N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxo-2-[[2-(trifluoromethyl)phenyl]azo]butyramide

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

Study period:

From 27 JUL 2011 to 23 AUG 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 473) and according to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - 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

Reference substance name:

N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxo-2-[[2-(trifluoromethyl)phenyl]azo]butyramide

EC Number:

268-734-6

EC Name:

N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxo-2-[[2-(trifluoromethyl)phenyl]azo]butyramide

Cas Number:

68134-22-5

Molecular formula:

C18H14F3N5O3

IUPAC Name:

3-oxo-N-(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2-{[2-(trifluoromethyl)phenyl]diazenyl}butanamide

Test material form:

solid: bulk

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: minimal essential medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital / beta-naphtoflavone induced rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 0.4, 1.0, 2.5, 6.1 , 15.4, 38.4, 96.0, 240.0, 600.0 µg/mL
Experiment II: 1.2, 2.4, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0 µg/mL

Without metabolic activation:
Experiment I: 0.4, 1.0, 2.5, 6.1 , 15.4, 38.4, 96.0, 240.0, 600.0 µg/mL
Experiment II: 1.2, 2.4, 4.7, 9.4, 18.8, 37.5, 75.0, 150.0 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of test item

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium (minimal essential medium)

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 18 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: At least 100 well spread metaphases per culture were evaluated for cytogenetic damage on coded slides, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell numbers


OTHER EXAMINATIONS:
- Determination of polyploidy: 500 per culture
- Determination of endoreplication: 500 per culture

Evaluation criteria:

Evaluation of the cultures was performed according to the OECD Guideline using NIKON microscopes with 100x objectives. 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 aberration rates. At least 100 well spread metaphases per culture were evaluated for cytogenetic damage on coded slides, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated.
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) and relative cell numbers were determined.

In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells scored at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases).

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours (Exp. I & II) after the start of treatment with the test item.
In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated.
No relevant influence of the test item on pH value or osmolarity was observed.
Precipitation of the test item in culture medium was observed in Experiment I at preparation interval 18 hours with 96.0 µg/mL and above. In this experiment severe precipitation could be observed on the prepared slides beginning with 38.4 µg/mL in the absence and presence of S9 mix. In Experiment II after 18 hours continuous treatment precipitation occurred with 4.7 µg/mL and above without S9 mix and in the presence of S9 mix with 9.4 µg/mL and above. Severe precipitation on the slides was observed in the absence of S9 mix beginning with 18.8 µg/mL and 37.5 µg/mL in the presence of S9 mix. Evaluation of the mentioned slides was impossible due to interference of the cells by the precipitate.
In this study, neither reduced mitotic indices nor reduced cell numbers could be observed up to the highest evaluated concentrations of the test item, except in Experiment I, in the presence of S9 mix. In this part of the study, the cell numbers were slightly reduced after treatment with 6.1, 15.4 and 38.4 µg/mL (56.4, 72.8 and 76.5 % of control).
In Experiment I, in the absence and presence of S9 mix and in Experiment II in the absence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (1.0 - 3.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (2.0 - 3.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical control data: 0.0 -4.0 % aberrant cells, excluding gaps. In Experiment II in the presence of S9 mix one single value slightly exceeded the range (6 % aberrant cells, excluding gaps) of the laboratory historical solvent control data (0.0 - 4.0 % aberrant cells, excluding gaps). However, this value was not statistically significant and no dose-dependency was observed with the evaluated concentrations. No relevant effect occurred in Experiment I and thus the finding is regarded as being without biological relevance.
In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (2.0 - 5.0 %) as compared to the rates of the solvent controls (2.7 - 3.2 %).
A slightly increased number of endomitotic cells was observed in both experiments (0.0 -1.5 %). As these effects were less frequent and not dose dependent they are regarded as biologically irrelevant.
In both experiments, either EMS (1000.0 or 600.0 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.

Any other information on results incl. tables

Summary of results of the chromosome aberration study with the test substance

Exp.	Preparation	Test item	Endomitotic	Polyploid	Cell numbers	Mitotic indices	Aberrant cells
	interval	concentration	cells	cells	in %	in %	in %
		in µg/mL	in %	in %	of control	of control	incl. gaps*	excl. gaps*	with exchanges
Exposure period 4 hrs without S9 mix
I	18 hrs	Solvent control^1	0.1	3.2	100.0	100.0	3.5	3.5	1.0
		Positive control^2	n.d.	n.d.	n.d.	116.1	25.0	25.0^S	17.5
		2.5	0.0	5.0	108.5	101.6	1.0	1.0	0.5
		6.1	0.0	3.7	123.2	92.5	2.5	2.0	0.0
		15.4	0.0	3.1	100.8	112.9	2.5	2.0	1.5
Exposure period 18 hrs without S9 mix
II	18 hrs	Solvent control^1	0.0	3.1	100.0	100.0	3.5	2.5	0.0
		Positive control^#^3	n.d.	n.d.	n.d.	68.9	52.0	51.0^S	15.0
		1.2	0.0	2.9	88.0	112.4	1.5	1.0	0.0
		2.4	0.0	2.8	94.9	111.6	4.0	3.5	0.5
		4.7^P	0.0	2.0	95.4	90.0	3.0	2.0	0.0
Exposure period 4 hrs with S9 mix
I	18 hrs	Solvent control^1	0.1	3.1	100.0	100.0	2.0	2.0	1.5
		Positive control^4	n.d.	n.d.	n.d.	88.5	23.0	22.5^S	9.5
		6.1	1.1	3.5	56.4	121.8	2.5	2.5	1.0
		15.4	1.0	2.7	72.8	101.7	1.5	1.5	0.5
		38.4	0.3	2.3	76.5	108.1	2.0	1.0	1.0
II	18 hrs	Solvent control^1	0.0	2.7	100	100	4.0	3.5	1.0
		Positive control^4	n.d.	n.d.	n.d.	48.6	16.5	15.0^S	6.5
		1.2	1.5	4.0	110.6	115.6	2.5	1.5	0.0
		2.4^##	0.2	2.6	111.9	103.8	6.5	6.0	1.3
		4.7	0.1	2.9	103.7	93.4	3.5	3.0	1.0
		9.4^P	0.2	2.4	100.1	120.5	4.0	4.0	0.5

*     Inclusive cells carrying exchanges

^#     Evaluation of 50 metaphases per culture

^##    Evaluation of 200 metaphases per culture

n.d. Not determined

^P     Precipitation occurred at the end of treatment

^S     Aberration frequency statistically significant higher than corresponding control values

¹     DMSO    0.5 % (v/v)

²         EMS 1000.0 µg/mL

³         EMS   600.0 µg/mL

⁴         CPA       1.4 µg/mL

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

In conclusion, the test substance is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to precipitating or the highest evaluable concentrations.

Executive summary:

The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79cells of the Chinese hamster in vitro in two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I	Exp. II
Exposure period	4 hrs	18 hrs	4 hrs	4 hrs
Recovery	14 hrs	-	14 hrs	14 hrs
Preparation interval	18 hrs	18 hrs	18 hrs	18 hrs

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were evaluated.

The highest applied concentration (600.0 µg/mL; approx. 1.5 mM) was chosen with regard to the ability to formulate a homogeneous suspension of the test item in an appropriate solvent.

In the evaluable slides neither in the absence nor in the presence of S9 mix the mitotic index and/or cell numbers were reduced indicating no cytotoxicity.

In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item.

No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

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