Ethyl 2-methylbutyrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No effect found in any of the three studies, therefore substance not classified.

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:

19 May to 22 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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

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

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.

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'.

- 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.

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.

Reference 2

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:

27 September 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Control: 0 µg/plate
Solvent control: 0 µg/plate
Ethylmethylbutyrate-2: 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

no

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-amino anthracene

Details on test system and experimental conditions:

CONTROL SUBSTANCES
2-Aminoanthracene (A3880-0, Aldrich, Steinheim), 9-aminoacridine (801112, Merck, Darmstadt), mitomycin C (lot 01073C, Serva, Heidelberg), 2-nitrofluorene (N1675-4, Aldrich, Steinheim), sodium azide (S2002, Sigma, Taufkirchen).

9-Aminoacridine, mitomycin C, and sodium azide were dissolved in distilled water. 2-Aminoanthracene, and 2-nitrofluorene were dissolved in DMSO.

REAGENTS AND NUTRIENT BROTH
Ampicillin trihydrate: Bayer, Leverkusen; L-histidine HClH2O: A. Jinomoto, Tokyo, Japan; dimethylsulfoxide (DMSO) and D-biotin: Merck, Darmstadt; crystal violet: Sigma, Deisenhofen; NADP (disodium salt) Lot No 8087C and glucose-6-phosphate (disodium salt) Lot No. 66664: ICN Biomedicals Inc., Ohio U.S.A.; Oxoid nutrient broth No.2 Lot No. 29143781: Oxoid, England; Bacteriological Agar Lot No. 84513: ICN Biomedicals, Aurora, Ohio, U.S.A..

METABOLIC ACTIVATION SYSTEM
The rat liver homogenate fractions used were prepared by King & Harnasch GmbH (lot KH3799, KH3900), 79199 Kirchzarten. The preparation of the 9000g supernatant of liver homogenates (S9) from Sprague-Dawley male rats (8-10 weeks old) induced with Aroclor 1254 (500 mg/kg body weight) is in accordance with the method recommended by Ames et al. (1975).

Each S9 lot was tested for
- protein concentration (Lowry Method)
- ability of enzymatic stimulation of benzo(a)pyrene and 2-aminoanthracene induced mutagenesis with tester strains TA98, TA100, and TA102.
- sterility

The S9 preparations (in 0.15 M KCI) were stored in liquid nitrogen.
S9-mix containing 10% S9 was freshly prepared for each mutagenicity assay. The concentration of the cofactors in the S9-mix (per ml) are: distilled water, 0.335 ml; phosphate buffer (0.2 M, pH 7.4), 0.5 ml; NADP (0.1 M), 0.04 ml; glucose-6-phosphate (1 M), 0.005 ml; MgCI2/KCI (0.4M/1.65M), 0.02 ml; S9 fraction, 0.1 ml. The S9-mix was kept on ice.

TEST PROCEDURE
Tests were performed by the standard plate-incorporation assay with and without liver homogenate activation as described by Ames et al. (1975). To each culture tube containing 2 ml of top agar * 0.1mI of bacteria was added followed by the test solution and 0.5 ml of S9-mix or phosphate buffer in the assays without metabolic activation. The test components were mixed thoroughly with a vortex and immediately poured onto coded minimal agar plates and carefully spread to achieve an uniform distribution of the top agar on the surface of the plate. The minimal agar plates contain 20 to 25 ml of 1.5% agar in Vogel-Bonner medium E with 2% glucose. Three parallel plates were prepared for each experimental point. Plates were kept for 48 to 72 h at 37°C in the dark and then examined. Besides the counting of the number of revertant colonies (his+ revertants), the plates were examined for the existence of a normal background lawn and/or precipitates and microscopically for microcolony growth.

When there is any question about the nature of colonies scored as revertants and when positive mutagenic results are obtained, the genotype of revertant colonies are spot-checked by picking and streaking on histidine free plates.

To confirm the reversion properties and specificity of the bacterial strains as well as the activity of the liver homogenates used, positive mutagenesis controls were run simultaneously. The genotypes of the tester strains (histidine requirement, spontaneous reversion frequency, sensitivity to UV-light, crystal violet and ampicillin) were checked in each experiment. The Vogel-Bonner minimal plates, the test compound, and the S9-mix were checked for sterility.

