presence of S-9. Evidence of toxicity ranging from a slight thinning of the background lawn to complete killing of the test bacteria was observed following the top two dose treatments of strain TA98, the top three dose treatments of strain TA102 and the top four dose treatments of strains TA100 and TA1537 in the presence of S-9. No statistically significant, dose-related and reproducible increases in revertant numbers were observed following any strain treatments in the absence or presence of metabolic activation.The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.

 

Under the test conditions, Myraldyl acetate is not considered as mutagenic in this bacterial system.