Reaction mass of [2,2,4-trimethyl-1-(2-methylpropanoyloxy)pentan-3-yl] benzoate and [2,2,4-trimethyl-3-(2-methylpropanoyloxy)pentyl] 2-methylpropanoate and (3-benzoyloxy-2,2,4-trimethylpentyl) benzoate

Genetic toxicity: in vitro

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

Test material information

Constituent 1

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

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

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 / 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:

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 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:

valid

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:

valid

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:

valid

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:

valid

Positive controls validity:

valid

Test results 5

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.