Dibutyl fumarate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD Guideline 471 (Bacterial Reverse Mutation Assay):

DIBUTYL FUMARATE is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 up to 5000 µg/plate, which is the limit concentration for this kind of test.

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test):

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.

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

There was no evidence of induction of gene mutation in any of these experiments either in the presence or absence of metabolic activation. In each of these experiments, the respective positive controls produced a statistically significant increase in the frequencies of mutants, under identical conditions.

The study indicated that the test item, Di-n-butylfumarate does not have the potential to cause gene mutation at thehprtlocus at the concentrations tested and under the conditions of testing.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

not applicable

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.

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.

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'.

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.

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.

Reference 2

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:

19 April 2010 to 4 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Fully GLP- and guideline compliant study

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

Target gene:

hprt locus of Chinese Hamster Ovary (CHO) cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275) with a model chromosome number 20 and a population doubling time of 10 to 14 hours was used. Batch No. 2 of this CHO-K1 cell line was tested for the absence of mycoplasma contamination at Mycoplasma Laboratory, Statens Serum Institut, Denmark and certified free of mycoplasma contamination on July 17, 2009.Established CHO cell line is useful in in vitro gene mutation testing because it is easily cultured in standard medium, has a small number of large chromosomes each with a more or less distinctive morphology and a relatively short cycle time. Cells were grown in tissue culture flasks at 37±1 °C in a carbon dioxide incubator (5±0.2% carbon dioxide in air).

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:

Initial Gene Mutation Assay:Presence of Metabolic Activation a) 72 b) 228 c) 722 and d) 2280 µg/mL (factor of v10)Absence of Metabolic Activation a) 4.5 b) 9 c) 18 and d) 36 µg/mL (factor of 2)Confirmatory Gene Mutation AssayPresence of Metabolic Activation a) 104 b) 290 c) 814 and d) 2280 µg/mL (factor of 2.8)Absence of Metabolic Activation a) 3 b) 7 c) 16 and d) 36 µg/mL (factor of 2.2)

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: Bap (5 µg/mL) for cultures with a metabolic activation system, EMS (600 µg/mL) for cultures without a metabolic activation system

Details on test system and experimental conditions:

Stock cultures of the CHO-K1 cell line are stored at the test facility as frozen permanents in liquid nitrogen.Hams F-12 medium supplemented with sodium bicarbonate, antibiotics and L-glutamine was the basic medium. Basic medium supplemented with 10% fetal bovine serum (FBS) was the complete medium and was used for the growth and multiplication of cells as well as in detaching and diluting the cells. Basic medium without serum was the treatment medium and was used for target cell exposure to the test item and controls. Cloning medium was basic medium supplemented with 20% FBS and was used for the determination of cell viability or plating/cloning efficiency. Selective medium was basic medium supplemented with 20% FBS and the selective agent 6-Thioguanine (6-TG) at 35 µg/mL and was used for the selection of mutants. Dulbeccos Phosphate buffered saline (PBS) (pH: 7.4)Trypsin: EDTA solutionFollowing chemicals were used in the study:Name / Lot/Batch No. / ManufacturerPotassium chloride / 24076802-1 / Qualigens Fine Chemicals Navi Mumbai 400 710, INDIAAmphoterecin B / 067K4159 & 128K4046 / Sigma Aldrich Inc. St. Louis, MO63103, USA 6-Thioguanine / 036K 0173 / Sigma Aldrich Inc. St. Louis, MO63103, USA Trypsin / 039K7013 / Sigma Aldrich Co. St. Louis, M063103, USA Magnesium chloride / MF6M561553 / Merck Specialities Pvt. Ltd. Worli, Mumbai 400 018, INDIAFetal Bovine Serum / 41F9293K & 41F7596K / Invitrogen Corporation Grand Island, NY14072, USAHams F-12 medium / 713870 / Invitrogen Corporation Grand Island, NY14072, USAL-Glutamine / 1329739 / Invitrogen Corporation Grand Island, NY14072, USADMSO / 3255979 / Spectrochem Pvt. Ltd. Mumbai, INDIAMethanol / SK 9F 590599 / Merck specialities pvt. Ltd., Worli, Mumbai 400 018, India

