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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Chemical Name (IUPAC) : Fumaric acid bis-(2-ethylhexyl)ester
Physical Appearance : Liquid
Purity as per Certificate of Analysis: 99.5%
Batch No.: LEDF1B1004
Manufactured Date: Jan. 2010
Expiry Date: Jan. 2011
Retest Date: no retest before Jan. 2011
Recommended Storage Condition: Ambient (+18 to +36º)

The identity of the test item was provided by the study sponsor by a Certificate of Analysis (CoA). The responsibility for the correct identity and purity rests with the sponsor.

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese Hamster (Cricetulus griseus) Ovary (CHO-K1) cell line, (ATCC CCL-61, Lot 4765275) hypodiploid with a modal chromosome number 20 and a population doubling time of 10 to 14 hours was used.
(American Type Culture Collection, P. O. Box 1549, Manassas, VA 20108, USA.)

Batch No. 2 of this CHO-K1 cell line was tested for the absence of mycoplasma contamination at Mycoplasma Laboratory, Statens Serum Institut, Artillerivej 5, Copenhagen S, Denmark and certified free of mycoplasma contamination on July 17, 2009.

Test approaches currently accepted under the OECD, guidelines for the assessment of mammalian cell clastogenicity involve the use of Chinese Hamster Ovary (CHO) cell line. This cell line has been demonstrated to be sensitive to the clastogenic activity of a variety of chemicals.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 homogenate, prepared from male Wistar rats induced with a single intraperitoneal injection of Aroclor 1254 (200 mg/mL) at 500 mg/kg body weight
Test concentrations with justification for top dose:
The guideline calls for testing, where there is cytotoxicity, to employ a high dose which causes a significant reduction in the growth of cells.
Based on the observations of the preliminary cytotoxicity test, the following concentrations of the test item were selected for testing in the chromosome aberration assay:

Experiment 1 (with S9) and 2 (without S9): 4-hour Exposure
a) 340 b) 1080 and c) 3400 µg/mL (factor of v10)

Experiment 3 (without S9): 21-hour Exposure
a)15 b) 30 and c) 60 µg/mL (factor of 2)
Vehicle / solvent:
Ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
150 µL ethanol was used as the solvent control in each of the experiments.
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
CPA for cultures with metabolic activation, EMS for cultures without metabolic activation
Details on test system and experimental conditions:
Stock cultures of the CHO-K1 cell line were stored in the test facility as frozen permanents in liquid nitrogen.
Cells were grown in T-75 cm2 flasks at 37±1 °C in a carbon dioxide incubator (5% CO2 in air).

Test medium, reagents and other chemicals:
Ham‘s F-12 medium supplemented with L-glutamine, sodium bicarbonate, antibiotics and 5 or 10 % of fetal bovine serum (F-12 FBS 5/10)
Dulbecco‘s Phosphate Buffered Saline (PBS), pH 7.4
Trypsin-EDTA solution

The following chemicals were used in the study:
Name / Lot/Batch No. / Manufacturer
Giemsas stain / G07A/9066/0607/72 / s.d. fine chem ltd.Worli Road Mumbai 400 030, INDIA
Potassium Chloride / 24076802-1 / Qualigens Fine Chemicals Navi Mumbai 400 710, INDIA
Colchicine / 097K1247 / Sigma Chemical Co. St. Louis, MO63103 USA
Trypsin / 039K7013 / Sigma Chemical Co. St. Louis, MO63103 USA
Amphoterecin B / 128K4046 / Sigma Chemical Co. St. Louis, MO63103 USA
DPX Mountant / 0000043008 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIA
EDTA / 4-0091 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIA
Glucose-6-phosphate / 0000065463 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIA
NADP / 0000055489 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIA
Penicillin / 0000044077 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIA
Phosphate Buffered Saline / 0000080644 & 0000069887 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIA
Sodium bicarbonate / 0000002983 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIA
Streptomycin / 0000065241 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIA
Magnesium chloride / MF6M561553 / Merck Specialities Pvt. Ltd. Worli, Mumbai 400 018, INDIA
Methanol / SK9F590599 / Merck Specialities Pvt. Ltd. Worli, Mumbai 400 018, INDIA
Fetal Bovine Serum / 41F7596K & 41F9293K / Invitrogen Corporation Grand Island, NY14072, USA
Hams F-12 medium / 713870 / Invitrogen Corporation Grand Island, NY14072, USA
L-Glutamine / 1329739 / Invitrogen Corporation Grand Island, NY14072, USA
Acetic acid / 83566905-2 / Thermo Electron LLS India Pvt. Ltd. Sion (East), Mumbai 400 022, INDIA
Xylene / B112A08 / RFCL A 3, Okhla Industrial Area Phase 1, New Delhi 110 020, INDIA
Ethanol / K35664003604 / Merck K Ca A Darmstadt, Germany

Reference Materials:
Chemical (CAS No.) / Lot No. / Manufacturer
Ethyl methanesulphonate (62-50-0) / 1423147 / Sigma Aldrich Co. St. Louis, MO 63103, USA
Cyclophosphamide monohydrate (6055-19-2) / 076K 1050 / Sigma Aldrich Co. St. Louis, MO 63103, USA
Evaluation criteria:
Cytotoxicity (preliminary test)
21 hours after the start of the treatment, medium from each flask (set 1, 2 and 3) was removed by aspiration, the cell monolayer was trypsinized and the cells were suspended in 10 mL F12 FBS5. The effect of the test item on cell multiplication was estimated by expressing the number of cells in each treated culture as a percentage of the number in the Ethanol control.

