Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key 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)

Link to relevant study records

Referenceopen allclose all

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
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
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) 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
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.
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

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.

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.

Endpoint:
in vitro gene mutation 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
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
S9 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
Both in the presence and absence of metabolic activation:
a) 155 b) 434 c) 1214 and d) 3400 µg/mL (factor of 2.8)

Confirmatory Gene Mutation Assay
Both in the presence and absence of metabolic activation:
a) 108 b) 340 c) 1080 and d) 3400 µg/mL (factor of v10)
Vehicle / solvent:
One hundred fifty microlitres (150 µL) Ethanol were 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:
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.

Ham‘s 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.
Dulbecco‘s Phosphate buffered saline (PBS) (pH: 7.4)
Trypsin: EDTA solution
Following chemicals were used in the study:
Name / Lot/Batch No. / Manufacturer
Potassium chloride / 24076802-1 / Qualigens Fine Chemicals Navi Mumbai 400 710, INDIA
Magnesium chloride / MF6M561553 & SK9F590599 / Merck Specialities Pvt. Ltd. Worli, Mumbai 400 018, INDIA
Fetal Bovine Serum / 41F9293K & 41F7596K / 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
EDTA / 4-0091 / Hi-Media Laboratories Pvt. Ltd., Mumbai 400 086, INDIA
Glucose-6-phosphate / 00000065463 / Hi-Media Laboratories Pvt. Ltd., Mumbai 400 086, INDIA
NADP / 00000055489 / 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
Methylene blue / 0000073631 / Hi-Media Laboratories Pvt. Ltd., Mumbai 400 086, INDIA
Ethanol / K35664003604 / Merck K Ca A Darmstadt, Germany
Amphoterecin B / 128K4046 / Sigma Aldrich Co. St. Louis, MO63103, USA
6-Thioguanine / 036K 0173 / Sigma Aldrich Co. St. Louis, MO63103, USA
Trypsin / 039K7013 / Sigma Aldrich Co. St. Louis, MO63103, USA
Ethylmethane sulphate (62-50-0) / 1423147 / Sigma Aldrich Co. St. Louis, M063103, USA
Benzo (a) pyrene (50-32-6) / 017K1044 / Sigma Aldrich Co. St. Louis, M063103, USA
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 formula

Y = (X + A) B
where,
Y = transformed mutant frequency
X = observed mutant frequency
and 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

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.

 

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 either in the presence or in the absence of metabolic activation up to the highest tested concentration of 3406 µg/mL.

 

Stability Test and Dose Formulation Analysis

Stability of dose formulations of Dioctylfumarate at the concentrations 1.5 mg/mL and 340 mg/mL in Ethanol were found to be stable for 5 hours when stored at room temperature.

The samples sent for the dose formulation analysis confirmed to the presence of Dioctylfumarate and were well within the limits

 

Parallel Cytotoxicity Assessment

In the initial assay, in the presence of metabolic activation, at the highest concentration tested, the RCE was 56%. In the absence of metabolic activation, at the highest concentration tested, the RCE was 39%. In the confirmatory assay, at the highest concentration tested, the RCE was 51 and 45% both in the presence and absence of activation, respectively

 

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 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:
It is concluded that the test item, Dioctylfumarate, 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 Dioctylfumarate 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).

 

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 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 3406 µg/mL either in the presence or in the absence of metabolic activation. The test item did not precipitate the test solutions up to 3406 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 155, 434, 1214 and 3400 µg/mL of the medium for 4 hours in the presence and absence of metabolic activation. In the confirmatory gene mutation assay, CHO cells were exposed to the test item in duplicate at concentrations of 108, 340, 1080 and 3400 µg/mL of the medium for 4 hours in the presence and absence of metabolic activation. 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, Dioctylfumarate, does not have the potential to cause gene mutation at the hprt locus at the concentrations tested and under the conditions of testing. 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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.