2-ISOPROPYL-1,3-DIOXANE-5,5-DICARBOXYLIC ACID DIETHYL ESTER

Genetic toxicity: in vitro

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:

2002

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ISOPROPYL-1,3-DIOXANE-5,5-DICARBOXYLIC ACID DIETHYL ESTER

EC Number:

609-063-4

Cas Number:

35113-48-5

Molecular formula:

C13H22O6

IUPAC Name:

2-ISOPROPYL-1,3-DIOXANE-5,5-DICARBOXYLIC ACID DIETHYL ESTER

Test material form:

other: Liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other:

Species / strain 2

Species / strain / cell type:

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

Test concentrations with justification for top dose:

The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

dimethyl sulphoxide

Controls

Untreated negative controls:

yes

Remarks:

spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

Mutation Study - Experiment 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation Study - Experiment 2
The second experiment was performed using methodology as described for Experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 µg/plate).

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. Acceptable ranges are presented in the standard test method section 3 with historical control ranges for 1999 and 2000 in Appendix 1.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.

Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. The historical control ranges for 1999 and 2000 are presented in Appendix 1.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

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.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction.The method was designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods.Salmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 andEscherichia colistrain WP2uvrA^-were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results.The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A slight, oily precipitate was observed under an inverted microscope only at 5000 µg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.The test material was considered to be non-mutagenic under the conditions of this test.