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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES test

A bacterial reverse mutation test was performed to determine the mutagenic nature of the test substance. The study was performed using Salmonella typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as a preincubation assay at six different concentration with 10 mg/plate being the maximum concentration.Cells were pre-incubated with both the test chemical and S9-mix for 20 min. The number of revertant (His+) colonies was scored after 48 hours of incubation. The result was considered positive if the number of revertant colonies was found twice the number of the controls(exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system. Hence,Ethyl butyrate is considered asNon-mutagenic (negative)in vitro in the test system used under the certain experimental conditions.

Mammalian chromosomal aberration test

An in vitro mammalian chromosomal aberration study was performed to determine the mutagenic nature of test chemical. The cells were exposed to the test material at three different doses with 2 mg/ml being the maximum concentration for 48 hr. Colcemid (0.2 µg/ml in final concentration) was added to the culture 2 hrs before cell harvesting. The slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation (liver microsome solution, S9 mix) systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others were recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9 %, equivocal if it was between 5.0 and 9.9 %, and positive if it was more than 10.0 %. The results showed thatEthyl butyrate did not give a rise of the number of cells with chromosome aberrations and/or induce polyploid cells when CHL cells were exposed to 2 mg/ml of test chemical in the absence of S9-mix. Thus, Ethylbutyrate is considered asNon-mutagenic (negative)when CHL cells are exposed to the test chemical without metabolic activation under the presented experimental conditions.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
A salmonella/microsome test (Ames tests) was performed on the test substance to evaluate its mutagenic effect.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
other: TA92, TA1535,TA100, TA1537, TA94 and TA98
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data available
Metabolic activation:
with and without
Metabolic activation system:
The liver microsome fraction (S-9) was prepared from the liver of Fischer rats
Test concentrations with justification for top dose:
Six different concentrations were used with 10 mg/plate as the maximum concentration.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available
Rationale for test conditions:
No data available
Evaluation criteria:
The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated).
Statistics:
No data available
Species / strain:
other: TA92, TA1535,TA100, TA1537, TA94 and TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
The maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment.
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce a doubling of His + revertant colonies over the control using S. typhimurium TA92, TA1535, TA100, TA1537, TA94, TA98 strains in the presence and absence of metabolic activation system. Hence, Ethyl butyrate is considered as Non-mutagenic (negative) in vitro in the test system used under the certain experimental conditions.
Executive summary:

A bacterial reverse mutation test was performed to determine the mutagenic nature of the test substance. The study was performed using Salmonella typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as a preincubation assay at six different concentration with 10 mg/plate being the maximum concentration.Cells were pre-incubated with both the test chemical and S9-mix for 20 min. The number of revertant (His+) colonies was scored after 48 hours of incubation. The result was considered positive if the number of revertant colonies was found twice the number of the controls(exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system. Hence, Ethyl butyrate is considered as Non-mutagenic (negative)in vitro in the test system used under the certain experimental conditions.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
A Chromosomal aberration tests in vitro was performed on the test substance to evaluate its mutagenic effect.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
No data available
Species / strain / cell type:
other: Chinese hamster fibroblast cell line
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum.
- Properly maintained: Yes, by 4 day passages
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data available
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
At three different doses with 2 mg/mL being the maximum dose concentration.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO
Untreated negative controls:
yes
Remarks:
Untreated cells served as negative controls.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: No data available
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): Giemsa solution (1.5%, pH 6.8)
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: 100 well spread metaphases

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No data available
- Other: No data available
Rationale for test conditions:
No data available
Evaluation criteria:
The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. In which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%,and positive if it was more than 10.0%.
Statistics:
No data available
Species / strain:
other: Chinese hamster fibroblast cell line
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer.

