Ethyl 2-methylbutyrate

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:

19 May to 22 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

Ethyl 2-methylbutyrate

EC Number:

231-225-4

EC Name:

Ethyl 2-methylbutyrate

Cas Number:

7452-79-1

Molecular formula:

C7H14O2

IUPAC Name:

ethyl 2-methylbutanoate

Constituent 2

Reference substance name:

Ethyl 2-methylbutyrate/Ethyl 2-methylbutanoate

IUPAC Name:

Ethyl 2-methylbutyrate/Ethyl 2-methylbutanoate

Constituent 3

Reference substance name:

Butanic acid, 2-methyl-, ethyl ester

IUPAC Name:

Butanic acid, 2-methyl-, ethyl ester

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Ethyl 2-methylbutyrate
- Analytical purity: 99.89% (See Appendix 9 of the attached report for details)
- Lot/batch No.: H3-H-5
- Expiration date of the lot/batch: August 04, 2014
- Storage condition of test material: (at JRF) - As per the instruction received from the Sponsor on storage of the test item, the test item will be stored in its original container as supplied by the Sponsor in refrigerator in the Test Item Control Office (TICO). The stability of the test item in storage is the responsibility of the Sponsor.
- Other:
- Date of manufacture: August 05, 2011
- Supplied by: Toyo Gosei Co., Ltd.
- Manufactured by: Toyo Gosei Co., Ltd.
- Appearance/colour/Odour: Colourless clear liquid/Fruity odour

Method

Species / strain

Species / strain / cell type:

other: human peripheral blood lymphocytes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiments 1 and 2: 81.875, 163.75, 327.5, 655 and 1310 µg/mL

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Remarks:

I. In the Absence of Metabolic Activation: Mitomycin-C; II. In the Presence of Metabolic Activation: Cyclophosphamide

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

STAIN (for cytogenetic assays): 5% Giesma

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: >1000

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Assay Evaluation Criteria: The test item was considered to have shown clastogenic activity in this study if the following criteria were met:
i. Increased frequency of metaphases with aberrant chromosomes observed at one or more test concentrations (concentration-related).
ii. Increases were reproducible between replicate cultures and between tests (when treatment conditions are the same).
iii. Increases in percent aberrant metaphases were statistically significant.
iv. Increases in percent aberrant metaphases were not associated with large changes in pH and osmolality of the treatment medium.
- Historical control data for this laboratory was also considered in the evaluation.
- An increase in the number of polyploidy cells indicates that the test item has the potential to inhibit mitotic processes and to induce numerical chromosome aberration. Biological relevance was considered first. The test item was considered non-mutagenic if the results did not meet the above-mentioned criteria.

Statistics:

Statistical Analysis: Gaps and polyploidy were not be included in the calculation of total aberration frequency. Data on mitotic index, polyploidy and percent aberrant cells were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s t-test (Gad. and Weil, 1994). Where the data did not meet the homogeneity of variance, Student’s t-test was performed to determine the level of significant difference between the negative control and three selected test concentrations (selected based on the mitotic index data) and positive controls.

Results and discussion

Test results

Species / strain:

mammalian cell line, other: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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

- Solubility, Precipitation, pH and Osmolality Tests: The test item was insoluble in culture medium, but soluble in dimethyl sulfoxide (DMSO) at the concentration 131 mg/mL. Therefore DMSO was selected as vehicle for this study.

 

A significant change in pH (±1 unit) or osmolality (≥ 50 mOsm/kg H₂O) was not observed at 0h and 3h in any of the tested concentrations of 163.75, 327.50, 655 and 1310 µg/mL of culture medium. Precipitation was not observed in any of the tested concentrations. The results of pH, osmolality and precipitation tests are provided in Appendix 2 of the attached report.

 

Therefore, the guidelines limit concentration of 1310 µg/mL (10 mM) was selected as the highest concentration for the cytotoxicity experiment.

 

- Cytotoxicity test: The cytotoxicity was assessed based on the results of precipitation pH and osmolality tests at the concentrations of 163.75, 327.5, 655 and 1310 µg/mL (approx. 10 mM) of ethyl 2-methybutyrate.

 

Precipitation was not observed in any of the tested concentrations. The pH and osmolality at the beginning of the treatment of a concentration of 1310 µg/mL were 7.31 and 451 mOsm/kg H₂O, respectively (compared to 7.30 and 445 mOsm/kg H₂O in the negative control) in the absence of metabolic activation. pH and osmolality of a concentration of 1310 µg/mL were 7.32 and 448 mOsm/kg H₂O, respectively (compared to 7.33 and 448 mOsm/kg H₂O in the negative control) in the presence of metabolic activation (see Appendix 3 of the attached report). Hence, a significant change in the pH or osmolality was not observed up to the guidelines limit concentration of 1310 µg/mL, noth in the absence and presence of metabolic activation.

