Benzoic acid, 2-chloro-5-isocyanato-, 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:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

Dose selection: due to technical reasons slightly higher doses were applied in experiment IB.

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-isocyanatobenzoyloxy)-2-methylpropanoate

EC Number:

695-953-8

Cas Number:

204918-22-9

Molecular formula:

C15H14ClNO5

IUPAC Name:

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

Method

Target gene:

V79

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Source: Laboratory of Mutagenicity Testing, LMP, Technical University Darmstadt
- Storage/Location: RCC Cytotest Cell Research GmbH / liquid nitrogen
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Propagation: 37°C in 80 ml plastic flasks. 5x10 5 cells per flasks seeded with 15 ml of MEM and 10 FCS.
- Subculture: 37°C in a humidified atmosphere with 4.5 % carbon dioxide (95.5% air).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Main experiment I:
25 µg/ml (without S9-mix)
300 µg/ml (with S9-mix)

Experiment IB:
30 µg/ml (without S9-mix)
87.5 µg/ml (with S9-mix) were chosen as top treatment concentrations, according to toxicity data obtained in experiment I.

Experiment II:
4 µg/ml (without S9-mix)
80 µg/ml (with S9-mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative non-toxicity to the cells.

Controls

Untreated negative controls:

yes

Remarks:

Culture medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

Without metabolic activation: EMS. With metabolic activation: CPA

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Range finding:
A pre-test on cell growth inhibition with 4hour and 24 hour treatment was performed in order to determine the toxicity of the test item. Cytotoxicity was determined using concentrations separated by no more than a factor of 2 – 3.33. The general experimental conditions in this pre-test were the same as for the cytogenetic main experiment.
In a quantitative assessment, exponentially growing cell cultures (seeding 41000 cells/slide with regard to the culture time of 48 hours) were treated with the test item for simulating the conditions of the main experiment. A qualitative evaluation of cell number and cell morphology was made 4 hours and 24 hours start of treatment. 24 hours after start of treatment the cells were stained. Using 400 fold microscopic magnification the cells were counted in 10 coordinate defined fields of the slides (2 slides per treatment group). The cells number of the treatment groups given as % cells in relation to the control.

Dose selection:
The highest concentration used in the pre-test was chosen with regard to the current OECD guidelines for in-vitro mammalian cytogenetic tests. With respect to the molecular weight of the test item, 3240 µg/ml (~ 10mM) was chosen as top treatment concentration.
Test item concentrations between 25.3 and 3240 µg/ml (with and without S9-mix) were chosen for the evaluation of cytotoxicity in the range finder. Using reduced cell numbers as an indicator for toxicity, toxic effects were observed after 4 hours treatment with 25.3 µg/ml and above (without S9-mix) and with 202.5 µg/ml and above (with S9-mix). Therefore, 25 µg/ml (without S9-mix) and 300 µg/ml (with S9-mix) were chosen as top concentrations for the 4 hours exposure in the main experiment I.
To verify the slight increased aberration frequencies observed in experiment I aberrant cells excluding gaps; a confirmatory experiment IB was performed. In experiment IB, 30 µg/ml (without S(-mix) and 87.5 µg/ml (with S9-mix) were chosen as top treatment concentrations, according to toxicity data obtained in experiment I.
Dose selection in experiment II was also influenced by test item toxicity. In the range finding experiment clearly reduced cell numbers were observed after 24 hours exposure with 25.3 µg/ml and above. Therefore, 30 µg/ml was originally chosen as top treatment concentration for continuous exposure in the absence of S9-mix. However, due to strong test item induced toxicity indicated by reduced mitotic indicies, this experiment was repeated using 4 µg/ml as top treatment concentration. In the presence of s9-mix 80 µg/ml were chosen as top treatment concentration with respect to the results obtained in experiment I.

Seeding of the cultures:
Exponentially growing stock cultures more than 50 % confluent were treated with trypsin at 37°C for approximately 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration was 0.2 % in Ca-Mg–free solution.
Prior to the trypsin treatment cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA.
The cells were seeded into Quadriperm dishes which contained microscopic slides (at least 2 per dish) and test group). In each chamber cells were seeded with regard to preparation time. The medium was MEM + 100 % FCS.

Treatment: Exposure period 4 hours:
The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50µl/ml S9 mix per ml culture medium were added. Concurrent negative, solvent and positive controls were performed. After 4 hours the cultures were washed twice with Saline G and then the cells were cultured in complete medium for the remaining culture time.

