Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 September 2008 - 23 December 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethoxycarbonylmethyl ethyl phthalate
EC Number:
201-555-3
EC Name:
Ethoxycarbonylmethyl ethyl phthalate
Cas Number:
84-72-0
Molecular formula:
C14H16O6
IUPAC Name:
ethyl 2-[(2-ethoxy-2-oxoethoxy)carbonyl]benzoate
Test material form:
liquid
Remarks:
clear, colourless, slightly viscous liquid
Details on test material:
Batch number: N-00103
Test material received: 01 September 2008

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Preliminary Toxicity test: 0 / 0.15 / 0.5 / 1.5 / 5 / 15 / 50 / 150 / 500 / 1500 / 5000 µg/plate
Experiment 1 - Range finding Test: 50 / 150 / 500 / 1500 / 5000 µg/plate
Experiment 2 - Main Test: 50 / 150 / 500 / 1500 / 5000 µg/plate
Vehicle / solvent:
Dimethyl sulphoxide
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: Benzo(a)pyrene, 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- in agar (plate incorporation)
- all tester strain cultures in the range of 1-9.9 x 10^9 bacteria per mL


NUMBER OF REPLICATIONS:
The test concetrations of the test material were assayed in triplicates against each tester strain.

Experiment 1 and 2:
Measured aliquotes (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 contens of each test tube were mixed and equally distributed onto the surface of Vogler- Bonner Minimal agar plates (one tube per plate). this procedure was repeated, in triplicate, for each bacterial strain and for each concetration 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.



Evaluation criteria:
Positive result: Dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

Negative result: A test material will be considered non-mutagenic (negative) if the criteria for positive result are not met.

If no clear positive or negative results are generated, the results are reported as equivocal.


The Test is 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 (within historical control ranges).
- 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 range of 1-9.9 x 10^9 bacteria per mL.
- Each mean positive control value should be at least twice 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.
- There should be a minimum of four non-toxic test material dose levels.
- There should be no evidence of excessive contamination.

Results and discussion

Test resultsopen allclose all
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:
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:
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
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 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

Applicant's summary and conclusion

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

Bacterial Reverse Mutation Assay (using Salmonella typhimurium and Escherichia coli) with ethoxycarbonylmethyl ethyl phthalate

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli 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 test item was dissolved in dimethyl sulfoxide (DMSO). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a seperate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (DMSO) 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 or 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 on 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. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9 -mix. 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.

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