Dimethyl 3-oxoglutarate

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:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl 3-oxoglutarate

EC Number:

217-385-8

EC Name:

Dimethyl 3-oxoglutarate

Cas Number:

1830-54-2

Molecular formula:

C7H10O5

IUPAC Name:

dimethyl 3-oxoglutarate

Details on test material:

- Name of test material (as cited in study report): LZ409, Batch No. 46332
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance):
- Substance type: organic
- Physical state: yellow liquid
- Analytical purity:
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.:
- Expiration date of the lot/batch:
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions:
- Storage condition of test material:
- Other:

Method

Target gene:

Histidine

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:

Aroclor induced / rat liver S9

Test concentrations with justification for top dose:

Toxicity study
17, 50, 167, 500, 1667 and 5000 microgram/plate

Mutation tests
17, 50, 167, 500, 1667 and 5000 microgram/plate

Quality control
- DMSO vehicle was tested at 0.1 ml/plate in both the presence and absence of S9 mix
- With S9 mix:
2-Aminoanthracene (2-AAN) was tested at 20 mikrogram/plate with E. Coli,
2 mikrogram/plate with TA 1535 and TA 1537 and
0.5 microgram/plate with TA 98 and TA 100.

- Without S9 mix:
N-Ethyl-N-nitro-N-nitrosoguanidine (ENNG) wastested at 2 mikrogram/plate with E. Coli.
Sodium azide was tested at 1 mikrogram/plate with TA 1535 and TA 100
2-nitrofluorene (2-NF) was tested at 1 mikrogramplate with TA 98
9-Aminoacridine (9-AA) was tested at 80 mikrogram/plate with TA 1537.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

sterile, ultra pure water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-ANN)

Controls 2

Untreated negative controls:

yes

Remarks:

sterile, ultra pure water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: NaN3

Controls 3

Untreated negative controls:

yes

Remarks:

sterile, ultra pure water

Negative solvent / vehicle controls:

no

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: 9-AA

Controls 4

Untreated negative controls:

yes

Remarks:

sterile, ultra pure water

Negative solvent / vehicle controls:

no

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Migrated to IUCLID6: 2-NF

Controls 5

Untreated negative controls:

yes

Remarks:

sterile, ultra pure water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Migrated to IUCLID6: ENNG

Details on test system and experimental conditions:

Direct Plate Method
Volumes of soft agar (2 ml) were dispensed into small sterile tubes. To this, 0.5 ml of S9 mix or 0.05 M phosphate buffer, pH: 7.4 were added, followed by 0. 1ml of bacteria. The solvent of the test solution (0.1 ml) was added last.
The tube contents which were continually cooling, were mixed and poured onto minimal medium plates, prepared in-house. These plates contained 25 ml of 1.5 % purified agar in Vogel-Bonner medium E with 2 % glucose. When the soft agar had set, the plates were inverted and incubated at 37 C for 2 or 3 days.

The Pre-Incubation method
Volumes of S9 mix or 0.05 M phosphate buffer, pH 7.4 (0.5 ml) were dispensed into small sterile tubes. This was followed by 0.1 ml of bacetria per tube and, finally the solvent or test solution (0.1 ml per tube). The tube tops were then screwed on tightly and the tubes placed in a shaking incubator at 37 C for 20 min. After this the tube tops were removed and 2 ml of soft agar added to each tube. The tube contents which were continually cooling, were mixed and then poured onto agar plates, as above. Than the soft agar had set, the plates were inverted and incubated at 37 C for 2 or 3 days.
After incubation, the colonies were counted using a Cardinal Colony counter (Perceptive Instruments, UK) set at maximum sensitivity, i.e. colonies of 0.1 mm or more in diameter were counted. The plates were also examined for precipitates and microscopically for microcolony growth.

Evaluation criteria:

Well established, according to Guideline.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity to the bacteria was observed as a thinning of the background lawn of microcolonies in the second mutation assay only (pre-incubation method).
Toxicity was observed with all 5 strains at 5000 mikrogramm/per plate, in the presence of S9 mix only.
Precipitation of the test item was observed only in the second mutation assay at 5000 mikrogramm/plate in the presence of S9 mix.

All tests were acceptable according to the study criteria.

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

The substance did not induce mutagenic activity in any of the 5 bacterial strains used, in either activation condition.