Ethyl acetoacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key 1 – In vitro bacterial reverse mutation assay

The assay was performed 1988 according to OECD-guidelines no. 471 and 472 with and without liver microsomal activation and used strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E.coli WP2uvrA. The test item was tested at the following concentrations: 4, 20, 100, 500, 2500, 5000 (only 2nd exp.)and 10000 (only 1st exp.) microg/plate.The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants occurred in the test groups with and without metabolic activation. Precipitation was not observed. EAA was found to be non-mutagenic in this test system.

Key 2 – In vitro chromosome aberration assay

The assay was performed 1999 according to OECD-guideline no. 473. The test was performed with and without liver microsomal activation and used V79 cells of Chinese hamster lung fibroblasts. The test item was tested at the following concentrations: 325.0, 650.0 and 1301.4 ug/ml. Treatment time was 3 and 20 hours.In the main experiment, no reduction of mitotic index was observed with and without metabolic activation at the 3 h treatment time. In the repeat experiment at the 20 h treatment time in the absence of S9-mix the mitotic index was dose dependent reduced (indication of toxicity) reaching 33 % of the solvent control value at the highest dose level tested, 1301.4 ug/ml.EAA was found to be non-clastogenic in this test system.

Key 3 – In vitro gene mutation assay

The study was performed 2011/2012 in accordance with OECD guideline no. 476. The test was performed with and without liver microsomal activation and used V79 cells.The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of the pre-experiment and the main experiments (1300 μg/mL) was equal to a molar concentration of about 10 mM. Neither genotoxicity nor cytotoxicity was observed. Ethyl acetoacetate is considered to be non-mutagenic in this HPRT assay.

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

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

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Source: Cell bank of "Genetic toxicology", HMR Germany, ProTox

Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permitting repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters. Thawed stock cultures were kept at approx. 37 "C and approx. 4 % CO, in 175 cm2 plastic flasks. About 5 x 1090 1 x lo6 cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (vlv) FCS (fetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (wlv) neomycinsulfate. The cells were subcultured twice a week.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-liver fractions of Sprague-Dawley rats, induced with Aroclor 1254

Test concentrations with justification for top dose:

TREATMENT TIME 3h:

1. With S9-mix:
- Solvent control: 0.0 ug/ml
- Positive control: CPA 3.0 ug/ml
- Test group 1: 325.0 ug/ml
- Test group 2: 650.0 ug/ml
- Test group 3: 1301.4 ug/ml

2. Without S9-mix:
- Solvent control: 0.0 ug/ml
- Positive control: EMS 1500.0 ug/ml
- Test group 1: 325.0 ug/ml
- Test group 2: 650.0 ug/ml
- Test group 3: 1301.4 ug/ml


TREATMENT TIME 20h:

1. Without S9-mix:
- Solvent control: 0.0 ug/ml
- Positive control: EMS 400.0 ug/ml
- Test group 1: 325.0 ug/ml
- Test group 2: 650.0 ug/ml
- Test group 3: 1301.4 ug/ml

Vehicle / solvent:

Cell culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: EMS (Ethyl methane sulfonate) / With metabolic activation: CPA (Cyclophosphamide - Endoxan)

Details on test system and experimental conditions:

Details are given in thge report

Evaluation criteria:

The evaluation of the results was performed as follows:
- The test compound is classified as mutagenic if there is a concentration-related increase in the aberration rate (without gaps).
- The test compound is classified as non-mutagenic if the tests are negative both with and without metabolic activation.
- The test compound is classified as mutagenic if it induces a statistically significant increase in the aberration rate (without gaps) with one or more of the concentrations tested as compared with the solvent controls.

Statistics:

Statistical evaluation was not necessary, because all dose groups were in the range of the solvent controls.

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Additional information on results:

No precipitation observed.

Remarks on result:

other: strain/cell type: V79

Remarks:

Migrated from field 'Test system'.

Solubility and preliminary toxicity testing:

Acetoacetic acid ethyl ester was dissolved in cell culture medium. Evaluation of the solubility in cell culture medium showed that 1301.4 ug/ml was the highest practicable concentration and produced no precipitate. This concentration corresponds to 10 mM, which is the highest dose level tolerated for the test system. Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 1301.4 ug/ml and a range of lower dose levels down to 10 ug/ml.