Treatment: Exposure period 18 and 28 hours:
The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells.
All cultures were incubated at 37°C in a humidified atmosphere with 4.5 % CO2 (95.9% air).

Preparation of the cultures:
16 hours and 26 hours, respectively after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2 hours later, the cells on the slides were treated in the chambers with hypotonic solution (0.4% KCl) for 20 minutes at 37°C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid. Per experiment both slides per group were prepared. After preparation the cells were stained with Giemsa.
Additionally, two cultures per test item and solvent control treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The toxicity of the test item is given as reduction of % cells as compared to solvent control.

Analysis of Metaphase cells:
Evaluation of the cultures was performed. Breaks , fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberrations rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploidy cells was determined (% polyploidy metaphases; in case of this aneuploidy means a near tetraploid karyotype).

Evaluation criteria:

The chromosome aberration assay performed in the test laboratory is considered acceptable if it meets the following criteria:
a) The number of structural aberrations (excl. gaps) found in the negative and/or solvent controls falls within the range of the historical
laboratory control data.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations
(excl. gaps), which are within the range of the laboratory historical data.

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 relevant influences of the test item on the pH values or osmolarity was observed (solvent control 390 mOsm, pH7.3 versus 339 mOsm and pH 7.2 at 3240 µg.ml).
Test item precipitation in culture medium was observed in the range finder after 4 hours treatment with 405 µg/ml and above in the absence and the presence of S9 mix.

In this study, in the absence of S9 mix reduced miotic indices and/or reduced cell numbers blow 50% of control were observed at least at the highest evaluated concentrations. In the presence of S9 mix, reduced cell numbers below 50 % of control were observed in experiment IA and experiment II whereas the mitotic index was not reduced.

In both experiments, in the absence and presence of S9 mix, no boilogically relevant increase in the number of cells carrying structural chromosome aberrations was observed. However, in experiment IA, aberration frequencies slightly exceeded the historical control data range were observed. In the absence of S9 mix, cultures after treatment with 6.3, 12.5 and 25 µg/l revealed an apparent dose related increase (2.0, 4.0 and 4.5 % aberrant cells excluding gaps) without a statistical significance compared to the solvent control data. This result could not be reproduced in experiment IB and is therefore regarded to be biologically irrelevant. In the presence of S9 mix, cultures after treatment with 18.8 and 37.5 µg/ml showed aberration frequencies (4.5 and 7.0 % aberrant cell excluding gaps) beyond the historical data range. This result could not be reproduced in experiment IB and is therefore regarded to be biologically irrelevant.

In all experiments, no biologically relevant increases in the rate of polyploidy metaphases was found after treatment with the test item as compared to the rates of the solvent controls.

Remarks on result:

other: strain/cell type: V79

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, he test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, CA 2814 B (Intermediate of CGA 276854) is considered to be non-clastogenic in the chromosome aberration test.

Executive summary:

The test item CA 2814 B (Intermediate of CGA 276854), dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in three independent experiments with the following study design. 

 

	Without S-9 mix			With S-9 mix
	Exp. 1A and B	Exp. II		Exp. 1A and B	Exp. II
Exposure period	4 hours	18 hours	28 hours	4 hours	4 hours
Recovery	14 hours	-	-	14 hours	24 hours
Preparation interval	18 hours	18 hours	28 hours	18 hours	28 hours

 

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. 

With respect to the molecular weight of the test item concentrations between 25.3 and 3240 µg/ml (10 mM) were chosen for the evaluation of Cytotoxicity in a range finding pre-experiment. 

Dose selection of the cytogenetic experiments was performed considering the toxicity data of the range finder. 

Toxic effects indicated clearly reduced cell numbers and/or mitotic indices below 50 % of control were observed in all experimental parts in the absence and the presence of S9 mix except in experiment IB in the presence of S9 mix. 

In this study, no biological relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. The marginal increases in experiment IA in the absence and presence of S9 mix as compared to our historical control data range (0 – 4 % aberrant cells excluding gaps) were not reproduced in experiment IB and II. Therefore, these findings are regarded to be purely fortuitous events of no relevance. 

No increase in the frequencies of polyploidy metaphases was found after treatment with the test item as compared to the frequencies of the controls. 

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations

 

Conclusion:

In conclusion, it can be stated that under the experimental conditions reported, he test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. 

Therefore, CA 2814 B (Intermediate of CGA 276854) is considered to be non-clastogenic in the chromosome aberration test.