Evaluation criteria:

There are several criteria for determining a positive result, such as a concentration related, or a reproducible increase in mutant frequency. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determining factor for a positive response. A test item, for which the results do not meet the above criteria is considered non mutagenic in this system.Positive results for an in vitro mammalian cell gene mutation test indicate that the test substance induces gene mutations in the cultured mammalian cells used. A positive concentration response that is reproducible is most meaningful. Negative results indicate that, under the test conditions, the test substance does not induce gene mutations in the cultured mammalian cells used. The Cloning Efficiency of the solvent/vehicle controls should not be less than 60 %.The mean mutant frequency of the solvent/vehicle controls in each experiment should fall within a range of 0 to 20 mutants per 106 clonable cells.The positive controls must induce a statistically significant response.

Statistics:

Power transformation procedure by Snee and Irr (1981) with which, the observed mutant frequency was transformed using the formulaY = (X + A) Bwhere, Y =transformed mutant frequencyX =observed mutant frequencyand A, B = constants.Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p=0.05, a Dunnetts test was conducted, comparing each treatment group and the positive control to the solvent control (p=0.05).

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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'.

Cytotoxicity Testand 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.

 

No evidence of significant cell growth inhibition as Relative Cloning Efficiency (10 to 20% RCE compared to solvent control) was seen at any of the tested concentrations in the presence of metabolic activation and up to 36 µg/mL in the absence of metabolic activation. At and above 71 µg/mL, dead and disfigured cells were observed in the absence of metabolic activation.

 

Stability Test and Dose Formulation Analysis

Stability of dose formulations of Di-n-butylfumarate at the concentrations 0.3 mg/mL and 228 mg/mL in DMSO were fround to be stable for 5 hours when stored at room temperature.

The samples sent for the dose formulation analysis confirmed to the presence ofDioctylfumarateand were well within the limits.

 

Parallel Cytotoxicity Assessment

In the initial assay, at the highest concentrations tested, the RCE was 60% and 59% in the presence and absence of metabolic activation, respectively. In the confirmatory assay, at the highest concentration tested, the RCE was 80% both in the presence and absence of activation.

 

Mutation Assay

The test concentrations and the positive controls showing mutant frequency (mutants/10⁶surviving cells) in the presence and absence of the metabolic activation were compared with the solvent control using the statistical method described earlier.

 

Initial Mutation Assay: Presence of Metabolic Activation

The frequency of mutants in the negative control was within the range of the in-house historical control data.

 

The test item did not cause a significant increase in the frequencies of mutants compared to the solvent control in the presence of metabolic activation at any of the tested concentrations.

 

Under similar conditions the positive control Benzo (a) pyrene induced a significant increase in the mutant frequency as compared with the solvent control.

 

Initial Mutation Assay: Absence of Metabolic Activation

The frequency of mutants in the negative control was within the range of the in-house historical control data.

 

The test item did not cause a significant increase in the frequencies of mutants compared to the solvent control in the presence of metabolic activation at any of the the tested concentrations.

 

Under similar conditions the positive control Ethylmethane sulphonate induced a significant increase in the mutant frequency as compared with the solvent control.

 

Confirmatory Mutation Assay: Presence of Metabolic Activation

The frequency of mutants in the negative control was within the range of the in-house historical control data.

 

The test item did not cause a significant increase in the frequencies of mutants compared to the solvent control in the presence of metabolic activation at any of the the tested concentrations.

 

Under similar conditions the positive control Benzo (a) pyrene induced a significant increase in the mutant frequency as compared with the solvent control.

 

Confirmatory Mutation Assay: Absence of Metabolic Activation

The frequency of mutants in the negative control was within the range of the in-house historical control data.

 

The test item did not cause a significant increase in the frequencies of mutants compared to the solvent control in the presence of metabolic activation at any of the tested concentrations.

 

Under similar conditions the positive control Ethylmethane sulphonate induced a significant increase in the mutant frequency as compared with the solvent control.

 

 

Discussion

No evidence for the induction of gene mutation was observed in any of the concentrations of the test item either in the presence or in the absence of metabolic activation.

 

Taken together, the results of the initial and confirmatory assays suggest that the test item does not have the potential to cause gene mutation either in the presence or in the absence of metabolic activation.

