Ethyl acetoacetate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Test material form:

other: non-viscous clear liquid

Details on test material:

- Molecular formula: C6H10O3
- Molecular weight: 130.14
- Physical state: liquid

Method

Target gene:

not applicable

Species / strain

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

Controls

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.