Repetition: The experiment was repeated in full after an interval of at least 3 days.

*composition: 0.6% agar, 0.5% NaCl, 10% of 0.5 mM stock solution of L-histidine and D-biotin. The molten agar was kept at 45°C in portions of 2ml in sterile culture tubes.

Evaluation criteria:

No details provided in report

Statistics:

Estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level, using a X2-test (Mohn and Ellenberger, 1977).

Mohn G.R., and J. Ellenberger (1977) The use of E. coli K12/343/113 (lambda) as a multi-purpose indicator strain in various mutagenicity testing procedures, in: B.J. Kilbey (Ed.), Handbook of mutagenicity test procedures, Elsevier, Amsterdam, pp. 95-118.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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'.

The results of the mutagenicity tests of the substance ETHYLMETHYLBUTYRATE-2 are summarized in Tables 1 to 4 of attached report. The tables show individual plate counts and the mean number of revertant colonies per plate from two independent experiments.

 

The number of spontaneous revertants observed using each of the five strains was close to those previously established in our laboratory and was within the range obtained by Ames et al. (1975) as well as reported by De Serres and Shelby (1979).

 

Similarly, the results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the full activity of the metabolizing system.

 

The experiments were done with and without metabolic activation by rat liver homogenate (S9). ETHYLMETHYLBUTYRATE-2 was tested as solution in DMSO in concentrations of 15 to 5000 µg per plate in the presence and absence of S9. In the absence and in the presence of S9-mix ETHYLMETHYLBUTYRATE-2 was not bacteriotoxic. Precipitation of the test compound on the plates was not observed.

 

As shown in the Tables 1 to 4 of the attached report, the test compound ETHYLMETHYLBUTYRATE-2 failed to induce a significant or dose related increase in the mutation frequency of the tester strains in the absence and presence of a metabolic activation system. Similarly, the estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level, using a X²-test (Mohn and Ellenberger, 1977), did not reveal a significant effect at any of the test points.

 

In conclusion, these results indicate that ETHYLMETHYLBUTYRATE-2 under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system. Ames, B.N., J, McCann and E Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 31, 347-364.De Serres, FJ., and M.D. Shelby (1979) Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay, Mutation Res., 64, 9-165. Mohn G.R., and J. Ellenberger (1977) The use of E. coli K12/343/113 (lambda) as a multi-purpose indicator strain in various mutagenicity testing procedures, in: B.J. Kilbey (Ed.), Handbook of mutagenicity test procedures, Elsevier, Amsterdam, pp. 95-118.

Conclusions:

Interpretation of results (migrated information): negative Not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

In conclusion, these results indicate that ETHYLMETHYLBUTYRATE-2 under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

Executive summary:

The mutagenicity of the substance ETHYLMETHYLBUTYRATE-2 was studied with five mutant strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100, and TA102) The investigations were carried using the standard plate incorporation assay with and without liver homogenate (S9) from Aroclor 1254 pretreated male rats as metabolic activation system.

 

ETHYLMBTHYLBUTYRATE-2 was dissolved in DMSO and tested in concentrations of 15 to 5000 µg per plate in the presence and absence of S9. In the absence and presence of S9-mix ETHYLMETHYLBUTYRATE-2 was not bacteriotoxic. Precipitation of the test compound on the plates was not observed.

 

Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

 

In the concentration range investigated, ETHYLMETHYLBUTYRATE-2 did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system.

 

In conclusion, these results indicate that ETHYLMETHYLBUTYRATE-2 under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

Reference 3

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 May to 04 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Experiment 1: 81.875, 163.75, 327.5, 655 and 1310 µg/mL
Experiment 2: 100, 200, 400, 800 and 1310 µg/mL

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Evaluation criteria:

Assay Evaluation Criteria: A test item was considered positive in the mutation assay if:
i. Concentration-related biologically significant increase in mutant frequency observed in comparison with concurrent negative control and the test item causes a three-fold increase (Nestmann, E.R. et al., 1991). in the number of 6-thioguanine resistant colonies relative to concurrent negative control and such increases were statistically significant and outside of the laboratory historical negative (solvent used) control range.
ii. A net increase in mutant colonies of treated above the concurrent control was observed in at least two of the concentrations tested.