 

In each of these assays, the positive control chemicals produced a statistically significant increase in the mutant frequencies, demonstrating that the system was able to detect the effect of known mutagens.

 

Conclusions:

Interpretation of results: negativeIt is concluded that the test item, Di-n-butylfumarate, does not have the potential to induce gene mutation in CHO cells at the tested concentrations and under the conditions of testing employed.

Executive summary:

The genotoxic potential of the test item Di-n-butylfumarate to induce gene mutation in mammalian cells was evaluated using Chinese hamster ovary (CHO) cells.

 

The study consisted of a preliminary toxicity test, an initial gene mutation assay and a confirmatory gene mutation assay comprising four independent experiments, two each in the presence and absence of metabolic activation system (S-9 fraction prepared from Aroclor 1254 induced rat liver).

 

Di-n-butylfumarate formed a solution indimethyl 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 did not cause a significant cell growth inhibition as Relative Cloning Efficiency (10 to 20% RCE compared to solvent control) up to the highest tested concentration of 2283 µg/mL in the presence of metabolic activation and up to 36 µg/mL in the absence of metabolic activation. The test item did not precipitate the test solutions up to 2283 µg/mL, and did not cause any appreciable change in the pH of the test medium at the end of 4-hour exposure to treatment either in the presence or in the absence of metabolic activation.

 

In the initial gene mutation assay, CHO cells were exposed to the test item in duplicate at concentrations of 72, 228, 722 and 2280 mg/mL of the medium and at 4.5, 9, 18 and 36 µg/mL for 4 hours in the presence and absence of metabolic activation, respectively. In the confirmatory gene mutation assay, CHO cells were exposed to the test item in duplicate at concentrations of 104, 290, 814 and 2280 µg/mL of the medium and at 3, 7, 16 and 36 µg/mL for 4 hours in the presence and absence of metabolic activation, respectively. In a similar way, a concurrent solvent control and appropriate positive controls viz., Benzo(a)pyrene in the presence of metabolic activation and Ethyl methanesulphonate in the absence of metabolic activation were also tested in duplicate.

 

There was no evidence of induction of gene mutation in any of these experiments either in the presence or absence of metabolic activation. In each of these experiments, the respective positive controls produced a statistically significant increase in the frequencies of mutants, under identical conditions.

 

The study indicated that the test item, Di-n-butylfumarate does not have the potential to cause gene mutation at the hprt locus at the concentrations tested and under the conditions of testing.

Reference 3

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:

8 April 2004 to 19 October 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Study

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

Target gene:

his- (s. typhimurium)

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

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 March 2004. 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.Additionally to the counting of colonies the bacterial background of the plates was inspected visually. The following signs of toxicity, if present, were recorded: A reduced bacterial background lawn (mottled instead of homogeneous). Microcolonies of bacteria instead of a homogeneous background lawn. No background lawn. Clearly reduced numbers of revertant colonies.

Statistics:

Means and standard deviation were calculated for the number of mutants in every concentration group.

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

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

Key result

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

Key result

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:

A precipitate was visible when the test substance was mixed with the agar at the 5000 and 1667 µg/plate samples. When the plates were counted, no precipitate was seen.

Conclusions:

Interpretation of results: negativeAccording to these results, "DIBUTYL FUMARATE" is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 up to 5000 µg/plate, which is the limit concentration for this kind of test.

Executive summary:

Method

"DIBUTYL FUMARATE" 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 to5000 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 of the test substance to the bacteria was observed up to 5000 µg per plate.

 

Solubility:

At the 5000 and 1667 µg/plate samples a precipitate was visible when the test substance was mixed with the agar. When the plates were counted, no precipitate was seen.

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, "DIBUTYL FUMARATE" 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.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Short description of key information:
Bacterial reverse mutation test (Ames test), OECD 471: not mutagenic.
In vitro chromosome aberration test, OECD 472: not clastogenic.
In vitro mammalian cell gene mutation test, OECD 476: not mutagenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification is derived from the results a bacterial reverse mutation assay (Ames test), a mammalian cell gene mutation test (HPRT) and an in vitro chromosome aberration test. All three tests gave negative results..