Registration Dossier

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:
from 2012-09-28 to 2012-11-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study without deviations and test procedure according to GLP standards

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his (Salmonella), trp (E. coli)
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Doses selected for the preliminary test: 1.2; 4.9; 20; 78; 313; 1250; and 5000 µg/plate
Doses selected for all strains both with and without metabolic activation in the main test. : 10; 20; 39; 78; 156; 313 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: from the result of the solubility test, the test substance was insoluble at 50 mg/mL in water, and was dissolved at 50 mg/mL in DMSO and at 100 mg/mL in acetone. And neither exothermic reaction nor generation of gas was observed.
Controlsopen allclose all
Negative controls:
yes
Remarks:
treated with only the DMSO used to prepare the test solution
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
Remarks:
Positive control for: strain TA100 without metabolic activation at 0.01 µg/plate, strain WP2 uvrA without metabolic activation at 0.01 µg/plate and strain TA98 without metabolic activation at 0.1 µg/plate.
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive control for strain TA1535 without metabolic activation at 0.5 µg/plate
Positive controls:
yes
Positive control substance:
other: 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine.2HCl (ICR-191)
Remarks:
Positive control for strain TA1537 without metabolic activation at 1.0 µg/plate
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
Positive control for strain TA1535 with metabolic activation at 2.0 µg/plate and for strain WP2 uvrA with metabolic activation at 10 µg/plate.
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Positive control for strain TA100 with metabolic activation at 5.0 µg/plate, for strain TA98 with metabolic activation at 5.0 µg/plate and for strain TA1537 at 5.0 µg/plate.
Details on test system and conditions:
Pre-culture procedure:
A bacterial suspension of each strain (20 µL of S. typhimurium TA strains, 5 µL of E.coli WP2 uvrA) was inoculated into an L-form culture tube (35 mL capacity) containing 10 mL of Nutrient Broth. This culture tube was left at 4 °C until strating incubation, and then incubated while shacking (100 rpm) in a water bath at 37 °C for 8 hours. After incubation, the optical density was measured and the number of viable cells was calculated by growth curve for each strain. The bacterial cultures were stored at room temperature until strarting of the test.

Test procedures: preincubation method
Evaluation criteria:
In the two main tests, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on teice as many as that of the negative control), and dose-response and reporoducibility were also observed, the test substance was judged to be positive. The results at each concentration were demonstrated with the mean and the standard deviation.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
growth inhibition was observed at 313 µg/plate in all strains
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
In the two main tests, neither an increase of revertant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.
The revertant colonies of the positive control showed an increase of more than twice that of the negative controls and they were within limits of controls (mean ± 3SD) in historical data, indicating that this study was performed correctly.
From these results, mutagenicity of the test substance was judged negative.
The growth inhibition of the test strains by the test substance was observed was observed at the doses marked with "*" in the tables. And the precipitate of the test substance on the plates was not observed either with or without metabolic activation.

Any other information on results incl. tables

The results are shown in tables 1 to3 and figures 1 and 2. The figures are based on table 3

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

There was no evidence of induced mutant colonies over background in any of the tester strains in the presence or absence of mammalian metabolic
activation in this study.
Executive summary:

Mutagenicity potential of 3,6 -dimethylheptan-2 -ol was assessed with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Eschrichia coli WP2 uvrA. In this study, neither an increase of revertant colonies more than twice in comparison with that of the negative control nor a dose-related response was observed in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.

From the above, it is judged 3,6 -dimethylheptan-2 -ol has no mutagenicity forward to bacteria under the described study conditions.