Diethyl succinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 26,2010 to June 22,2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl succinate

EC Number:

203-419-9

EC Name:

Dimethyl succinate

Cas Number:

106-65-0

Molecular formula:

C6H10O4

IUPAC Name:

dimethyl butanedioate

Details on test material:

- Name of test material:Dimethyl succinate
- Molecular formula :C6H10O4
- Molecular weight : 146.14 g/mol
- Smiles notation : O=C(OC)CCC(=O)OC
- Physical state:clear , colourless liquid
- Analytical purity:99.81 %
- Batch number:T14B208/335
- Expiration date of the lot/batch:2010-12-01
- Storage condition of test material:room temperature, protected from moisture and light
- Density: 1.1190 g/cm^3 (20°C)
- Water solubility: 102 g/L (25°C)
- Melting point 19°C
- Boiling point : 196°C

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK+/- (Clone 3.7.2C) mouse lymphoma cells from American Type Culture Collection, Rockville, Maryland. The generation time and mutation rates (spontaneous and induced) are checked in this laboratory. The cells are checked at regular intervals for the absence of mycoplasmal contamination.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Two independent assays for mutation to trifluorothymidine resistance were performed using the dose levels described in the following table:

Assay No.: S9 Treatment Dose level (µ/ml)
Time (hours)
1 - 3 1460, 730, 365, 183 and 91.3
1 + 3 1460, 730, 365, 183 and 91.3
2 - 24 1460, 730, 365, 183 and 91.3
2 + 3 1460, 973, 649, 433, 288 and 192

Vehicle / solvent:

- Solvent used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: in the absence of S9 metabolism

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: in the presence of S9 metabolism

Details on test system and experimental conditions:

Preparation of test cell cultures

A cell suspension (10^6 cells/ml) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes.
The treatment media were prepared as follows:

Without S9 metabolism – 3-hour treatment time

Cell suspension (10^6 cells/ml in complete medium 5%) 10.0 ml
Complete medium (5%) 9.8 ml
Control or Test item solution 0.2 ml


Without S9 metabolism – 24-hour treatment time

Cell suspension (10^6 cells/ml in complete medium 10%) 3.0 ml
Complete medium (10%) 16.8 ml
Control or Test item solution 0.2 ml


With S9 metabolism – 3-hour treatment time

Cell suspension (1E6 cells/ml in complete medium 5%) 10.0 ml
S9 mix 9.8 ml
Control or Test item solution 0.2 ml


The cultures were incubated at 37°C. At the end of the incubation period, the treatment medium was removed and the cultures centrifuged and washed twice with Phosphate Buffered Saline (PBS).

Cytotoxicity assay
A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined.
Treatments were performed in the absence and in the presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. After washing in Phosphate Buffered Saline (PBS), cells were resuspended in 20 ml RPMI minimal medium A. Cell concentrations were adjusted to 8 cells/ml using complete medium (20%) and, for each dose level, 0.2 ml was plated into 96 microtitre wells. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. Wells containing viable clones were identified by the eye using background illumination and then counted.

Mutation assay
Treatment of cell cultures
Experiments were performed including vehicle and positive controls, in the absence and presence of S9 metabolising system.

Preparation of test cell cultures was performed as described . Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture.

In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained in the first experiment without metabolic activation, the second experiment in the absence of S9 metabolism, was performed using a long treatment time (24 hours).

After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and cell densities were determined. The number of cells was adjusted to give 2 x 105 cells/ml. The cultures were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

Determination of survival
Following adjustment of the cell densities, samples of the cultures were diluted to 8 cell/ml using complete medium A (20%). A 0.2 ml aliquot of each diluted culture was placed into each well of two 96-well plates. The plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 8 days. After incubation, wells containing viable clones were identified by the eye using background illumination and then counted.

Expression period
During the expression period (two days after treatment) the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period the cell densities of each culture were determined and adjusted to give 2 x 10^5 cells/ml.


Plating for 5-trifluorothymidine resistance
After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 µg/ml) and an estimated 2 x 10^3 cells were plated in each well of four 96-well plates.

Plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as described in section 4.3.2 and counted. In addition, the number of wells containing large colonies and the number containing small colonies were scored.

Plating for viability
After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted.

Evaluation criteria:

The assay was considered valid if the following criteria were met:

(i) The cloning efficiencies at Day 2 in the untreated control cultures in the absence of S9 metabolic activation fell within the range of 65- 120%.

(ii) The untreated control growth factor in the absence of S9 metabolic activation over 2 days fell within the range of 8 – 32.

(iii) The mutant frequencies in the untreated control cultures fell within the range of 50 200 x 106 viable cells.

(iv) The positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).

Statistics:

Statistical analysis was performed according to UKEMS guidelines (Robinson W.D.,1990)

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At the highest dose level of 1460 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Effects of pH: The addition of the test item solutions generated a slight dose-dependent decrease in pH (7.69 - 7.24).
- Effects of osmolality: The solvent, DMSO, causes an increase in the apparent osmolality of the treatment solution, since it depresses the freezing po int. However, since it passes freely in and out of cells, this increase is only apparent. A small but real increase in osmotic pressure may be caused only during the period of equilibration after the addition of the solvent. The increase due to the solvent (approx. 150 mOsm/kg) can therefore be
subtracted from the treatment medium values, to assess the effects of the test substance. Since the test item displaces the solvent in the test item solution, lower apparent osmotic pressures may be observed at higher dose levels.
- Solubility: A preliminary solubility trial was performed and the test item was found to be soluble in DMSO at a dose level of 146 mg/ml. This solvent is considered adequate for treatment since it is compatible with the survival of the cells and the S9 metabolic activity An aliquot of the solution at 1 46 mg/ml was added to RPMI complete medium (10%) in the ratio 1 : 100 and generated a clear solution. This permitted a final concentration in the treatment medium of 1460 µg/ml corresponding to 0.01 M (the upper limit to testing indicated in the Study Protocol).
- Precipitation: not observed

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

In the absence of S9 metabolic activation, using a short and long treatment time, no statistically significant increases in mutant frequency were observed. In the first experiment, a significant dose-relationship was indicated by the linear trend analysis (p<5%). However, no statistically significant increases in mutant frequency were observed and the effect was not reproduced in the second experiment. Hence, the linear trend indicated in Experiment 1 was considered to be attributable to a chance event not related to the action of the test item and to be of no biological significance.

 

In the presence of S9 metabolic activation, in the first experiment, a statistically significant increase in mutation frequency was observed at 1460 μg/ml (p<1%) and a significant dose‑relationship was indicated by the linear trend analysis (p<1%). In the second experiment, using a modified dose-range, a statistically significant increase in mutant frequency was observed at 1460 μg/ml (p<5%) and a significant dose-relationship was indicated by the linear trend analysis (p<1%). However, the mutant frequencies observed at these dose levels were within the historical control range observed at RTC and the IMF were lower than the GEF. Hence, the linear trend and the observed increases were considered to be of no biological significance.

Applicant's summary and conclusion

Conclusions:

It is concluded that Dimethyl Succinate (DMS) does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

Mutagenic activity has been assessed by assaying for the induction of trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells according to OECD / EU test methods. Dimethyl Succinate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in the absence or presence of S9 metabolic activation.