Benzoic acid, 2-chloro-5-nitro-, 1,1-dimethyl-2-oxo-2-(2-propen-1-yloxy)ethyl 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:

2000

Report date:

2000

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

prop-2-en-1-yl 2-(2-chloro-5-nitrobenzoyloxy)-2-methylpropanoate

EC Number:

696-317-2

Cas Number:

174489-76-0

Molecular formula:

C14H14ClNO6

IUPAC Name:

prop-2-en-1-yl 2-(2-chloro-5-nitrobenzoyloxy)-2-methylpropanoate

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Cell line:
ATCC (American Type Culture Collection) CCL 61 (ovary, Chinese hamster, CHO K1)
- Maintenance:
The cell line CCL 61 (Chinese hamster ovary cells, CHO) was maintained in culture medium consisting of Nutrient mixture-F12 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.

Metabolic activation:

with and without

Metabolic activation system:

Post mitochondrial superntant (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: 6.26, 12.50 and 25.00 µg/ml
- Confirmatory experiment
21 h treatment: 6.25, 12.50 and 25.00 µg/ml
45 h treatment: 1.56, 3.13 and 6.25 µg/ml

Higher concentrations could not be scored due to cytotoxicity. Cytotoxicity was observed at the highest concentration. Mitomycin C (0.2 µg/ml) was used as a positive control in the 21 hours experiments.
Experiment with metabolic activation:
- Original experiment:
3 h treatment/18 h recovery: 12.50, 25.00 and 50.00 µg/ml
- Confirmatory experiment
3 h treatment/18 h recovery: 12.50, 25.00 and 50.00 µg/ml
3 h treatment/24 h recovery: 12.50, 25.00 and 50.00 µg/ml
Final concentrations higher than 50 µg/ml of culture medium could not be tested due to solubility limitations. Cyclophosphamide (20.0 µg/ml) was used as a positive control in the 3 hours/18 hours experiments.

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

supplemented with the respective volume of the vehicle.

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.2 µg/ml without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

supplemented with the respective volume of the vehicle.

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

20.0 µg/ml with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
2 replicate cultures evaluated per concentration.

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


Solubility of test substance:
CA 2218 A was dissolved in DMSO at room temperature. The highest soluble concentration was 500 mg/ml. This solution caused the formation of strong precipitates after 100 fold dilution with culture medium. The highest substance concentration in DMSO (stock solution) resulting in a tolerable homogeneous turbidity after 100 fold dilution with culture medium was 50 mg/ml. Lower concentrations were prepared by serial dilution of the stock solution with DMSO. The respective solutions were added (1:100) to the cell cultures. The final concentration of the vehicle in the culture medium was 1%.

The addition of the stock solutions to the cell cultures produced turbidity in the culture medium at the final concentrations of 50 µg/ml in the experiments with and without activation.

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: 119/2, 119/4, 119/20,) 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. Th substance in DMSO was added (1:100) to the cells in culture medium. 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.
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, 24 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.

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:

Scoring the slides:
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:

Statistical Analysis:
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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity test / selection of concentrations:

The highest concentration of 25.00 µg/ml selected for chromosome analysis in experiment 1 (without metabolic activation, 3 hours treatment/18 hours recovery) caused 19% suppression of mitotic activity. Due to toxicity, at the next higher concentration of 50.00 µg/ml no scorable cells were found.
The highest concentration of 50.00 µg/ml selected for chromosome analysis in experiment 2 (with metabolic activation 3 hours treatment/18 hours recovery) caused practically no suppression of mitotic activity.
In experiment 3 (without metabolic activation, 21 hours treatment) the highest concentration of 25.00 µg/ml selected for chromosome analysis caused 43 % suppression of mitotic activity. The next higher concentration of 50.00 µg/ml suppressed mitotic activity by 98 %.
In experiment 4 (with metabolic activation, 3 hours treatment/18 hours recovery) the highest concentration of 50.00 µg/ml selected for chromosome analysis caused no suppression of mitotic activity.
The highest concentration of 6.25 µg/ml selected for chromosomal analysis in experiment 5 (without metabolic activation, 45 hours treatment) caused 23 % suppression of mitotic activity. The next higher concentration of 12.50 µg/ml suppressed mitotic activity by 86 % and was not scored according to the criteria for selection of concentrations.
In experiment 6 (with metabolic activation, 3 hour treatment/42 hours recovery) the highest concentration of 50.00 µg/ml selected for chromosomal analysis caused no suppression of mitotic activity.

Original mutagenicity study:

In the experiment performed without metabolic activation (experiment 1), 0.5% of metaphases with specific chromosomal aberrations were detected in the negative control. At the concentration of 6.25 µg/ml, 12.50 µg/ml, and 25.00 µg/ml 2.0%, 1.5%, and 2.0% of cells with specific chromosomal aberrations were found.
In the experiments performed with metabolic activation (experiment 2), 2.0% of metaphases with specific chromosomal aberrations were seen in the negative control. At the concentrations of 12.50 µg/ml, 25.00 µg/ml and 50.00 µg/ml the respective values were 2.5%, 0.0% and 4.5%.