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce chromosomal aberrations when CHL cells were exposed to 2 mg/ml of test chemical in the absence of metabolic activation thus, Ethyl butyrate is considered as Non-mutagenic (negative) substance under the presented experimental conditions.
Executive summary:

An in vitro mammalian chromosomal aberration study was performed to determine the mutagenic nature of test chemical. The cells were exposed to the test material at three different doses with 2 mg/ml being the maximum concentration for 48 hr. Colcemid (0.2 µg/ml in final concentration) was added to the culture 2 hrs before cell harvesting. The slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation (liver microsome solution, S9 mix) systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others were recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9 %, equivocal if it was between 5.0 and 9.9 %, and positive if it was more than 10.0 %. The results showed thatEthyl butyrate did not give a rise of the number of cells with chromosome aberrations and/or induce polyploid cells when CHL cells were exposed to 2 mg/ml of test chemical in the absence of S9-mix. Thus, Ethylbutyrate is considered asNon-mutagenic (negative)when CHL cells are exposed to the test chemical without metabolic activation under the presented experimental conditions.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in vitro:

Various publications available were reviewed to determine the mutagenic nature of the test chemical. The studies are as summarised below:

#1

A bacterial reverse mutation test was performed to determine the mutagenic nature of the test substance. The study was performed using Salmonella typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as a preincubation assay at six different concentration with 10 mg/plate being the maximum concentration. Cells were pre-incubated with both the test chemical and S9-mix for 20 min. The number of revertant (His+) colonies was scored after 48 hours of incubation. The result was considered positive if the number of revertant colonies was found twice the number of the controls (exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system. Hence, Ethyl butyrate is considered as Non-mutagenic (negative)in vitro in the test system used under the certain experimental conditions. 

#2

An in vitro mammalian chromosomal aberration study was performed to determine the mutagenic nature of test chemical. The cells were exposed to the test material at three different doses with 2 mg/ml being the maximum concentration for 48 hr. Colcemid (0.2 µg/ml in final concentration) was added to the culture 2 hrs before cell harvesting. The slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation (liver microsome solution, S9 mix) systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others were recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9 %, equivocal if it was between 5.0 and 9.9 %, and positive if it was more than 10.0 %. The results showed that Ethyl butyrate did not give a rise of the number of cells with chromosome aberrations and/or induce polyploid cells when CHL cells were exposed to 2 mg/ml of test chemical in the absence of S9-mix. Thus, Ethyl butyrate is considered as Non-mutagenic (negative)when CHL cells are exposed to the test chemical without metabolic activation under the presented experimental conditions.

 

#3

A bacterial gene mutation test was performed on test chemical using Salmonella typhimurium strains TA102 and TA97. The bacterial cells were preincubated with the test substance at the dose levels of 0.01 to 1 mg/plate and withS9 mix or phosphate buffer (pH 7.4) for 20 min. The test chemical was solved in DMSO. The plates were observed for a dose-dependent increase in the number of revertant/plate. As seen by the results, no tested concentrations of the target chemical induced gene mutation in S. typhimurium strains TA97 and TA102 in the presence or absence of S9 metabolic activation system. Hence, Ethyl butyrate is considered as Non-mutagenic (negative) in bacterial reverse mutation test in vitro.

 #4

An in vitro mammalian chromosome aberrations test was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster lung cell line CHL / IU in the presence and absence of metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 0.075, 0.15, 0.30, 0.60, 1.2 mg/ml. The doses for the main study were based on the preliminary dose range finding study. The experiment was performed either by a short-term treatment method with exposure duration of 6 hrs or by a continuous treatment method with 24 hrs of exposure time. Based on the observations made, the test chemical did not induceanincrease in the number of cells with chromosome aberrationswhen CHL cell were exposed for 6 or 24 hrs to the test chemical with or without S9 metabolic activation. No effect ofEthyl butyrateon the number of polyploidy cells was observed. Thus, Ethylbutyrate is considered asNon-mutagenic (negative)when CHL cells are exposed to the test chemical at the concentration ≤ 1.2 mg/ml under the presented experimental conditions.

 

Based on the in vitro experimental data available, the test chemical of Ethyl butyrate ( CAS 105-54-4) does not induce gene mutation in vitro. Hence the test chemical is not likely to classify as a mutagenic substance.

Justification for classification or non-classification

Based on the in vitro experimental data available, the test chemical of Ethyl butyrate ( CAS 105-54-4) does not induce gene mutation in vitro. Hence the test chemical is not likely to classify as a mutagenic substance according to CLP regulations.