Registration Dossier

Diss Factsheets

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:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
In the pre - incubation assay in the tester strain TA100, the test item solutions were incubated for 29 minutes. The deviation had no effect on the study integrity.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-benzoyloxy-2,2,4-trimethylpentyl isobutyrate
EC Number:
245-054-8
EC Name:
3-benzoyloxy-2,2,4-trimethylpentyl isobutyrate
Cas Number:
22527-63-5
Molecular formula:
C19H28O4
IUPAC Name:
[2,2,4-trimethyl-1-(2-methylpropanoyloxy)pentan-3-yl] benzoate
Constituent 2
Chemical structure
Reference substance name:
2,2,4-trimethylpentane-1,3-diyl dibenzoate
EC Number:
268-316-3
EC Name:
2,2,4-trimethylpentane-1,3-diyl dibenzoate
Cas Number:
68052-23-3
Molecular formula:
C22H26O4
IUPAC Name:
(3-benzoyloxy-2,2,4-trimethylpentyl) benzoate
Constituent 3
Chemical structure
Reference substance name:
1-isopropyl-2,2-dimethyltrimethylene diisobutyrate
EC Number:
229-934-9
EC Name:
1-isopropyl-2,2-dimethyltrimethylene diisobutyrate
Cas Number:
6846-50-0
Molecular formula:
C16H30O4
IUPAC Name:
[2,2,4-trimethyl-3-(2-methylpropanoyloxy)pentyl] 2-methylpropanoate
Test material form:
liquid
Details on test material:

- Density: 1.027
- Moisture content: 0.02%
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Eastman Chemical Company, lot V046212201
- Expiration date of the lot/batch: 30 April 2014
- Purity test date: 07 May 2012
- Storage conditions: Room temperature in dark

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and the WP2uvrA, both with and without S9-mix. Ten concentrations 0, 0.15, 0.5, 1.5, 5, 15, 50,150, 500, 1500 and 5000 μg/plate were tested in triplicate.
Vehicle / solvent:
The vehicle of the test item was dimethyl sulfoxide.
Controls
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation methods at five dose concentrations

DURATION
- Preincubation period: 20 minutes
- Exposure duration:48 h

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
There are several criteria determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study.
1. A dose related increase in mutant frequency over the dose range tested
2. A reproducible increase at one or more concentrations
3. Biological relevance against in-house historical control ranges
4. Statistical analysis of data determined by UKEMS
5. Fold increase greater than two times the concurrent solvent control for any tester strain
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. If no conclusion on mutagenicity can be made the result will be reported as equivivocal

Results and discussion

Test resultsopen allclose all
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (oily in appearance) was noted at and above 1500ug//plate, this observation did not prevent scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA) in all concentrations tested (0 - 5000ug/plate).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: All positive control chemicals used in the test induced marked increase in the frequency of revertant colonies thus confriming the activity of the S9 mix and the sensititivity of the bacterrial strains.
- Negative (solvent/vehicle) historical control data: Results for the negative control (spontaneous mutation rates) were considered to be acceptable.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed at any dose level tested up to the maximum dose level of 5000ug/plate

Applicant's summary and conclusion

Conclusions:
All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. 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. The test item, Reaction Mass of 1-isopropyl-2,2 dimethyltrimethylene diisobutyrate and 3-benzoyloxy-2,2,4-trimethyl pentyl isobutyrate and 2,2,4-trimethylpentane-1,3-diyl dibenzoate, was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The objective of this study was to determine the potential of the test substance and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli(E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. The study procedures described in this report were based on the most recent OECD and EC guidelines. The test item was dissolved in dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item precipitate was noted at and above 1500ug/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the first mutation experiment, the test item was tested up to concentrations of 5000μg/plate in the strains TA1535, TA1537, TA98, TA100 and WPuvrA. The test item precipitate was noted at and above 1500ug/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test item was tested up to concentrations of 5000μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item precipitate was noted at and above 1500ug/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

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.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four 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.

In conclusion, based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.