Dibutyl fumarate

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:

21 April 2010 to ...

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully GLP- and guideline compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

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:

Dibutyl fumarate

EC Number:

203-327-9

EC Name:

Dibutyl fumarate

Cas Number:

105-75-9

Molecular formula:

C12H20O4

IUPAC Name:

dibutyl but-2-enedioate

Details on test material:

Common Name: DBFCAS No.: 105-75-9Chemical Name (IUPAC): Fumaric acid di-n-butylesterPhysical Appearance: LiquidPurity as per Certificate of Analysis : 98.5%Batch No.: LEDB2A6041Manufactured Date: Nov. 2009Expiry Date: May 2011Retest Date: no retest before May 2011Recommended 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): 4-hour Exposurea) 80 b) 253 and c) 800 µg/mL (factor of v10)Experiment 2 (without S9): 4-hour Exposure a) 10 b) 20 and c) 40 µg/mL (factor of 2)Experiment 3 (without S9): 21-hour Exposure a) 7.5 b) 15 and c) 30 µg/mL (factor of 2)

Vehicle / solvent:

DMSO: One hundred fifty microlitres (150 ¿L) DMSO was used as the solvent control in each of the experiments.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

One hundred fifty microlitres (150 µL) Ethanol were used as the solvent control in each of the experiments.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: 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:Hams F-12 medium supplemented with L-glutamine, sodium bicarbonate, antibiotics and 5 or 10 % of fetal bovine serum (F-12 FBS 5/10)Dulbeccos Phosphate Buffered Saline (PBS), pH 7.4Trypsin-EDTA solutionThe following chemicals were used in the study:Name / Lot/Batch No. / ManufacturerGiemsas stain / G07A/9066/0607/72 / s.d. fine chem ltd.Worli Road Mumbai 400 030, INDIAPotassium Chloride / 24076802-1 / Qualigens Fine Chemicals Navi Mumbai 400 710, INDIAColchicine / 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, INDIAEDTA / 4-0091 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIAGlucose-6-phosphate / 0000065463 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIANADP / 0000055489 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIAPenicillin / 0000044077 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIAPhosphate Buffered Saline / 0000080644 & 0000069887 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIASodium bicarbonate / 0000002983 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIAStreptomycin / 0000065241 / Hi-Media Laboratories Pvt. Ltd. Mumbai 400 086, INDIAMagnesium chloride / MF6M561553 / Merck Specialities Pvt. Ltd. Worli, Mumbai 400 018, INDIAMethanol / SK9F590599 / Merck Specialities Pvt. Ltd. Worli, Mumbai 400 018, INDIAFetal Bovine Serum / 41F7596K & 41F9293K / Invitrogen Corporation Grand Island, NY14072, USAHams F-12 medium / 713870 / Invitrogen Corporation Grand Island, NY14072, USAL-Glutamine / 1329739 / Invitrogen Corporation Grand Island, NY14072, USAAcetic acid / 83566905-2 / Thermo Electron LLS India Pvt. Ltd. Sion (East), Mumbai 400 022, INDIAXylene / B112A08 / RFCL A 3, Okhla Industrial Area Phase 1, New Delhi 110 020, INDIADMSO / 3255979 / Spectrochem Private Limited, Mumbai, IndiaReference Materials:Chemical (CAS No.) / Lot No. / ManufacturerEthyl methanesulphonate (62-50-0) / 1423147 / Sigma Aldrich Co. St. Louis, MO 63103, USACyclophosphamide monohydrate (6055-19-2) / 076K 1050 / Sigma Aldrich Co. St. Louis, MO 63103, USA

Evaluation criteria:

Cytotoxicity (preliminary test)Twenty one 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

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

not determined

Remarks:

non mutagenic

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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 6.85 and 6.98 with 6.92 in the DMSO control while in the absence of metabolic activation, ranged from 7.09 to 7.59 with 7.09 in the DMSO control.

At the end of 4-hour exposure period, Osmolarity of the test solutions in the presence of metabolic activation, ranged from 0.420 to 0.444 OSMOL/kg with 0.434 OSMOL/kg in the DMSO control while in the absence of metabolic activation, ranged from 0.407 to 0.430 OSMOL/kg with 0.439 OSMOL/kg in the DMSO control.

There was evidence of significant inhibition in the growth of CHO cells at and above 1141 µg/mL compared to the DMSO control in the presence of metabolic activation.

In the absence of metabolic activation with 4-hour exposure, no evidence of significant reduction in the growth of CHO cells observed up to 36 µg/mL compared to the DMSO control. However, there was evidence of significant growth inhibition at 36 µg/mL compared to the DMSO control with 21-hour exposure. Dead and disfigured cells were observed at and above 71 µg/mL.

Chromosome Aberration Assay

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

At the highest concentration tested (800 µg/mL), the reduction in the cell growth was 46 % compared to the DMSO control.

The incidence of aberrations in the DMSO 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 (40 µg/mL), the reduction in the cell growth was 46 % compared to the DMSO control.

The incidence of aberrations in the DMSO 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 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 (30 µg/mL), the reduction in the cell growth was 45 % compared to the respective DMSO control.

The incidence of aberrations in the DMSO 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 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 Di-n-butylfumarate, either in the presence or absence of metabolic activation.

Taken together, the results of the three experiments support a conclusion that the test item, Di-n-butylfumarate 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:

Interpretation of results: negativeIt was concluded that the test item, Di-n-butylfumarate, 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, Di-n-butylfumarate, 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 (S-9 fraction prepared from Aroclor 1254 induced rat liver).

 

Di-n-butylfumarate formed a solution in dimethyl sulphoxide (DMSO) at the required concentration of 250 mg/mL and was found to be stable at the concentrations of 0.3 mg/mL and 228 mg/mL (equivalent to 10 mM test item concentration) in DMSO for 5 hours when stored at room temperature.

 

In a preliminary cytotoxicity test for the selection of test doses, Di-n-butylfumarate showed evidence of significant growth inhibition (>50 %) at and above 1141 µg/mL compared to the DMSO control in the presence of metabolic activation. In the absence of metabolic activation with 4-hour exposure, no evidence of significant reduction in the growth of CHO cells observed up to 36 µg/mL compared to the DMSO control. However, there was evidence of significant growth inhibition at 36 µg/mL compared to the DMSO control with 21-hour exposure. Dead and disfigured cells were observed at and above 71 mg/mL.

 

 In the definitive chromosome aberration assay, CHO cells were exposed to the test item in triplicate at concentrations of 80, 253 and 800 µg/mL of the medium in the presence of metabolic activation for 4 hours, at 10, 20 and 40 mg/mL and at 7.5, 15 and 30 µg/mL of the medium in the absence of metabolic activation for 4 and 21 hours, respectively. 

  

Similarly, concurrent solvent (DMSO) 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 (DMSO control, test concentrations or positive controls) and slides were prepared for chromosome analysis.

 

At the highest concentration tested (800 µg/mL), in the presence of metabolic activation with 4-hour, the reduction in the cell growth was 46 %. Similarly in the absence of metabolic activation with 4-hour exposure and at the highest concentration tested (40 µg/mL), the reduction in cell growth was 46 %. At the highest concentration tested (30 µg/mL) in the absence of metabolic activation with the 21-hour exposure, the reduction in cell growth was 45%.

 

A total of 200 metaphases per dose level from triplicate cultures from the DMSO 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 DMSO 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, Di-n-butylfumarate is not clastogenic at the concentrations tested and under the conditions of testing.