Confirmatory mutagenicity study:

In the experiment performed without metabolic activation (experiment 3), 0.5 % of metaphases with specific chromosomal aberrations were detected in the negative control. At the concentration of 6.25 µg/ml, 12.50 µg/ml and 25.00 µg/ml, 1.0 %, 1.5 % and 3.0 % of cells with specific chromosomal aberrations were registered.
In the experiment performed with metabolic activation (experiment 4), 1.5 % of metaphases with specific chromosomal aberrations were seen in the negative control. At the concentration of 12.50 µg/ml, 25.00 µg/ml and 50.00 µg/ml, 1.0 %, 2.0 % and 3.5 % of cells showed specific chromosomal aberrations.
In the experiment performed without metabolic activation after 45 hours treatment (experiment 5), 1.0 % of metaphases with specific chromosomal aberrations were detected in the negative control cultures. At concentrations of 1.56 µg/ml, 3.13 µg/ml and 6.25 µg/ml, the corresponding values were 0.5 %, 3.0 % and 1.0 %.
In the experiment performed with metabolic activation after 3 hours treatment/42 hours recovery (experiment 6), 1.0 % of metaphases with specific chromosomal aberrations were registered in the negative cultures. At the concentrations of 12.50 µg/ml, 25.00 µg/ml and 50.00 µg/ml, 1.0 %, 1.5 % and 2.0 % of cells with specific chromosomal aberrations ere found.

Discussion:

In the confirmatory experiment without metabolic activation (3 hours treatment/18 hours recovery) at the concentrations of 25.00 µg/ml, a slight, but statistically significant increased number of metaphases with chromosomal aberrations was observed. Since this incidence of metaphases with aberrations (3.0%) is clearly within the historical negative control range and does not meet the criteria for a positive response (frequency of aberrant metaphases ≥ 6 %), it is considered to be of spontaneous origin and not related to treatment with the test material.
None of the other values showed a statistically significant difference when compared with their respective negative control.
Unspecified chromosomal aberrations in the form of chromatid gaps found in all experiments were within the frequency generally observed.
In the confirmatory experiment without metabolic activation (45 hours treatment), the percentage of polyploid metaphases was slightly increased at the highest concentration. This effect occurred at the limit of cytotoxicity after long treatment only. It is attributed to general cytotoxicity rather than to an effect on the spindle.

CA 2218 A (Intermediate of CGA 276854) was devoid of any clastogenic activity.

Positive Controls:
The treatment of the cultures with mitomycin-C (0.2 µg/ml) and cyclophosphamide (20.0 µg/ml) respectively, was followed by a high incidence of specific chromosomal aberrations (40 to 66 %).

Remarks on result:

other: strain/cell type: CCL 61

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

It is concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in-vitro treated with CA 2218 A (Intermediate of CGA 276854).

Executive summary:

CA 2218 A (Intermediate of CGA 276854), identified as crystals, 96.8% purity, batch OP.802002, 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 DMSO and tested at each of the following conditions. 

Experiment without metabolic activation:

-      Original experiment:

3 h treatment/18 h recovery: 6.26, 12.50 and 25.00 µg/ml

-      Confirmatory experiment

21 h treatment: 6.25, 12.50 and 25.00 µg/ml

45 h treatment: 1.56, 3.13 and 6.25 µg/ml

 

Higher concentrations could not be scored due to cytotoxicity. Cytotoxicity was observed at the highest concentration. Mitomycin C (0.2 µg/ml) was used as a positive control in the 21 hours experiments. 

Experiment with metabolic activation:

-      Original experiment:

3 h treatment/18 h recovery: 12.50, 25.00 and 50.00 µg/ml

-      Confirmatory experiment

3 h treatment/18 h recovery: 12.50, 25.00 and 50.00 µg/ml

3 h treatment/24 h recovery: 12.50, 25.00 and 50.00 µg/ml

Final concentrations higher than 50 µg/ml of culture medium could not be tested due to solubility limitations. Cyclophosphamide (20.0 µg/ml) was used as a positive control in the 3 hours/18 hours experiments. 

 

In both experiments performed with and without metabolic activation no biological relevant increase I the number of metaphases containing specific chromosomal aberrations was observed. The incidence of aberrant cells was within historical control range at all doses assessed. 

In the experiment without metabolic activation using the long treatment time (45 hours) the percentage of polyploidy metaphases was slightly increased at the highest concentration. 

 

It is concluded that under the given experimental conditions no evidence of clastogenic effects was obtained in Chinese hamster ovary cells in-vitro treated with CA 2218 A (Intermediate of CGA 276854).