N-[4-(aminocarbonyl)phenyl]-4-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azo]benzamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

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:

Dimethyl 2-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azo]terephthalate

EC Number:

252-650-1

EC Name:

Dimethyl 2-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azo]terephthalate

Cas Number:

35636-63-6

Molecular formula:

C21H19N5O7

IUPAC Name:

dimethyl 2-({2-oxo-1-[(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)carbamoyl]propyl}diazenyl)terephthalate

Test material form:

solid: nanoform

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Cytokinesis block (if used):

Colchicine was used as the spindle inhibitor at 0.2 μg/mL

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from male Wistar rats induced with a single intraperitoneal injection of Aroclor 1254 (0.7 mL/rat ready to use solution), 5 days prior to sacrifice.
The S9 homogenate batch prepared and stored in the test facility either at -68 to -86 °C or in liquid
nitrogen until its use. Each batch of S9 homogenate is assessed for sterility, protein content (modified Lowry Assay, Sword and Thomson, 1980) and for its ability to metabolize the pro-mutagens 2-Aminoanthracene and Benzo (a) pyrene to mutagens using Salmonella typhimurium TA100 strain.

S9 homogenate was thawed immediately before use and mixed with the co-factor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in PBS.

7.2.1 S9 Mix
The co-factor solution was prepared by dissolving the following co-factors in 18 mL PBS for the preliminary cytotoxicity test and the chromosome aberration assay.

NADP (4 mM) 57 mg
Glucose-6-phosphate (5 mM) 31 mg
Magnesium chloride (8 mM) 29 mg
Potassium chloride (33 mM) 44 mg

The S9 mix was prepared by mixing 4 mL S9 homogenate with 16 mL of the co-factor solution, for the preliminary cytotoxicity test and the chromosomal aberration assay.

Test concentrations with justification for top dose:

Test Concentrations
Following concentrations of the test item were used in the preliminary cytotoxicity test:
a) 7.81, b) 15.63, c) 31.25, d) 62.5, e) 125, f) 250, g) 500, h) 1000 and i) 2000 μg/mL (limit concentration as per OECD 473).
At the beginning and end of 3-hour exposure period, there was no precipitation of the test item, both in the presence and absence of metabolic activation at the test concentrations of 7.81, 15.63, 31.25, 62.5 and 125 µg/mL.
At the beginning and end of the 3-hour exposure, both in the presence and absence of metabolic activation, there was slight precipitation of the test item at 250 µg/mL and moderate precipitation of the test item at 500, 1000 and 2000 µg/mL.
Based on the guideline requirement, the test concentrations which exhibited moderate precipitation of the test item were not processed further and 250 µg/mL, which exhibited slight precipitation of the test item was selected as the top concentration in the preliminary cytotoxicity test.

Based on observations and preliminary cytotoxicity test results, it was decided to test up to a maximum of 250 µg/mL in the presence and absence of metabolic activation with the 3-hour exposure and in the absence of metabolic activation with the 21-hours exposure period in the chromosomal aberration assay.

Main test
Based on the observations of the preliminary cytotoxicity test, following concentrations of the test item are selected for testing in the chromosomal aberration assay

a) 15.63 b) 62.5 and c) 250 μg/mL

Vehicle / solvent:

Sterile water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 63830396) with a doubling time approximately 12 -14 hours and modal chromosome number of 20 was used as the test system.
The cell line was tested for mycoplasma in the testing facility. The karyotype analysis of this cell line was periodically performed and documented.
Cells were grown in T- 25 cm2 and T-75 cm2 flasks at 37 ± 1 °C in a carbon dioxide incubator (5 ± 0.2 % CO2 in air)

The study consisted of a preliminary cytotoxicity test and a chromosome aberration assay. Chromosome aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic
activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21-hour exposure.
The test item formed a free flowing suspension in sterile water at 200 mg/mL and a pasty suspension in DMSO at 200 mg/mL. Hence, sterile water was selected as the vehicle of choice for the chromosomal aberration test.
In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, the test item did not exhibit the required level of cytotoxicity (reduction in the cell growth by 55 ± 5 % of the concurrent vehicle control) up to the highest tested concentration of 250 μg/mL, both, in the presence and absence of metabolic activation (short term treatment) and in the absence of metabolic activation (long term treatment).
Based on these observations and preliminary cytotoxicity test results, it was decided to test up to a maximum of 250 μg/mL in the presence and absence of metabolic activation with the 3-hour exposure and in the absence of metabolic activation with the 21-hours exposure period in the chromosomal aberration assay.