 

The reduction in mitotic index observed was -6.50, 18.57, -3.69, 27.42% and 9.99, 15.65, 19.17, 29.71% at the concentrations of 163.75, 327.5, 655 and 1310 µg/mL of ethyl 2-methylbutyrate, in the absence and presence of metabolic activation (2% v/v S9), respectively.

 

Hence, 1310 µg/mL was selected as the highest concentration to be tested in the main study experiment i.e. concentration above the limit of solubility, and more than one concentration with visible precipitation. The mean percent mitotic index and individual values of replicate culture for percent reduction in mitotic index and percent reduction in mitotic index are provided in Table 1 of the attached report.

 

- Main study: No relevant influence of the test item on pH value or osmolality was observed in the absence (Phase I and II) and presence of metabolic activation (Phase I and III).

 

Precipitation was not observed at any of the tested concentrations.

 

Phase I [Absence and presence (2% v/v S9) of metabolic activation and exposure for 3h and 30 minutes]: The mitotic indices of cultures treated with various concentrations of ethyl 2-methylbutyrate, with the positive and negative (DMSO) controls are provided in Table 2 of the attached report. The reduction in percent mitotic index observed was 6.67, 15.13, 1.81, 6.24, 26.74% and 9.71, 5.29, 9.86, 20.39, 31.27% at the test concentrations of 81.875, 163.75, 327.5, 655 and 1310 µg/mL of ethyl 2-methybutyrate, in the absence and presence of metabolic activation, respectively.

 

Hence, a significant level of cytotoxicity was not observed in phase I (both in the absence and presence of metabolic activation) up to the guidelines limit concentration of 1310 µg/mL. Therefore, dose concentrations selected for scoring of chromosome aberrations both in the absence and presence of metabolic activation were: 327.5, 655 and 1310 µg/mL culture medium.

 

Both in the absence and presence of metabolic activation, ethyl 2-methylbutyrate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (see Table 3 and Appendix 1 of the attached report).

 

Both in the absence and presence of metabolic activation, ethyl 2-methybutyrate did not increase the number of cells with endoreduplicated chromosomes (see Appendix 1 of the attached report). Ethyl 2-methybutyrate did not induce increases in the number of polyploidy cells both in the absence and presence of metabolic activation (2% v/v S9) (see Table 3 and Appendix 1 of the attached report).

 

Phase II [Absence of metabolic activation and exposure for 24h] and Phase III [Presence (4% v/v S9) of metabolic activation and exposure for 3h and 30 minutes]: The mitotic indices of cultures treated with various concentrations of ethyl 2-methybutyrate, with the positive and negative (DMSO) controls both in the absence (Phase II) and presence (Phase III) of metabolic activation system are presented in Table 2 of the attached report. The reduction in mitotic index observed was 22.53, 24.04, 14.79, 11.15, 33.89% and 21.79, 19.58, 12.26, 14.95, 20.22% at the test concentrations of 81.875, 163.75, 327.5, 655 and 1310 µg/mL of ethyl 2-methylbutyrate, in the absence (Phase II) and presence (Phase III) of metabolic activation, respectively.

 

Hence the desired level of cytotoxicity i.e. reduction of the mitotic index (≥ 50%), was not observed both in the absence and presence of metabolic activation at the dose level of 1310 µg/mL. Therefore, dose levels selected for scoring of chromosome aberrations were:

 

Phase II (In the absence of metabolic activation system) and Phase III (In the presence of metabolic activation system): 327.5, 655 and 1310 µg/mL culture medium.

 

Both in the absence and presence of metabolic activation, ethyl 2-methylbutyrate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (see Table 3 and Appendix 1 of the attached report).

 

Both in the absence and presence of metabolic activation, ethyl 2-methylbutyrate did not increase the number of cells with endoreduplicated chromosomes (see Appendix 1 of the attached report). Ethyl 2-methylbutyrate did not induce increases in the number of polyploidy cells both in the absence and presence of metabolic activation (4% v/v S9) (see Table 3, Appendix 1 of the attached report).

