Reaction mass of 1-methyl-4-(4-methylpentyl)cyclohex-3-ene-1-carbaldehyde and 1-methyl-3-(4-methylpentyl)-3-cyclohexene-1-carboxaldehyde

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:

May 15, 2003 - June 05, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guideline and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L1362000, Annex 4D, dated May 19, 2000

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-methyl-4-(4-methylpentyl)cyclohex-3-ene-1-carbaldehyde

EC Number:

266-314-7

EC Name:

1-methyl-4-(4-methylpentyl)cyclohex-3-ene-1-carbaldehyde

Cas Number:

66327-54-6

Molecular formula:

C14H24O

IUPAC Name:

1-methyl-4-(4-methylpentyl)cyclohex-3-ene-1-carbaldehyde

Constituent 2

Reference substance name:

1-methyl-3-(4-methylpentyl)-3-Cyclohexene-1-carboxaldehyde

Cas Number:

693252-50-5

Molecular formula:

C14H24O

IUPAC Name:

1-methyl-3-(4-methylpentyl)-3-Cyclohexene-1-carboxaldehyde

impurity 1

Reference substance name:

Cyclohexanecarboxaldehyde, 1-methyl-4-(4-methylpentyl)-, trans-

Cas Number:

76169-46-5

Molecular formula:

C14H26O

IUPAC Name:

Cyclohexanecarboxaldehyde, 1-methyl-4-(4-methylpentyl)-, trans-

impurity 2

Reference substance name:

Cyclohexanecarboxaldehyde, 1-methyl-4-(4-methylpentyl)-, cis-

Cas Number:

76169-45-4

Molecular formula:

C14H26O

IUPAC Name:

Cyclohexanecarboxaldehyde, 1-methyl-4-(4-methylpentyl)-, cis-

impurity 3

Reference substance name:

1-methyl-4-(4-methylpentylidene)cyclohexanecarbaldehyde

Molecular formula:

C14H24O

IUPAC Name:

1-methyl-4-(4-methylpentylidene)cyclohexanecarbaldehyde

impurity 4

Reference substance name:

1-methyl-4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

EC Number:

257-942-2

EC Name:

1-methyl-4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbaldehyde

Cas Number:

52475-86-2

Molecular formula:

C14H22O

IUPAC Name:

1-Methyl-4-(4-methylpent-3-en-1-yl)cyclohex-3-ene-1-carbaldehyde

impurity 5

Reference substance name:

1-methyl-3-(4-methylpentylidene)cyclohexanecarbaldehyde

Molecular formula:

C12H24O

IUPAC Name:

1-methyl-3-(4-methylpentylidene)cyclohexanecarbaldehyde

Specific details on test material used for the study:

- Name of test material (as cited in study report): VERNALDEHYDE
- Description: colourless to pale yellow liquid
- Lot/batch No.: 9000505038
- Expiration date of the lot/batch: August 27, 2003
- Storage condition of test material: approx. 4°C

Method

Target gene:

- S. typhimurium: Histidine gene

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Preliminary test (without and with S9) all 5 strains: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment 1 (without and with S9) TA1535, TA1537, TA98, TA100 and TA102 (without S9): 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment 1 TA102 (with S9): 3, 10, 33, 100, 333 and 1000 µg/plate
Experiment 2 (without and with S9) all 5 strains: 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: methyl methane sulfonate in water deionised 4.0 µL/plate for TA102

Remarks:

without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine in DMSO, 10 µg/plate for TA98 and 50 µg/plate for TA1537

Remarks:

without S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: sodium azide in water deionised, 10 µg/plate for TA100 and for TA1535

Remarks:

without S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO, 2.5 µg/plate for TA98, TA100, TA1535, TA1537 and 10 µg/plate for TA102

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: exp.1 in agar (plate incorporation); exp.2 preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 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 than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such as an increase is not considered biologically relevant.

Statistics:

Not required.

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

Remarks:

Exp.1: TA1537 without S9 at 5000 µg/plate and TA102 with S9 at 333 and 1000 µg/plate. Exp.2: TA1537 without S9 at 333 µg/plate and with S9 at 1000-5000 µg/plate, TA1535 with S9 at 5000 µg/plate and TA100 with S9 at 1000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate. Except in strain TA102 with S9, toxicity was observed at the dose of 333 µg/plate and above.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The historical control data in experiment 1 were slightly above the historicalcontrol range in strain TA98 (with S9, negative control) and TA100 (with S9, negative and solvent control). Since these deviations are rather small, these effects are considered to be based upon biologically irrelevant fluctuations in the number of colonies and have no detrimental impact on the outcome of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Exp.1: TA1537 without S9 at 5000 µg/plate and TA102 with S9 at 333 and 1000 µg/plate.
Exp.2: TA1537 without S9 at 333 µg/plate and with S9 at 1000-5000 µg/plate, TA1535 with S9 at 5000 µg/plate and TA100 with S9 at 1000 µg/plate.

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay, performed according to OECD 471 and GLP principles.

Executive summary:

The genetic toxicity of the substance was assessed using Salmonella typhimurium TA98, TA100, TA102, TA1535 and TA1537 strains, in accordance with OECD 471 guideline and GLP principles. No precipitation was observed up to and including the top dose of 5000 µg/plate. Slight toxicity was observed in several strains.

All bacterial strains showed negative responses over the entire dose range, i.e. no biologically significant dose-related increase in the number of revertants in two independently repeated experiments with and without metabolic activation up to the highest concentration of 5000 µg/plate. Based on the results it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.