Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and test guideline compliant study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
liquid
Details on test material:
Sponsor's Identification: Linalyl Isobutyrate
Description: Colourless liquid
Chemical name: 3,7-Dimethyl-1,6-octadien-3-yl 2-methyl propanoate
Storage conditions: Approximately 4 °C in the dark

Method

Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell lines (if applicable):
Not applicable
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-fraction; S9 fraction prepared from liver homogenates of male Sprague-Dawley rats induced with three consecutive daily oral doses of phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day)
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
- 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA100 strain, with and without S9 mix using the direct plate incorporation method

Mutation Test- Experiment 1 (Range-finding Test):
- 100, 333, 1000, 2500 and 5000 µg/plate in all tester strains, with and without S9 mix using the direct plate incorporation method

Mutation Test- Experiment 2 (Main Test):
- 100, 333, 1000, 2500 and 5000 µg/plate in all tester strains, with and without S9 mix using the direct plate incorporation method

Vehicle:
Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in DMSO at the same concentration in solubility checks performed in-house. DMSO was therefore selected as the vehicle.
- Test substance preparation: The test material was accurately weighed and approximate half-log dilutions prepared in DMSO by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (1.6 %) of the test material. Prior to use, the solvent was dried using molecular sieves (sodium alumino-silicate) i.e., 2 mm pellets with a nominal pore diameter of 4 x 10^-4 microns.
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate for TA100 and 5µg/plate for TA1535; 9-Aminoacridine: 80 µg/plate for TA1537; 4-Nitroquinoline-1-oxide: 0.2 µg/plate for TA98; Mitomycin C: 0.5 µg/plate for TA102
Remarks:
without S9-mix
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537; Benzo(a)pyrene: 5 µg/plate for TA98; 1,8-dihydroxyanthraquinone: 10 µg/plate for TA102
Remarks:
with S9-mix
Details on test system and conditions:
SOURCE OF TEST SYSTEM: Salmonella typhimurium strains were obtained from the University of California at Berkeley on culture discs on 4 August 1995 and were stored at -196°C in a Statebourne liquid nitrogen freezer, model SXR 34.

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Incubation period: Plates were incubated at 37 °C for approximately 48 h

NUMBER OF REPLICATIONS:
- Preliminary Toxicity Test: Single plate/dose for treatment and vehicle control
- Experiment 1 (Range-finding Test) and Experiment 2 (Main Test): 3 plates/dose for treatment, vehicle and positive controls

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was determined by growth of the bacterial background lawn.

OTHER:
- Revertant colonies were counted using a Domino colony counter.
Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression (5)) significant increase in the revertant count in at least one strain of bacteria.
Statistics:
no data

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:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Under the test conditions, Linalyl Isobutyrate is not considered as mutagenic in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 & TA 102 strains.