Ethyl 2-methylvalerate

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 23 August 2012

Reliability:

1 (reliable without restriction)

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-methylvalerate (Ethyl 2-methylpentanoate)

IUPAC Name:

Ethyl 2-methylvalerate (Ethyl 2-methylpentanoate)

Constituent 2

Reference substance name:

Ethyl 2-methylvalerate

EC Number:

254-384-1

EC Name:

Ethyl 2-methylvalerate

Cas Number:

39255-32-8

Molecular formula:

C8H16O2

IUPAC Name:

ethyl 2-methylpentanoate

Constituent 3

Reference substance name:

Pentanoic acid, 2-methyl-, ethyl ester

IUPAC Name:

Pentanoic acid, 2-methyl-, ethyl ester

Details on test material:

- Name of test material (as cited in study report): Ethyl 2-methylvalerate (Ethyl 2-methylpentanoate)
- Analytical purity: 99.94 (GC%) [Refer Certificate of Analysis in Appendix 9 of the attached report]
- Lot/batch No.: H3-G-26
- Expiration date of the lot/batch: July 25, 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 was 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: July 26, 2011
- Manufactured by: Toyo Gosei Co. Ltd.
- Supplied by: Toyo Gosei Co. Ltd.
- Appearance: Colorless liquid/fruity odour

Method

Species / strain

Species / strain / cell type:

other: human peripheral blood lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- In the first mutagenicity experiment (Phase I), human peripheral blood lymphocyte cultures were exposed to ethyl 2-methylvalerate (ethyl 2-methylpentanoate) at the test concentrations of 90.625, 181.25, 362.5, 725, and 1450 µg/mL of culture media, both in the absence and presence of metabolic activation (2% v/v S9) for a period of 3 h 30 minutes.
- In the second mutagenicity experiment, human peripheral blood lymphocyte cultures were exposed to ethyl 2-methylvalerate (ethyl 2-methylpentanoate) at the dose levels of 90.625, 181.25, 362.5, 725, and 1450 µg/mL of culture media both in the absence (Phase II) and presence of (Phase III) metabolic activation (4% v/v S9).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Selection time (if incubation with a selection agent): up to 24h

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

OTHER EXAMINATIONS:
- Determination of endoreplication: none

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:

lymphocytes: Human blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:no change observed
- Effects of osmolality: no change observed
- Water solubility: insoluble
- Precipitation: none observed

Remarks on result:

other: strain/cell type: peripheral human blood lymphocytes

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 145000 µg/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 181.25, 362.5, 725 and 1450 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 1450 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 181.25, 362.5, 725 and 1450 µg/mL (approx. 10 mM) of ethyl 2-methyvalerate (ethyl 2-methylpentanoate).

 

Precipitation was not observed in any of the tested concentrations.

 

The pH and osmolality at the beginning of the treatment of a concentration of 1450 µg/mL were 7.24 and 440 mOsm/kg H₂O, respectively (compared to 7.25 and 447 mOsm/kg H₂O in the negative control) in the absence of metabolic activation. pH and osmolality of a concentration of 1450 µg/mL were 7.28 and 439 mOsm/kg H₂O, respectively (compared to 7.26 and 446 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 higher concentration of 1450 µg/mL, both in the absence and presence of metabolic activation.

 

The reduction in mitotic index observed was -2.05, -7.15, 5.05, -28.65% and 15.06, 14.18, 26.15, 31.72% at the concentrations of 181.25, 362.5, 725 and 1450 µg/mL of ethyl 2-methylvalerate (ethyl 2-methylpentanoate), in the absence and presence of metabolic activation (2% v/v S9), respectively.

 

Hence, 1450 µg/mL (10 mM) was selected as the highest concentration to be tested in the main study experiment. The mean percent mitotic index and individual values of replicate culture for percent 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-methylvalerate (ethyl 2-methypentanoate), with the positive and negative (DMSO) controls are provided in Table 2 of the attached report. The reduction in percent mitotic index observed was -9.12, 16.83, 21.66, 9.31, 9.12% and 14.65, 10.06, 16.09, 9.92, 30.94% at the test concentrations of 90.625, 181.25, 362.5, 725 and 1450 µg/mL of ethyl 2-methylvalerate (ethyl 2-methylpentanoate), 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 1450 µg/mL. Therefore, dose concentrations selected for scoring of chromosome aberrations both in the absence and presence of metabolic activation were: 362.5, 725 and 1450 µg/mL culture medium.