Definitions of chromosome aberrations are given in the report.
Statistics:
Statistical analyses of the experimental data were carried out using validated SYSTAT Statistical package Ver.12.0. Data were analysed for proportions of aberrant metaphases in each sample, including and excluding gaps as aberrations. Pooled data from each test concentration and the positive control are compared with the solvent control using the one-tailed Fisher exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Cytotoxicity Test and Justification for the Selection of Test Doses

No precipitation of test solutions observed at any of the test concentrations either in the presence or in the absence of metabolic activation.

At the end of 4-hour exposure period, pH of the test solutions in the presence of metabolic activation, ranged between 7.05 and 7.23 with 7.08 in the Ethanol control while in the absence of metabolic activation, ranged from 7.00 to 7.44 with 6.97 in the Ethanol controls.

At the end of 4-hour exposure period, Osmolarity of the test solutions in the presence of metabolic activation, ranged from 0.428 to 0.467 OSMOL/kg with 0.431 OSMOL/kg in the Ethanol control while in the absence of metabolic activation, ranged from 0.378 to 0.443 OSMOL/kg with 0.440 OSMOL/kg in the Ethanol control.

There was no evidence of significant inhibition in the growth of CHO cells up to the highest tested concentration of 3406 µg/mL compared to the Ethanol  control both in the presence and absence of metabolic activation with 4-hour exposure.

In the absence of metabolic activation with 21-hour exposure, evidence of significant reduction in the growth of CHO cells was observed at and above 54 mg/mL compared to the Ethanol control.

Chromosome Aberration Assay

Experiment 1 (with S9): (4-hour Exposure)

At the highest concentration tested (3400 µg/mL), the reduction in the cell growth was 31 % compared to the Ethanol control.

The incidence of aberrations in the Ethanol control was within the range of the in-house historical control data.

The incidence of aberrant metaphases both including and excluding gaps was statistically comparable to the solvent control value at all the concentrations tested.

The positive control, cyclophosphamide monohydrate caused a statistically significant increase in the aberrant metaphases both including and excluding gaps.

Experiment 2 (without S9): 4-hour Exposure

At the highest concentration tested (3400 µg/mL), the reduction in the cell growth was 41 % compared to the Ethanol control.

The incidence of aberrations in the Ethanol control was within the range of the in-house historical control data (Annexure 4).

The incidence of aberrant metaphases both including and excluding gaps was statistically comparable to the solvent control value at all the three test concentrations. 

The positive control, ethyl methanesulphonate caused a statistically significant increase in aberrant metaphases both including and excluding gaps.

Experiment 3 (without S9): 21-hour Exposure

At the highest concentration tested (60 µg/mL), the reduction in the cell growth was 54 % compared to the respective Ethanol control.

The incidence of aberrations in the Ethanol control was within the range of the in-house historical control data (Annexure 4).

The incidence of aberrant metaphases both including and excluding gaps was statistically comparable to the solvent control value at all the three test concentrations. 

Ethyl methanesulphonate caused a statistically significant increase in the aberrant metaphases both including and excluding gaps.

Discussion

No evidence for the induction of chromosome aberrations either including or excluding gaps was obtained in any of the experiments at any test concentrations of Dioctylfumarate, either in the presence or absence of metabolic activation.

Taken together, the results of the three experiments support a conclusion that the test item, Dioctylfumarate does not have the potential to cause chromosome damage (including or excluding gaps) either in the presence or absence of metabolic activation.

In each of these experiments, the respective positive controls produced a statistically significant increase in aberrant metaphases, demonstrating that the system was able to detect the effect of known mutagens.

Applicant's summary and conclusion

Conclusions:
It was concluded that the test item, Dioctylfumarate does not have the potential to induce chromosome damage in CHO cells at the tested concentrations and under the conditions of testing employed.
Executive summary:

The clastogenic potential of the test item, Dioctylfumarate, to induce chromosome aberrations in mammalian cells was evaluated using cultured Chinese Hamster Ovary (CHO) cells.

 

The study consisted of a preliminary toxicity test and a chromosome aberration assay comprising of three independent experiments: one each in the presence and absence of metabolic activation and a confirmatory experiment in the absence of metabolic activation (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

Dioctylfumarate formed a solution in Ethanol at the required concentration of 250 mg/mL and was found to be stable at the concentrations of 1.5 mg/mL and 340 mg/mL in Ethanol for 5 hours when stored at room temperature.

 

In a preliminary cytotoxicity test for the selection of test doses, Dioctylfumarate showed no evidence of significant growth inhibition up to the highest tested concentration of 3406 µg/mL (equivalent to 10 mM Dioctylfumarate) compared to the Ethanol control both in the presence and absence of metabolic activation with 4-hour exposure. In the absence of metabolic activation with 21-hour exposure, there was evidence of significant reduction in the growth of CHO cells at and above 54mg/mL compared to the Ethanol control.

 

In the definitive chromosome aberration assay, CHO cells were exposed to the test item in triplicate at concentrations of 340, 1080 and 3400 µg/mL of the medium in the presence and absence of metabolic activation for 4 hours, and at 15, 30 and 60mg/mL of the medium in the absence of metabolic activation for 21 hours. 

  

Similarly, concurrent solvent (Ethanol) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulphonate in the absence of metabolic activation) were also tested in triplicate.

 

In each case, the cells in C-metaphase were harvested at 21 hours after the start of the treatment (Ethanol control, test concentrations or positive controls) and slides were prepared for chromosome analysis.

 

A total of 200 metaphases per dose level from triplicate cultures from the Ethanol control, each treatment group and the positive control were evaluated for chromosome aberrations. The data from the treatment groups and the positive control were statistically compared with the Ethanol control.

 

There was no evidence of induction of chromosome aberrations, including or excluding gaps, either in the presence or absence of metabolic activation, in any of these three experiments. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases.

 

The study indicated that the test item, Dioctylfumarate is not clastogenic at the concentrations tested and under the conditions of testing.