Propanoic acid, 2-hydroxy-2-methyl-, 2-propen-1-yl ester

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

yes

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

yes

Principles of method if other than guideline:

Bidistilled water instead of DMSO was used as vehicle for the test substance.
In the original experiment without metabolic activation (experiment 1) four instead of three concentrations were evaluated for chromosomal
aberrations.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

prop-2-en-1-yl 2-hydroxy-2-methylpropanoate

EC Number:

700-908-3

Cas Number:

19444-21-4

Molecular formula:

C7H12O3

IUPAC Name:

prop-2-en-1-yl 2-hydroxy-2-methylpropanoate

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cell line: ATCC (Americam Type Culture Collection ) CCL 61 (ovary, Chinese hamster, CHO K1).

Metabolic activation:

with and without

Metabolic activation system:

Post mitochondrial supernatant (S9 fraction) from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

Experiment without metabolic activation:
- Original experiment:
3 h treatment/18 h recovery: 39.06, 78.13, 156.25 and 312.50 µg/ml
- Confirmatory experiment
21 h treatment: 625.00, 1250.00 and 2500.00 µg/ml
Higher concentrations could not be scored due to cytotoxicity. Mitomycin C (0.2 µg/ml) was used as a positive control.
Experiment with metabolic activation:
- Original experiment:
3 h treatment/18 h recovery: 9.77, 19.53 and 39.06 µg/ml
- Confirmatory experiment
3 h treatment/18 recovery: 9.77, 19.53 and 39.06 µg/ml
Higher concentrations could not be scored due to cytotoxicity. Cyclophosphamide (20.0 µg/ml) was used as a positive control.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Bidistilled water (Bidistilled water instead of DMSO was used as vehicle for the test substance).

Controls

Untreated negative controls:

yes

Remarks:

supplemented with the respective volume of the vehicle

Negative solvent / vehicle controls:

no

Positive controls:

yes

Remarks:

See comment below

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Mitomycin C (without metabolic activation) Cyclophosphamide (with metabolic activation)

Details on test system and experimental conditions:

Positive controls:
The treatnment of the culltures with mitomycin C, 0.2 µg/ml and cyclophosphamide, 20.0 µg/ml, respectively was followed by a high incidence of specific chromosomal aberrations in the experimets one and two of the original study (56.0% and 60.0%) and in the experiments one and two of the confirmatory study (70.0% and 70.0%).

The cytotoxicity test (measurement of mitotic test index) was performed as an integral part of the mutagenicity test. A series of glass slides in quadruple culture dishes (Quadriperm) was seeded with Chinese hamster ovary cells (preparation/passage number: 110/13, 110/15, 111/16, 111/14) at a density of at least 1 x 104 cells/ml (21 hour experiments) or 4 x 103 cells/ml (45 hour experiments). The preincubation time before treatment was about 24 hours. In the experiments in which the substance was metabolically activated, 0.5ml of an activation mixture was 0.5 ml of an activation mixture was added to 4.5 ml of Nutrient Mixture F-12. Mitomycin C (KTOWA HAKKO KOGYO Co. Ltd., Japan) 0.2 µg/ml, a mutagen not requiring S9 activation, and cyclophosphamide (CPA, SATA-WERKE, Germany) 20.0 µg/ml, which requires metabolic activation, were used as positive controls. In addition a negative control was set in each experiment, supplemented with the respective volume of vehicle. Quadruplicate cultures were prepared for each group in each assay.

Duration:
To ensure analysis of first post treatment mitoses a major sampling time of 1.5 times cell cycle was selected. This corresponds to about 21 hours for the CHO cells used. To ensure the above requirement even in case of a delayed cell cycle, which may be caused by the test substance, an additional sampling time was chosen, 42 hours after the first one. Treatment was performed throughout the whole period in the absence of metabolic activation. In the presence of metabolic activation treatment was shortened to three hours because prolonged exposure to S9-fraction would result in cytoxicity.
Two hours prior to harvesting, the cultures were treated with Colcemide (GIBCO) 0.4 µg/ml to arrest cells in metaphase. The experiment was terminated by hypotonic treatment (0.075 M KCl solution) of the cells, followed by fixation (methanol: acetic acid, 3:1). Slides were air dried and stained with orcein.

Maintenance of the cell line:
The cell line CCL 61 (Chinese hamster ovary cells, CHO) was maintained in culture medium consisting of Nutrient mixture-12 supplemented with 10% fetal calf serum + Penicillin/Streptomycin 100 units/ml/100 µg/ml (Gibco AG, Basel, Switzerland) in 75 cm2 tissue-culture (plastic) flasks. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. The cells were passaged twice weekly.
The cell cultures were periodically checked for mycoplasma contamination.

Solubility of test substance:
CA 2215 A was dissolved in bidistilled water at room temperature and sterilized by filtration through a 0.2 µm Acrodisc-CR filter. The highest concentration (stock solution) used for the assay was 50mg/ml. Lower concentrations were prepared by serial dilution of the stock solution with bidistilled water. The respective solutions were added (1:10) to the cell cultures. The final concentration of the vehicle bidistilled water in the culture medium was 10%.

NUMBER OF REPLICATIONS: 2 cultures evaluated per concentration.

