Acetic acid, esters with turpentine-oil myrcene fraction terpene alcs.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 25, 2007 - November 3, 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

(2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

p-menth-1-en-8-yl acetate

EC Number:

201-265-7

EC Name:

p-menth-1-en-8-yl acetate

Cas Number:

80-26-2

Molecular formula:

C12H20O2

IUPAC Name:

1-methyl-1-(4-methylcyclohex-3-en-1-yl)ethyl acetate

Constituent 2

Reference substance name:

Myrcenyl acetate

EC Number:

214-262-0

EC Name:

Myrcenyl acetate

Cas Number:

1118-39-4

Molecular formula:

C12H20O2

IUPAC Name:

1,1-dimethyl-5-methylenehept-6-en-1-yl acetate

Constituent 3

Reference substance name:

(E)-2-(3,3-dimethylcyclohexylidene)ethyl acetate

EC Number:

308-183-1

EC Name:

(E)-2-(3,3-dimethylcyclohexylidene)ethyl acetate

Cas Number:

97890-04-5

Molecular formula:

C12H20O2

IUPAC Name:

(E)-2-(3,3-dimethylcyclohexylidene)ethyl acetate

Constituent 4

Reference substance name:

(Z)-2-(3,3-dimethylcyclohexylidene)ethyl acetate

EC Number:

308-184-7

EC Name:

(Z)-2-(3,3-dimethylcyclohexylidene)ethyl acetate

Cas Number:

97890-05-6

Molecular formula:

C12H20O2

IUPAC Name:

(Z)-2-(3,3-dimethylcyclohexylidene)ethyl acetate

Constituent 5

Reference substance name:

Geranyl acetate

EC Number:

203-341-5

EC Name:

Geranyl acetate

Cas Number:

105-87-3

Molecular formula:

C12H20O2

IUPAC Name:

(E)-3,7-dimethylocta-2,6-dien-1-yl acetate

Constituent 6

Reference substance name:

1-methyl-4-(1-methylethylidene)cyclohexyl acetate

EC Number:

233-564-3

EC Name:

1-methyl-4-(1-methylethylidene)cyclohexyl acetate

Cas Number:

10235-63-9

Molecular formula:

C12H20O2

IUPAC Name:

4-isopropylidene-1-methylcyclohexyl acetate

Constituent 7

Reference substance name:

4-(5-methyl-1-methylene-4-hexenyl)-1-(4-methylpent-3-enyl)cyclohexene

EC Number:

208-546-3

EC Name:

4-(5-methyl-1-methylene-4-hexenyl)-1-(4-methylpent-3-enyl)cyclohexene

Cas Number:

532-87-6

Molecular formula:

C20H32

IUPAC Name:

4-(5-methyl-1-methylenehex-4-en-1-yl)-1-(4-methylpent-3-en-1-yl)cyclohexene

Constituent 8

Reference substance name:

cis-1-methyl-4-(1-methylvinyl)cyclohexyl acetate

EC Number:

244-028-3

EC Name:

cis-1-methyl-4-(1-methylvinyl)cyclohexyl acetate

Cas Number:

20777-47-3

Molecular formula:

C12H20O2

IUPAC Name:

4-isopropenyl-1-methylcyclohexyl acetate

Constituent 9

Reference substance name:

p-menth-1-en-8-ol

EC Number:

202-680-6

EC Name:

p-menth-1-en-8-ol

Cas Number:

98-55-5

Molecular formula:

C10H16O

IUPAC Name:

2-(4-methylcyclohex-3-en-1-yl)propan-2-ol

Constituent 10

Reference substance name:

Neryl acetate

EC Number:

205-459-2

EC Name:

Neryl acetate

Cas Number:

141-12-8

Molecular formula:

C12H20O2

IUPAC Name:

(Z)-3,7-dimethylocta-2,6-dien-1-yl acetate

Constituent 11

Reference substance name:

1-(3,3-dimethylcyclohex-1-en-1-yl)ethyl acetate

Cas Number:

150461-96-4

Molecular formula:

C12H20O2

IUPAC Name:

1-(3,3-dimethylcyclohex-1-en-1-yl)ethyl acetate

Constituent 12

Reference substance name:

Tentative monoterpene diacetate

IUPAC Name:

Tentative monoterpene diacetate

Constituent 13

Reference substance name:

Tentative oxygenated monoterpene acetate

IUPAC Name:

Tentative oxygenated monoterpene acetate

Constituent 14

Reference substance name:

Tentative Diterpene hydrocarbon

IUPAC Name:

Tentative Diterpene hydrocarbon

Constituent 15

Reference substance name:

Unresolved/non-elucidated mixed terpene/diterpene/oxygenated terpene/terpene acetate derivatives

IUPAC Name:

Unresolved/non-elucidated mixed terpene/diterpene/oxygenated terpene/terpene acetate derivatives

Test material form:

liquid

Specific details on test material used for the study:

Sponsor's identification: Neobergamate Forte
Description: Pale yellow liquid
Batch number: 867284455
Date received: 14 September 2007
Storage conditions: Room temperature in the dark

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

- Preliminary test:
TA100 and WP2uvrA:
Without and with S9-mix: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- Experiment 1:
TA 1535, TA 1537, TA 98 and TA 100:
Without and with S9-mix: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
E. coli WP2 uvr A:
Without and with S9-mix: 50, 150, 500, 1500 and 5000 µg/plate
- Experiment 2:
TA 1535, TA 1537, TA 98 and TA 100:
Without and with S9-mix: 15, 50, 150, 500, 1500 and 5000 µg/plate
E. coli WP2 uvr A:
Without and with S9-mix: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle: acetone
- Justification for choice of vehicle: the test material was immiscible in DMSO at 50 mg/mL but was fully miscible in acetone at the same concentration. Sterile distilled water was not selected following information supplied by the sponsor. Acetone was therefore selected.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Preliminary test: 1
- Experiment 1 and 2: 3

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn and the reduction of the revertant colonies.

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

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-releated increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evalaution, however, statistical significance will not be the only determining factor for a positive respons.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Kirkland DJ (Ed) (1989). Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report - Part III, Cambridge University Press.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(while tested up to limit concentrations)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 1500 μg/plate and above in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA 1535, TA 1537, TA 98 and TA 100: toxicity was observed at dose levels of 1500 μg/plate and above
WP2uvrA: No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Applicant's summary and conclusion

Conclusions:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments, both in the absence and presence of S9-mix. The substance was tested up to limit concentrations. Adequate negative, solvent and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.