Negative results were confirmed by a repeat test (short duration), using a modification in test concentrations.

Statistics:

Weighted regression analysis was performed to evaluate the dose response relationship (Li, A.P. et al, 1987; Hsie, A.W. et al., 1981) on ethyl 2-methylbutyrate treatment groups against negative control (excluding positive controls).

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:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

- Solubility, Precipitation, pH and Osmolarity Tests: The test item was found to be insoluble in the culture medium, while found to be completely soluble in dimethyl sulfoxide (DMSO) at a concentration of 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, 327.5, 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 5 of the attached report.

 

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

 

- Cytotoxicity Test: Cytotoxicity due to the test item was assessed by calculating the percent relative cloning efficiency following treatment.

 

Precipitation was not observed in any of the tested concentrations.

 

The pH and osmolality at the beginning of the treatment at 1310 µg/mL was 7.18 and 477 mOsm/kg H₂O, respectively (compared to 7.26 and 461 mOsm/kg H₂O in the negative control) in the absence of metabolic activation, while pH and osmolality at 1310 µg/mL was 7.13 and 469 mOsm/kg H₂O, respectively (compared to 7.25 and 479 mOsm/kg H₂O in the negative control) in the presence of metabolic activation (Appendix 6 of the attached report). Hence, significant change in the pH and osmolality was not observed up to the guidelines limit concentration of 1310 µg/mL, both in the absence and presence of metabolic activation.

 

Normal growth was observed in all the tested concentration, both in the absence and presence of metabolic activation. The percent relative cloning efficiency observed was 96.44, 85.16, 77.81, 77.81 and 71.20% in the absence of metabolic activation and 90.87, 92.18, 89.53, and 61.03% in the presence of metabolic activation (2% v/v S9), at the test concentrations of 163.75, 327.5, 655 and 1310 µg/mL of culture medium, respectively. Therefore significant level of cytotoxicity (relative survival of 10 - 20%) was not observed both in the absence and presence of metabolic activation up to the guidelines limit concentration of 1310 µg/mL. Based on the observed results, 1310 µg/mL (10mM) was selected as the highest concentrations both in the absence and presence of metabolic activation for the main study experiment.

 

- Mutagenicity Test: No relevant influence of the test item on osmolality and pH value was observed in the absence and presence of metabolic activation during either trial.

  - Absolute and relative cloning efficiency/survival following treatment: The percent mean absolute cloning efficiency (ACE) and percent relative cloning efficiency/survival following the treatment both in the absence and presence of metabolic activation in trial I and II are summarised in the attached report. The mean values are provided in Table 2 of the attached report with individual data in Appendix 1 of the attached report. The dose response curves for relative cloning efficiency in the presence and absence of metabolic activation are provided in Figure 1 of the attached report.

  - Absolute cloning efficiency at selection and mutant frequency: The mean absolute cloning efficiency (ACE) at selection and mean mutation frequency per 1 x 10⁶ cells (MF) in the absence and presence of metabolic activation for trial I and II are provided in the attached report. The values of absolute cloning efficiency at selection, number of mutant colonies and mutant frequency for each test concentrations and individual data are provided in the attached report. The dose response curves for mutant frequency both in the absence and presence of metabolic activation for trial I and II and provided in the attached report. The values of absolute cloning efficiency at selection, number of mutant colonies and mutant frequency for each test concentrations are provided in Table 3 of the attached report with individual data in Appendix 2 and Appendix 3 of the attached report. The dose response curves for mutant frequency both in the absence and presence of metabolic activation for trial I and II are provided in Figure 2 of the attached report.

 

A weighted regression analysis was performed to evaluate the significant dose-related effect in the mutation frequency of cultures treated with ethyl 2-methylbutyrate with the concurrent negative control group. Statistical analysis was not performed for the positive controls.