NUMBER OF CELLS EVALUATED: 200 (wherever possible; except positive controls) per concentration.

DETERMINATION OF CYTOTOXICITY
The highest concentration used or the lowest concentration which suppresses mitotic activity by approximately 50-80% compared to the control group was selected as the highest for the analysis of chromosome aberrations together with two lower concentrations. For the determination of the mitotic index (MI) the preparations from the various cultures were examined first, uncoded. The percentages of mitotic suppression in comparison with the controls were evaluated by counting at least 2000 cells from one slide each of the treatment groups and the negative control group. The determination of the mitotic coefficient was performed for each experiment separately. From the results of corresponding original run, suitable concentrations were determined for t experiments of the confirmatory study.

Evaluation criteria:

Prior to analysis the selected slides were coded, likewise the cultures treated with the vehicle alone as well as the positive control. Whenever possible two hundred well spread metaphase figures with 17 to 21 centromeres from two cultures (100 metaphases per replicate culture) in the vehicle control and in the treated groups were scored. At least fifty metaphases were scored in the positive controls (25 per replicate culture). The slides were examined for the following structural aberrations.
• Specific aberrations: chromatid and chromosome deletions (including breaks, deletions and fragment), chromatid exchanges (including triradials, quadriradials, endfusions, acentric rings), chromosome exchanges (including dicentrics, centric and acentric rings),
• Multiple aberrations: metaphases containing more than 10 aberrations of different types or more than 5 aberrations of one particular type (excluding gaps),
• Unspecific aberrations: gaps (chromatis- and chromosome-),
In addition the frequency of polyploidy metaphases (multiples of 2n > 30 centromers, including endoreduplication figures) was recorded.
Using a computerized coordinate reading system (Microptic system, Microptic Ltd, UK) attached to the vernier scale on the microscope stage, the coordinates of all metaphases were recorded.

Statistics:

The evaluated numbers of specific aberrations were subjected to statistical analysis. In the preliminary tests the data were assessed for flask affects (dependence of cells within each culture) using chi-squared test. The nonsignificant result of this test means there is no substantial evidence to conclude a flask effect (although a flask effect may still exist). Accordingly, a chi-squared test for trend was preformed modelling all cells in a given experimental unit. Consequently the power of the test is substantially increased, resulting in a rather safe judgement of the observed effects.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the presence of a metabolic activation system

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Discussion:
In both the original and confirmatory study with metabolic activation (experiments 2 and 4) the number of metaphases with specific chromosomal aberrations was slightly increased at the highest concentration, each. These effects fulfilled the criteria for a positive response (>6% aberrant metaphases and statistically significant difference from the respective value of the negative control). In the confirmatory experiment without activation (experiment 3) at the highest concentration and in the confirmatory experiment with activation (experiment 4) at the intermediate concentration, also increased numbers of aberrations metaphases were observed. These incidences of metaphases with aberrations (5.5% and 4.4%), however, did not meet criteria for a positive response (frequencies of aberrant metaphases are below 6%).
Unspecific chromosomal aberrations in the form of chromatid gaps showed an increase in the original experiment with metabolic activation.
Since clastogenic effects were observed in both experiments with metabolic activation, the third and fourth experiment of the confirmatory study (without metabolic activation, 45 hours treatment; with metabolic activation, 3 hours treatment/42 hours recovery) were not scored.
It is concluded that the metabolites of CA 2215 A exerted effects in Chinese hamster ovary cells in-vitro.

Remarks on result:

other: strain/cell type: CCL 61

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation

It is concluded that under the given experimental conditions CA 2215 A exerted clastogenic effects in Chinese hamster ovary cells in-vitro in the presence of a metabolic activation system.

Executive summary:

CA 2215 A (Intermediate of CGA 276854), identified as a liquid, 99% purity, batch P.608005, was investigated for clastogenic (chromosome-damaging) effects on Chinese hamster ovary cells in-vitro with and without extrinsic metabolic activation (S9). The compound was dissolved in bidistilled water and tested at each of the following conditions. 

Experiment without metabolic activation:

-      Original experiment:

3 h treatment/18 h recovery: 39.06, 78.13, 156.25 and 312.50 µg/ml

-      Confirmatory experiment

21 h treatment: 625.00, 1250.00 and 2500.00 µg/ml

Higher concentrations could not be scored due to cytotoxicity. Mitomycin C (0.2 µg/ml) was used as a positive control. 

Experiment with metabolic activation:

-      Original experiment:

3 h treatment/18 h recovery: 9.77, 19.53 and 39.06 µg/ml

-      Confirmatory experiment

3 h treatment/18 recovery: 9.77, 19.53 and 39.06 µg/ml

Higher concentrations could not be scored due to cytotoxicity. Cyclophosphamide (20.0 µg/ml) was used as a positive control. 

In the experiments performed without metabolic activation no biological relevant increase in the number of metaphases containing specific chromosomal aberrations was observed. 

In the experiments carried out with metabolic activation treatment with CA 2215 A led to a slight increase in the number of metaphases containing specific chromosomal aberrations at the highest concentration, each

It is concluded that under the given experimental conditions CA 2215 A exerted clastogenic effects in Chinese hamster ovary cells in-vitro in the presence of a metabolic activation system.