Dimethyl succinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 17, 2010 to June 14, 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.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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 succinate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot No.of test material: T14B208335
- Production date: 30 Nov 2008
- Expiration date of the lot/batch: 31 December 2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition at RTC of test material: Room temperature

Method

Target gene:

Lymphocytes cells

Species / strain

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

Whole blood was collected from two healthy male volunteer donors (one for each experiment). The volunteers were non-smokers and were not receiving any medication prior to the time of sampling.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

10.0, 5.00, 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781 mM corresponding to 1460, 730, 365, 183, 91.3, 45.6, 22.8, 11.4 µg/ml .

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO, batch no.1395037 52208P07.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

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:

cyclophosphamide

Remarks:

Migrated to IUCLID6: In the presence of S9.

Details on test system and experimental conditions:

ASSAY FOR CHROMOSOMAL ABERRATIONS

Two independent assays for chromosomal damage were performed.
For the first experiment, both in the absence and presence of S9 metabolism, the treatment time was 3 hours after which the cells were allowed to recover prior to harvesting. The harvest time of 24 hours, corresponding to approximately 1.5 cell cycles, was used.
As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using the same harvest time. A continuous treatment until harvest was used. For the present study, repetition of treatment in the presence of S9 metabolism was not considered necessary. In case of negative results in the presence of S9, repetition may be advisable when further investigation is required to focus on a specific dose-range byusing a narrow space interval. A repetition may also be suggested when a metabolic activation system, different from the standard (rodent liver S9), is considered more adequate. Since no relevant toxicity was observed at any dose level in the presence of S9 metabolism, a further treatment in the presence of S9 metabolism using a different concentration spacing was not regarded as necessary. In addition, no specific information on test item metabolism was available to justify the use of other metabolic systems.
Both for the first and second experiments the dose levels of 10.0, 5.00, 2.50, 1.25, 0.625, 0.313, 0.156 and 0.0781 mM corresponding to 1460, 730,365, 183, 91.3, 45.6, 22.8 and 11.4 µg/ml, were used in the absence or presence of S9 metabolism.
Appropriate negative and positive control cultures were included in each experiment. Positive control treated cultures received Mitomycin-C in the absence of S9 metabolism at dose levels of 0.75 and 0.50 µg/ml in the first main experiment. For the second experiment cultures received Mitomycin-C at dose levels of 0.45 and 0.30 µg/ml. In the presence of S9 metabolism cultures received Cyclophosphamide at dose levels of 18.0 and 23.0 µg/ml. Two cultures were prepared at each test point. Air-dried slides were prepared from each culture and stained 3% Giemsa.

Evaluation criteria:

In this assay, the test item is considered to have clastogenic properties if the following criteria are all fulfilled:
- statistically significant increases in the incidence of cells bearing aberrations are observed at any dose level over the concurrent control.
- the increases are reproduced in both replicate cultures.
- The increases must exceed historical controls. Any significant increase over the concurrent negative controls is therefore compared with the histor ical control.

Statistics:

For the statistical analysis, Fisher's Exact Test was used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. The analysis was performed using sets of data either including or excluding gaps. Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations including or excluding gaps over the control values, was observed in any experiment both in the absence or presence of S9 metabolism.

Results and discussion

Test results

Species / strain:

lymphocytes: Human

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Slight dose related reductions of pH of the treatment media were observed following treatment with the test item in the presence of S9 metabolism. No variation of the pH of treatment media was observed in the absence of S9 metabolism.
- Effects of osmolality: No remarkable variation of the osmolality of the treatment medium was observed at any dose level in the absence or presence of S9 metabolism.
- Solubility: It was considered appropriate to evaluate the solubility at the concentration of 146 mg/ml in DMSO. This concentration was chosen sinc e when an aliquot of stock solution was added to culture medium in the ratio 1:100, it gave a final concentration of 1460 µg/ml. The concentrat i on of 1460 µg/ml was selected as the maximum dose level for treatment, corresponded to 10 mM and was lower than 5000 µg/ml.
- Precipitation: No precipitation or opacity of the treatment medium was observed at the beginning or end of treatment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first experiment, following treatment with the test item no remarkable cytotoxicity was observed at any dose level in the absence or presence oS9 metabolism. In the second experiment, following the continuous treatment in the absence of S9 metabolism, slight toxicity was observed at the
two higher dose levels (10.0 and 5.00 mM), where the mitotic index was reduced to 84% and 76% of the control respectively. No toxicity was observedover the remaining dose-range.
The highest dose level selected for the scoring of aberrations should be a concentration causing moderate toxicity (ideally the reduction of mitotic
index should be approximately 50%). In the absence of toxicity the highest treatment level should be selected as the highest dose for scoring.

Any other information on results incl. tables

Assay results

One hundred metaphase spreads were scored for chromosomal aberrations from each culture. In addition, cells bearing chromosome numerical changes were also recorded separately. Following treatment with the test item, no increase in the incidence of cells bearing aberrations including or excluding gaps over the control value was observed in any experiment. Two endoreduplicated cells were observed in one replicate culture from the highest dose level selected for scoring in the presence of S9 metabolism. One endoreduplicated cell was also observed in one replicate culture selected for scoring in the absence of S9 metabolism from the low dose level (3 hours treatment) and intermediate dose level (continuous treatment) respectively. One polyploid cell was seen in one solvent control culture in the absence of S9 metabolism (continuous treatment). Due to the low incidence of cells bearing chromosome numerical changes, these observations were not considered biologically meaningful.

Marked increases in the frequency of cells bearing aberrations (including and excluding gaps) were seen in the cultures treated with the positive control substances, Mitomycin-C and Cyclophosphamide, indicating the correct functioning of the assay system.

Applicant's summary and conclusion

Conclusions:

On the basis of these results it is concluded that dimethylsuccinate (DMS) does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Executive summary:

The ability to cause chromosomal damage in cultured human lymphocytes has been investigated according to OECD / EU test methods. Dimethyl succinate does not induce chromosomal aberrations in human lymphocytes under the reported experimental conditions.