Mutagenicity test:

In the main experiment no reduction of mitotic index was observed with and without metabolic activation at the 3 h treatment time. i In the repeat experiment at the 20 h treatment time in the absence of S9-mix the mitotic index was dose dependent reduced (indication of toxicity) reaching 33 % of the solvent control value at the highest dose level tested, 1301.4 ug/ml. After treatment with the test compound there was no relevant increase in the number of polyploid cells as compared with the solvent controls. The test compound Acetoacetic acid ethyl ester was assessed for its mutagenic potential in vitro in the chromosome aberration test in two independent experiments. No relevant reproducible enhancement of metaphases with aberrations over the range of the solvent control was found with any of the concentrations used, either with or without metabolic activation by S9-mix. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Conclusions:

Interpretation of results (migrated information):
negative

EAA was not clastogenic in this chromosome aberration test system in vitro with cells of the V79 Chinese hamster cell line under the conditionsdescribed in this study.

Executive summary:

The assay was performed 1999 according to OECD-guideline no. 473. The test was performed with and without liver microsomal activation and used V79 cells of Chinese hamster lung fibroblasts. The test item was tested at the following concentrations:  325.0, 650.0 and 1301.4 ug/ml. Treatment time was 3 and 20 hours. In the main experiment, no reduction of mitotic index was observed with and without metabolic activation at the 3 h treatment time. In the repeat experiment at the 20 h treatment time in the absence of S9-mix the mitotic index was dose dependent reduced (indication of toxicity) reaching 33 % of the solvent control value at the highest dose level tested, 1301.4 ug/ml. EAA was found to be non-clastogenic in this test system.

Reference 2

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:

December 2011 - May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other 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

Target gene:

V79 cells of the Chinese hamster

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9 was used as the metabolic activation system

Test concentrations with justification for top dose:

Experiment I
- 4 hours (-S9): 40.6; 81.3; 162.5; 325.0; 650.0; 1300.0 ug/ml
- 4 hours (+S9): 40.6; 81.3; 162.5; 325.0; 650.0; 1300.0 ug/ml

Experiment II
- 24 hours (-S9): 40.6; 81.3; 162.5; 325.0; 650.0; 1300.0 ug/ml
- 4 hours (+S9): 40.6; 81.3; 162.5; 325.0; 650.0; 1300.0 ug/ml

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

DURATION
- Exposure duration: 2 and 24 hours
- Expression time (cells in growth medium): 7 days

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: solvent control: 7.55; test item (1300 ug/ml): 7.49
- Effects of osmolality: solvent control: mOsm = 389; test item (1300 ug/ml): mOsm = 382
- Evaporation from medium: no
- Precipitation: no

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

none

Conclusions:

Interpretation of results (migrated information):
negative

It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, EAA is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed 2011/2012 in accordance with OECD guideline no. 476. The test was performed with and without liver microsomal activation and used V79 cells.The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of the pre-experiment and the main experiments (1300 μg/mL) was equal to a molar concentration of about 10 mM. Neither genotoxicity nor cytotoxicity was observed. Ethyl acetoacetate is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-liver fractions of Sprague-Dawley rats, induced with Aroclor 1254

Test concentrations with justification for top dose:

Test substance concentrations of 4, 20, 100, 500, 2500, 5000 (only 2nd exp.)and 10000 (only 1st exp.) microg/plate were used in the main test.

Vehicle / solvent:

Aqua bidist.

Untreated negative controls:

yes

Remarks:

each strain alone or in presence of the solvent

Negative solvent / vehicle controls:

yes

Remarks:

aqua bidist.

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

further positive control substances were used: 9-Aminoacridine, 2-Nitrofluorene, N-Methyl-N-nitro-N-nitrosoguanidine, Benz[a]pyrene, 2-Aminoanthracene

Details on test system and experimental conditions:

The study was performed by the direct plate incorporation method w ith and without metabolic activation.

Evaluation criteria:

Not given in the report; reference to literature.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation observed.

Remarks on result:

other: all strains/cell types tested

Conclusions:

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not
induce gene mutations in the five strains tested.

Executive summary:

The assay was performed 1988 according to OECD-guidelines no. 471 and 472 with and without liver microsomal activation and used strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E.coli WP2uvrA. The test item was tested at the following concentrations:  4, 20, 100, 500, 2500, 5000 (only 2nd exp.)and 10000 (only 1st exp.) microg/plate.The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants occurred in the test groups with and without metabolic activation. Precipitation was not observed. EAA was found to be non-mutagenic in this test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data available the substance is not classified or labeled according to Directive 67/548/EEC (DSD) or Regulation 1272/2008/EC (CLP)