Reaction mass of 3,7-dimethyloct-7-en-1-yl 3-methylbut-2-enoate and 3,7-dimethyloctyl 3-methyl-2-butenoate and citronellyl 3-methylcrotonate

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:

From December 21, 1982 to January 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1983

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Citronellyl 3-methylcrotonate

EC Number:

244-019-4

EC Name:

Citronellyl 3-methylcrotonate

Cas Number:

20770-40-5

Molecular formula:

C15H26O2

IUPAC Name:

3,7-dimethyloct-6-en-1-yl 3-methylbut-2-enoate

Constituent 2

Reference substance name:

3,7-dimethyloctyl 3-methyl-2-butenoate

EC Number:

275-360-7

EC Name:

3,7-dimethyloctyl 3-methyl-2-butenoate

Cas Number:

71383-07-8

Molecular formula:

C15H28O2

IUPAC Name:

3,7-dimethyloctyl 3-methylbut-2-enoate

Constituent 3

Reference substance name:

3,7-dimethyloct-7-en-1-yl 3-methylbut-2-enoate

Cas Number:

74499-48-2

Molecular formula:

C15H26O2

IUPAC Name:

3,7-dimethyloct-7-en-1-yl 3-methylbut-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

A sample of the test product, a colourless liquid with a mild flavour, designated Sinodor (X-09648, 13.12.82), was received from the sponsor on December 21, 1982. The test material was dissolved in ethanol just prior to use.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9
S9 mix is prepared by mixing the thawed S9 with a NADPH generating system. The final concentrations of the various ingredients in the S9 mix are:
MgCl2 8 mM
KCl 33 mM
G6P 5 mM
NADP 4 mM
Sodium phosphate pH 7.4 100 mM
S9 10%
The S9 mix is prepared just prior to use with cold sterile solutions and is kept in ice water before and during use

Test concentrations with justification for top dose:

A preliminary test was carried out to assess the chemical toxicity of the test substance for the bacteria. The results show that 10 mg of the test substance per plate was slightly toxic whereas 1 mg per plate did not show a growth-inhibiting effect. However, at a dose of 1 mg per plate, the compound precipitated in the plate. Therefore 500 micrograms per plate was chosen as the highest dose level for the mutagenicity study.
Appropriate test solutions in ethanol containing 0, 0.06, 0.19, 0.56, 1.67 and 5.00 mg/ml were prepared immediately before use.

Vehicle / solvent:

Ethanol

Controls

Controls 1

Positive controls:

yes

Positive control substance:

sodium azide

Controls 2

Positive controls:

yes

Positive control substance:

other: hycanthone methanesulphonate (12.5 micrograms per 0.1 ml water per plate) for strains TA 1537, TA 1538 and TA 98 without S9 mix

Controls 3

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (0.5 micrograms per 0.1 ml DMSO per plate) for TA 1535, TA 1538, TA 98, TA 100 in the presence of S9 mix. For TA 1537 2.0 micrograms per plate was used in the presence of S9 mix.

Details on test system and experimental conditions:

Preparation and storage of stock cultures:
A fresh culture of each strain is prepared by inoculating nutrient broth with bacteria of the strain in question and incubating the broth for 16h (overnight) at 37°C while shaking. The cultures are then mixed with DMSO to a final DMSO concentration of 8.75%. Subsequently 0.5 ml portions are pipetted into sterile polypropylene vials which are then quickly frozen on dry ice and stored at -80°C.
Part of each culture is retained and examined for the number of spontaneous revertants, histidine requirement and sensitivity to ampicilline, crystal violet and UV radiation. In addition the reversion induced by reference mutagens is determined.

Preparation of cultures for the tests:
To obtain a culture for the mutagenicity test, nutrient broth is inoculated with a thawed aliquot of the stock culture in question (0.1 ml per 10 ml nutrient bouillon). The broth is incubated for 16h (overnight) at 37°C while shaking. The viable count of each culture is determined by plating appropriate dilutions of the culture on nutrient broth agar plates. The cultures are subsequently stored in a refrigerator at 5°C until use, but no longer than 5 days.

Evaluation criteria:

A positive response in the assay system is taken to be a two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed with the solvent, together with evidence of a dose response.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

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

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

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

Remarks on result:

other: High concentration based on precipitation.

Applicant's summary and conclusion

Conclusions:

It is concluded that SINODOR does not show any mutagenic activity in the Salmonella/mammalian microsome mutagenicity test under the conditions employed in this evaluation.

Executive summary:

Sinodor was examined for mutagenic activity in the Ames test using the histidine requiring S.typhimurium mutants TA 1535, TA 1537, TA 1538, TA98 and TA100 as indicator strains and a liver microsome fraction of Aroclor-induced rats for metabolic activation.

It is concluded that SINODOR does not show any mutagenic activity in the Salmonella/mammalian microsome mutagenicity test under the conditions employed in this evaluation.