In the chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate at the concentrations of 15.63, 62.5 and 250 μg/mL in Experiments 1, 2 and 3 of the chromosomal aberration assay. Concurrent vehicle (SW) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation at 21 hour exposure) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at approximately 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.
At the highest concentrations tested, the reduction in cell growth as RICC was 47, 43 and 53 % in experiments 1, 2 and 3, respectively, compared to the vehicle control.
A total of 300 metaphases each from the SW control, each treatment level and the positive controls were evaluated for chromosomal aberrations.

Rationale for test conditions:

Conditions according to guideline

Evaluation criteria:

Metaphases of all concentrations of the test item, the positive and vehicle control cultures were scored.

8.1 Definition for Aberrations

The chromosome and chromatid aberrations observed have been grouped into three categories - gaps, breaks (includes deletions and displacements) and exchanges.

Gap

Chromatid gap is a non-staining region of a single chromatid in which there is a minimal misalignment of the chromatid.

Chromosome gap is a non-staining region at the same locus in both chromatids of a single chromosome in which there is minimal misalignment of the chromatids.

Break

Chromatid break is discontinuity of a single chromatid in which there is clear misalignment of one of the chromatids.

Chromosome break is discontinuity at the same locus in both chromatids of a single chromosome, giving rise to an acentric fragment.

A chromatid deletion occurs when a bit of chromatid is missing as a result of a break.

A chromosome deletion occurs when bits of both chromatids are missing as a result of a break.

Chromatid displacement occurs when the fragment of the chromatid beyond the break point is not in alignment with the chromosome of origin.

Chromosome displacement occurs when the fragments of both chromatids beyond the break point are not in alignment with the chromosome of origin.

Exchange

Exchanges occur as a result of two or more chromatid lesions and the subsequent rearrangement of chromatid material.

Ring chromosomes are formed when a chromosome undergoes two breaks and the broken ends of the chromosome reunite in a ring structure either with or without a centromere.

Pulverization

In cells with pulverization, the chromosomes are reduced to masses of fragments. This represents the degree of damage inflicted on the chromosome. This aberration indicates the cytotoxic effect of the test item.

Polyploidy

Cell containing more than the diploid number (2n) of chromosomes,

Statistics:

Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fischer exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

TABLE 3.      Results of Preliminary Cytotoxicity Test

Treatment (µg/mL)	Presence of metabolic activation  (3-hour exposure)			Absence of metabolic activation  (3-hour exposure)			Absence of metabolic activation  (21-hour exposure)
	Final – initial cell count (1x10^6/flask)	Cell growth index RICC (%)	Cell growth inhibition (%)	Final – initial cell count (1x10^6/flask)	Cell growth index RICC (%)	Cell growth inhibition (%)	Final – initial cell count (1x10^6/flask)	Cell growth index RICC (%)	Cell growth inhibition (%)
Sterile water	0.78125	100	0	0.83125	100	0	0.71875	100	0
7.81	0.71875	92	8	0.79375	95	5	0.63125	88	12
15.63	0.63125	81	19	0.73125	88	12	0.55625	77	23
31.25	0.56875	73	27	0.68125	82	18	0.50625	70	30
62.5	0.51875	66	34	0.60625	73	27	0.43125	60	40
125	0.48125	62	38	0.55625	67	33	0.38125	53	47
250	0.41875	54	46	0.49375	59	41	0.31875	44	56

Base line cell count: 1.58125 X 10⁶/flask

 

TABLE 4.      Summary Results of Chromosomal Aberration Assay - Experiment 1

Treatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@						Total No. (%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges		Includi ng Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct
Sterile water	300	0	0	1 (0.33)	0	0	0	1 (0.33)	1 (0.33)	0
15.63	300	0	0	0	0	0	0	0	0	16
62.5	300	0	0	0	0	0	0	0	0	33
250	300	0	0	1 (0.33)	0	0	0	1 (0.33)	1 (0.33)	47
CPA 55	300	1 (0.33)	0	12 (4.00)	19 (6.33)	38 (12.67)	31 (10.33)	73 (24.33)	72+ (24.00)	35

*: Metaphase plate with one or more than one aberration considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %         

Cs: Chromosome type            Ct: Chromatid type       

CPA: Cyclophosphamide monohydrate                     +: Significantly higher than control (p <0.05) by Fischer exact test Note: There were no incidences of polyploidy and endoreduplicated cells