 

The absolute cloning efficiency in the negative control was above 60% during both the trials. The mutant frequency, in the negative control group

was less than 20 per 10⁶cells clonable cells during both the trials, validating the acceptability of the test item (Li, A.P. et al., 1987). A significant

dose-related increase in the mutation frequency was not observed in any of the treated concentrations, and the mutation frequency was comparable to that from the negative control group. The increased mutant frequency observed in the positive controls in trials I and II demonstrated the efficiency of the test system and suitability of the test procedures and conditions employed in the study.

Conclusions:

Interpretation of results (migrated information): negative

From these results it is concluded that ethyl 2-methylbutyrate does not have potential to induce gene mutations at the hgprt locus of CHO-K1 cells, both in the absence and presence of metabolic activation under the present experimental conditions.

Executive summary:

This study was performed to evaluate the mutagenic potential of ethyl 2-methylbutyrate (supplied by Toyo Gosei Co., Ltd.,) both in the absence and presence of metabolic activation using the CHO-K1 cell line. The methods followed were compliant with the OECD 476 test guidelines.

 

Dose selection for the gene mutation experiments was based on pre-tests -considering the cytotoxicity, the occurrence of precipitation, changes in the pH and osmolality.

 

Ethyl 2-methylbutyrate was tested in two independent experiments, with and without metabolic activation (2% v/v S9). The cultures treated with solvent used (DMSO) were kept both in the absence and presence (2% v/v S9) of metabolic activation and served as negative controls. Ethyl methanesulfonate (0.4 µL/mL) and Benzo(a)pyrene (6 µg/mL) were used as the positive control in the absence and presence of metabolic activation, respectively.

In the first mutagenicity experiment (Trial I), the CHO-K1 cells 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 4 hours. No significant dose-related effect was observed in any of the treated concentrations both in the absence and presence of metabolic activation (2% v/v S9) and the induced mutation frequency was comparable to that from the negative control group.

 

A second trial was conducted to confirm the negative results obtained in trial I in the absence and presence of metabolic activation (2% v/v S9). The proliferating cells were exposed to ethyl 2- methylbutyrate at the test concentrations of 100, 200, 400, 800 and 1310 µg/mL culture media both in the absence and presence of metabolic activation system (2% v/v S9) for a period of 4 hours. No significant dose-related effect was observed in any of the treated concentrations both in the absence and presence of metabolic activation (2% v/v S9) and the induced mutation frequency was comparable to that from the negative control group.

No significant dose-related effect was observed at any of the treated concentrations during either trial of the experiments.

 

No relevant influence of the test item on pH value and osmolality was observed both in the absence and presence of metabolic activation during both the trials.

 

The absolute cloning efficiency of the CHO-K1 cell line (negative control) was above 60% in both the trials. The spontaneous mutation level was (negative control) within the acceptable limit [less than 20 per 10⁶clonable cells (Li, A.P.et al., 1987)] in both the trials, validating the acceptability of the test system. The increased mutant frequency observed in positive controls in trial I and II demonstrated the efficiency of the test system and suitability of the test procedures and conditions employed in the study.

From these results, it is concluded that ethyl 2-methylbutyrate does not have potential to induce gene mutations at the hgprt locus of CHO-K1 cells both in the absence and presence of metabolic activation under the present experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

- Mutagenicity study of ETHYLMETHYLBUTYRATE-2 in the Salmonella typhimurium/mammalian microsome reverse mutation assay (Ames-Test): Not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA9S, TA100, and TA102 in the presence and absence of a metabolizing system.

- In vitro mammalian chromosome aberration test of ethyl 2-methylbutyrate in human peripheral blood lymphocytes: 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.

- In vitro mammalian cell gene forward mutation test at the hgprt locus of the Chinese hamster ovary (CHO)-K1 cell line using Ethyl 2-methylbutyrate: From these results it is concluded that ethyl 2-methylbutyrate does not have potential to induce gene mutations at the hgprt locus of CHO-K1 cells both in the absence and presence of metabolic activation under the present experimental conditions.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No effect found in any of the three studies, therefore substance not classified.