 

Both in the absence and presence of metabolic activation, ethyl 2-methylvalerate (ethyl 2-methylpentanoate) 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-methylvalerate (ethyl 2-methylpentanoate) did not increase the number of cells with endoreduplicated chromosomes (see Appendix 1 of the attached report). Ethyl 2-methylvalerate (ethyl 2-methylpentanoate) did not induce increases in the number of polyploid 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-methylvalerate (ethyl 2-methypentanoate), 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 -8.30, 3.68, -6.94, -11.37, 8.74% and -3.83, -3.03, 13.39, -0.64, 31.74% at the test concentrations of 90.625, 181.25, 362.5, 725 and 1450 µg/mL of ethyl 2-methylvalerate (ethyl 2-methypentanoate), in the absence (Phase II) and presence (Phase III) of metabolic activation, respectively.

 

Hence the desired level of cytotoxicity was not observed both in the absence (phase II) and presence (phase III) of metabolic activation up to the guidelines limit concentration of 1450 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): 362.5, 725 and 1450 µg/mL culture medium.

 

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

 

Statistically significant reduction in the mitotic index was observed in the culture treated at the test concentration of 1450 µg/mL as well as in the cultures treated with positive control chemical cyclophosphamide, in the presence (Phase III) of metabolic activation.

 

The number of cells with chromosome aberrations found in the negative control cultures was within the laboratory historical control data range. The positive control chemical mitomycin-C and cyclophosphamide produced either statistically significant or 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 polyploid 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-methylvalerate (ethyl 2-methypentanoate) did not show any potential to induce chromosomal aberrations, both in the absence and presence (2 and 4% v/v S9) of metabolic activation under the present experimental conditions.

Executive summary:

This study was conducted to evaluate the potential of ethyl 2-methylvalerate (ethyl 2-methylpentanoate) (supplied by Toyo Gosei Co., Ltd) to induce chromosomal aberrations in human peripheral blood lymphocyte cultures. The methods followed were compliant with the OECD No. 473 test guidelines.

                 

Dose selection for the cytogenetic experiments was based on pre-tests, considering the cytotoxicity, the occurrence of precipitation, changes in the pH and 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 1450 µg/mL. Hence, 1450 µg/mL (approx. 10 mM) was selected as the highest concentration to be tested in main study experiment i.e., concentration above the limit of solubility and more than one concentration with visible precipitation.

 

Ethyl 2-methylvalerate (ethyl 2-methylpentanoate) was tested in two independent experiments.

 

In the first mutagenicity experiment (Phase I), human peripheral blood lymphocyte cultures were exposed to ethyl 2-methylvalerate (ethyl 2-methylpentanoate) at the test concentrations of 90.625, 181.25, 362.5, 725, and 1450 µg/mL of culture media, both in the absence and

presence of metabolic activation (2% v/v S9) for a period of 3 h 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 (close to 50%), was not observed up to the tested concentration of 1450 µg/mL, both in the absence (Reduction - 9.12%) and presence of metabolic activation (Reduction - 30.94%).

 

In the second mutagenicity experiment, human peripheral blood lymphocyte cultures were exposed to ethyl 2-methylvalerate (ethyl 2-methylpentanoate) at the dose levels of 90.625, 181.25, 362.5, 725, and 1450 µg/mL of 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-methylvalerate (ethyl 2-methylpentanoate) was tested up to 1450 µg/mL for a 24 h continuous exposure time in the absence of metabolic activation (Phase II). In the presence of metabolic activation (Phase III), ethyl 2- methylvalerate (ethyl 2-methylpentanoate) was tested up to 1450 µg/mL for a 3 h and 30 minutes with increased concentration of metabolic activation (4% v/v S9). Significant reduction in the mitotic index (≥ 50%), was not observed up to the concentration of 1450 µg/mL, both in the absence (Phase II - 8.74%) and presence (Phase III - 31.74%) of metabolic activation.

The number of cells with chromosome aberrations found in the negative control cultures was within the historical control data range. 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-methylvalerate (ethyl 2-methylpentanoate) did not induce a statistically significant or 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-methylvalerate (ethyl 2-methylpentanoate) on the number of polyploid 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-methylvalerate (ethyl 2-methylpentanoate) did not show potential to induce chromosomal aberrations both in the absence and presence of metabolic activation under the present experimental conditions.