 

The number of cells with chromosome aberrations found in the negative control cultures was within the laboratory historical control data range except in phase II/-S9, where percent aberrant cells excluding gaps were 3.0%, against a historical range of 2.5%, through the aberrant cells were within the combined historical range of 0-3% for negative controls ±S9, in this laboratory. As the mean of percent aberrant cells in the phase II/-S9 was very close to the historical ranges, this value was considered as valid, as mentioned in the acceptance criteria of study plan. Considering the historical range in this laboratory these small increases were considered as chance events and not related with experimental conditions. The positive control chemical mitomycin-C and cyclophosphamide produced statistically significant and biologically relevant increases in the frequency of aberrant cells during all the phases of the main study experiment. Therefore it was concluded that the test conditions were adequate and that the metabolic activation system (S9 mix) functioned properly.

 

The summaries of % mitotic index are provided in Table 2 of the attached report for phase I, II and III. Summaries of % aberrant and polyploidy cells are provided in Table 3 of the attached report and types of aberrations in Phase I, II and III with individual values are provided in Appendix 1 of the attached report.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative

From the results of this study, it is concluded that ethyl 2-methylbutyrate did not show any potential to induce chromosomal aberrations, both in the absence and presence (2 and 4% v/v S9 of metabolic activation system under the present experimental conditions.

Executive summary:

This study was conducted to evaluate the potential of ethyl 2-methylbutyrate (supplied by Toyo Gosei Co., Ltd, Japan) to induce chromosomal aberrations in human peripheral blood lymphocyte cultures. The methods followed were compliant with the OECD 473 test guidelines.

 

Dose selection for the cytogenetic experiments was based on pre-tests, considering the cytotoxicity, the occurrence of precipitation and changes in the pH or osmolality. A significant level of cytotoxicity was not observed both in the absence and presence of metabolic activation up to the guidelines limit concentration of 1310 µg/mL. Hence, 1310 µg/mL (approx. 10 mM) was selected as the highest concentration to be tested in main study experiment.

Ethyl 2-methylbutyrate was tested in two independent experiments.

In the first mutagenicity experiment (Phase I), human peripheral blood lymphocyte cultures were exposed to ethyl 2-methylbutyrate at the test concentrations of 81.875, 163.75, 327.5, 655 and 1310 µg/mL of culture media, both in the absence and presence of metabolic activation (2% v/v S9) for a period of 3h and 30 minutes. The cultures treated with solvent (DMSO) were kept both in the absence and presence (2% v/v S9) of metabolic activation and served as negative controls. Significant reduction in the mitotic index (≥ 50%), was not observed up to the concentration of 1310 µg/mL, both in the absence (Reduction - 26.74%) and presence (Reduction - 31.27%) of metabolic activation.

In the second mutagenicity experiment, human peripheral blood lymphocyte cultures were exposed to ethyl 2-methylbutyrate at the dose levels of 8 1.875, 163.75, 327.5, 655, and 1310 µg/mL culture media both in the absence (Phase II) and presence of (Phase III) metabolic activation (4% v/v S9). The cultures treated with solvent (DMSO) were kept both in the absence and presence (4% v/v S9) of metabolic activation and served as negative controls. Ethyl 2-methylbutyrate was tested up to 1310 µg/mL for a 24h continuous exposure time in the absence of metabolic activation (Phase II). In the presence of metabolic activation (Phase III), ethyl 2-methylbutyrate was tested up to 1310 µg/mL for a 3 h 30 minutes exposure time with increased concentration of S9 (4% v/v S9). A desired level of cytotoxicity i.e. reduction in the mitotic index (≥ 50%), was not observed both in the absence (Phase II - 33.89%) and presence of metabolic activation (Phase III - 20.22) up to the concentration of 1310 µg/mL.

The number of cells with chromosome aberrations found in the negative control cultures was within or close to the historical control data range (0 - 3%). Positive control chemicals, mitomycin-C and cyclophosphamide both produced either a statistically significant or biologically relevant increases in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Hence, the increased frequency of aberrations observed in the positive controls demonstrated the sensitivity and robustness of the test system and suitability of the methods and conditions employed in the experiment.

 

Ethyl 2-methylbutyrate did not induce a statistically significant and biologically relevant increase in the number of cells with chromosome aberrations in the absence or presence of S9-mix in either of the two independently repeated experiments (i.e., Phases I, II or III).

 

No effects of the ethyl 2-methylbutyrate on the number of polyploidy cells or cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

From the results of this study, it is concluded that ethyl 2-methylbutyrate did not show potential to induce chromosomal aberrations both in the absence and presence of metabolic activation under the present experimental conditions.