 

TABLE 5.      Summary Results of Chromosomal Aberration Assay - Experiment 2 

Treatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@						Total No. (%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges		Including Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct
Sterile water	300	1 (0.33)	0	0	0	0	0	1 (0.33)	0	0
15.63	300	0	0	0	0	0	0	0	0	10
62.5	300	0	1 (0.33)	0	0	0	0	1 (0.33)	0	25
250	300	0	0	0	0	0	0	0	0	43

   

 *: Metaphase plate with one or more than one aberration considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %         

Cs: Chromosome type            Ct: Chromatid type                              

Note: There were no incidences of polyploidy and endoreduplicated cells

 

TABLE 6.      Summary Results of Chromosomal Aberration Assay - Experiment 3

 

Treatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@						Total No. (%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges		Including Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct
Sterile water	300	0	0	1 (0.33)	0	0	0	1 (0.33)	1 (0.33)	0
15.63	300	0	0	0	0	0	0	0	0	22
62.5	300	0	0	0	1 (0.33)	0	0	1 (0.33)	1 (0.33)	38
250	300	0	0	1 (0.33)	0	0	0	1 (0.33)	1 (0.33)	53
EMS 600	300	4 (1.33)	0	35 (11.67)	36 (12.00)	9 (3.00)	15 (5.00)	84 (28.00)	81+ (27.00)	28

*: Metaphase plate with one or more than one aberration considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %         

Cs: Chromosome type            Ct: Chromatid type       

EMS: Ethyl methanesulfonate            +: Significantly higher than control (p <0.05) by Fischer exact test

Note: There were no incidences of polyploidy and endoreduplicated cells

 

ANNEXURE 2. Historical Vehicle and Positive Control Data

                                Test System: CHO cells                                              No. of Studies: 56

 

                                Vehicle Control                                                                                  

Parameter	Total No. of Metaphases with Aberrations Excluding Gaps
	Presence of Metabolic Activation	Absence of Metabolic Activation
Range	0 - 5	0 - 4
Mean ± SD	0 ± 1	1 ± 1

 

Positive Control 

Parameter	Total No. of Metaphases with Aberrations Excluding Gaps
	Presence of Metabolic Activation	Absence of Metabolic Activation
Range	51 - 204	63 - 248
Mean ± SD	124 ± 37	121 ± 32

 

CHO: Chinese Hamster Ovary Cells

SD: Standard deviation

Applicant's summary and conclusion

Conclusions:

The test item, C.I. Pigment Yellow 175 was not clastogenic in CHO-K1 cells at the tested concentration and under the conditions of testing employed.

Executive summary:

The clastogenic potential of the test item, C.I. Pigment Yellow 175 to induce chromosomal aberrations in mammalian cells was evaluated using cultured Chinese Hamster Ovary (CHO-K1) cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

The study consisted of a preliminary cytotoxicity test and a chromosome aberration assay. Chromosome aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21-hour exposure.

 

The test item formed a free flowing suspension in sterile water at 200 mg/mL and a pasty suspension in DMSO at 200 mg/mL. Hence, sterile water was selected as the vehicle of choice for the chromosomal aberration test.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, the test item did not exhibit the required level of cytotoxicity (reduction in the cell growth by 55± 5 % of the concurrent vehicle control)up to the highest tested concentration of 250µg/mL,both, in the presence and absence of metabolic activation (short term treatment) and in the absence of metabolic activation (long term treatment).

 

Based on these observations and preliminary cytotoxicity test results, it was decided to test up to a maximum of 250 µg/mL in thepresence and absence of metabolic activation with the 3-hour exposure and in the absence of metabolic activation with the 21-hours exposure period in the chromosomal aberration assay.

 

In the chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate at the concentrations of 15.63, 62.5 and 250mg/mL in Experiments 1, 2 and 3 of the chromosomal aberration assay. Concurrent vehicle (SW) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation at 21 hour exposure) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at approximately 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.

 

At the highest concentrations tested, the reduction in cell growth as RICC was 47, 43 and 53 % in experiments 1, 2 and 3, respectively, compared to the vehicle control.

 

A total of 300 metaphases each from the SW control, each treatment level and the positive controls were evaluated for chromosomal aberrations.

 

There was no evidence of statistically significant induction of chromosomal aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases.

The study indicated that the test item, C.I. Pigment Yellow 175 was not clastogenic at the concentrations tested and under the conditions of testing