Methyl 2,3-dibromopropionate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2019-01-03 to 2019-01-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed to current OECD guidelines with no significant deviations and run in OECD GLP certified lab.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

In tester strain TA100 in absence of S9-mix no acceptable responses of the solvent control could be obtained, however due to the positive results observed in all other tester strains this has no effect on the study outcome.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 2,3-dibromopropionate

EC Number:

217-044-3

EC Name:

Methyl 2,3-dibromopropionate

Cas Number:

1729-67-5

Molecular formula:

C4H6Br2O2

IUPAC Name:

methyl 2,3-dibromopropanoate

Test material form:

liquid

Specific details on test material used for the study:

Purity: 98%
Batch No.: 800327680

Method

Target gene:

histidine locus and tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

Dose-range Finding Test: TA100 and WP2uvrA, both with and without S9-mix, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate.
Mutation Assay: TA1535, TA1537 and TA98, without S9-mix, 0.17, 0.54, 1.7, 5.4, 17, 52 and 164 μg/plate; TA1535, TA1537 and TA98, with S9-mix, 1.7, 5.4, 17, 52, 164 and 512 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (dimethyl sulfoxide)

- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test item formed a clear to light yellow solution in DMSO.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For TA1535, without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

For TA1537, without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

For TA98, without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

For TA100, without metabolic activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

For WP2uvrA, without metabolic activation

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

For TA1535, TA1537, TA98, TA100 and WP2uvrA, with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate in each strain for dose-range finding test and mutation assay
- Number of independent experiments: 1


METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E+09 cells/mL
- Test substance added in agar (plate incorporation)


TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

not determined

Remarks:

as no acceptable response of the solvent control could be obtained

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose-range Finding Test/First Mutation Experiment
- Precipitate: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain.
- Toxicity: Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strains TA100 in absence of S9-mix.
- Mutagenicity: In tester strain WP2uvrA in the absence and presence of S9-mix, the test item induced an up to 2.6- and 6-fold dose-related increase in the number of revertant colonies compared to the solvent control, respectively. In tester strain TA100 in the presence of S9-mix, the test item induced an up to 6.2-fold dose-related increase in the number of revertant colonies compared to the solvent control, respectively. In tester strain TA1535, the test item induced up to 4.6- and 5.3-fold dose-related increase in the number of revertant colonies in the absence and presence of S9-mix, respectively. In tester strain TA1537, the test item induced up to 3.5- and 3-fold dose-related increase in the number of revertant colonies in the absence and presence of S9-mix, respectively. In tester strain TA98, the test item induced up to 8.5- and 3.4-fold dose-related increase in the number of revertant colonies in the absence and presence of S9-mix, respectively.

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

This study was to determine the potential of the test item and/or its metabolites to induce reverse mutations according to OECD Guideline 471 (Bacterial Reverse Mutation Assay).

Strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA were treated with the test substance in the presence or absence of an exogenous mammalian metabolic activation system (S9).

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or presence of microcolonies, was observed in tester strain TA100 in the presence of S9-mix and in tester strain WP2uvrA absence of S9-mix.

In tester strain WP2uvrA in the absence and presence of S9-mix, the test item induced an up to 2.6- and 6-fold dose-related increase in the number of revertant colonies compared to the solvent control, respectively. The increases observed were above the laboratory historical control data range and more than two-fold the concurrent solvent control.

In tester strain TA100 in the presence of S9-mix, the test item induced an up to 6.2-fold dose-related increase in the number of revertant colonies compared to the solvent control, respectively. The increases observed were above the laboratory historical control data range and more than two-fold the concurrent solvent control.

Based on the results of the dose-range finding test, the test item was tested in the first assay at a concentration range of 0.17 – 164 μg/plate and 1.7 – 512 μg/plate in absence and presence of S9-mix, respectively, in the tester strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain TA98 in presence of S9-mix.

In tester strain TA1535, the test item induced up to 4.6- and 5.3-fold dose-related increase in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were above the laboratory historical control data range and more than three-fold the concurrent solvent control.

In tester strain TA1537, the test item induced up to 3.5- and 3-fold dose-related increase in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were three-fold or more of the concurrent solvent control but within the laboratory historical control data range.

In tester strain TA98, the test item induced up to 8.5- and 3.4-fold dose-related increase in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were above the laboratory historical control data range and more than three-fold the concurrent solvent control.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Except in tester strain TA100 in absence of S9-mix were no acceptable responses of the solvent control could be obtained.

 

In conclusion, based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.