Registration Dossier

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:
28 February 2002 to 1 August 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Diisopropyl succinate
EC Number:
213-110-0
EC Name:
Diisopropyl succinate
Cas Number:
924-88-9
Molecular formula:
C10H18O4
IUPAC Name:
diisopropyl succinate
Details on test material:
Name "DIISOPROPYL SUCCINATE".
Chemical name: Bernsteinsäurediisopropylester.
Trading name DIPS.
Molecular formula: C10H18O4.
Supplier: Sponsor.
Batch No.: EPLS 301.
CAS No.: 924-88-9.
Appearance: Clear, colourless liquid.
Density: 0.9847 g/cm3 at 20 °C.
Boiling point: 247 °C.
Solubility: 25 g/L at 20 °C.
Conditions of storage: In the refrigerator, in the dark, may be used under light.
Stability under conditions of storage: Until December 2002.
Date of expiry: 31 December 2002.

Method

Target gene:
his- (s. typhimurium)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA97a
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
5000, 1667, 556, 185 and 62 µg/plate
Vehicle / solvent:
DMSO
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylene-diamine; t-Butyl-hydroperoxide; 2- Aminoanthracene; 1,8-Dihydroxy-anthraquinone; 7,12-Dimethylbenz[a]anthracene; 2-Nitrofluorene; Sodium acide
Details on test system and experimental conditions:
Bacterial strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535, obtained from Prof. Bruce N. Ames, Berkeley, California, were used.
The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.
The actual batch of the strains was tested for ampicillin resistance (TA102: ampicillin/tetracycline resistance), UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances in September 2000. The bacteria were stored frozen since that time.

The test substance was tested without as well as with an external metabolising system (S9 mix). The results were verified by a second, independent experiment.
For metabolic activation (S9-Mix) the microsomal fraction of homogenised livers of rats treated once with 500 mg/kg of Aroclor 1254 was used. The preparation was done in September 2001. Four days after treatment, the feed was withdrawn for one night. The livers of the animals were removed and homogenised in cold 0.15 mol/l KCl. Three mL of homogenate were obtained per gram of liver. Then the homogenate was centrifuged for 10 minutes at 9000 x g. The supernatant contained the microsomes. Small portions of the microsomes were stored in liquid nitrogen. Immediately before use they were thawed and mixed with the cofactor solution. The metabolic activity of the microsomes was verified by the positive control substances of each study.

The exposure was performed according to the 'Plate Incorporation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state. The number of viable cells in the overnight-cultures is in the range of 2 x 108 cells per mL.
For each sample the following solutions were combined:
0.1 mL of the overnight culture of the bacteria,
0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
0.1 mL of the appropriate test- or reference substance solution and
2 mL of top agar.
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).
The plates with less than about 50 revertant colonies, i.e. the plates of TA98 and TA1535 with the exception of the positive controls, were counted visually by marking the colonies with a felt tipped pen. The other plates were photographed with a video camera and the picture files were scanned for colonies by a computer program.
Evaluation criteria:
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2½ fold of the amount of the spontaneous revertants.
For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1 2/3 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of our historic data of the Ames test.
Statistics:
Means and standard deviation were calculated for the number of mutants in every concentration group.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
only reduced numbers of revertants at 5000 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
only reduced numbers of revertants at 5000 µg/plate
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation of the test substance was seen in any of the concentration groups.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

According to the results obtained in this study, "DIISOPROPYL SUCCINATE" is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 up to 5000 µg/plate which is the limit concentration for this kind of test.
Executive summary:

Method

"DIISOPROPYL SUCCINATE" was tested for mutagenic activity with the"Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD-guideline 471 and directive 2000/32/EC, part B.13/14.

The test substance, dissolved in DMSO, was tested at concentrations ranging from 62 µg to 5000 µg per plate according to the"direct plate incorporation method" without external metabolisation as well as with an external metabolising system (S9-mix). As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included. An independent repetition of the experiment was performed.

 

Results

Positive controls:

All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system.

 

Test substance:

Toxicity:

No toxicity to the bacterial background lawn was seen up to 5000 µg/plate. In some samples with 5000 µg/plate reduced numbers of revertants were found.

 

Solubility:

No precipitation of the test substance was seen in any of the concentration groups.


Mutagenicity:

In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results.

  

Conclusion

According to the results obtained in this study,"DIISOPROPYL SUCCINATE" is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 up to 5000 µg/plate which is the limit concentration for this kind of test.