N-[2-[(2-cyano-4,6-dinitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

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:

12 October 2015 to 27 January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2016

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:

N-[2-[(2-cyano-4,6-dinitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

EC Number:

246-058-2

EC Name:

N-[2-[(2-cyano-4,6-dinitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

Cas Number:

24170-60-3

Molecular formula:

C19H19N7O5

IUPAC Name:

N-{2-[(2-cyano-4,6-dinitrophenyl)diazenyl]-5-(diethylamino)phenyl}acetamide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 20140804 (China)
- Expiration date of the lot/batch: 21 August 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary test: 0, 200, 600, 2000 μg/mL
Without S9 (4 h): 5, 10, 20, 40, 60, 80, 100 μg/mL
Without S9 (20 h): 0.25, 0.5, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10 μg/mL
Without S9 (4 h): 5, 10, 20, 40, 60, 80, 100 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Dimethylsulfoxide (DMSO) was used as the vehicle based on the solubility tests and compatibility with the target cells.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Toxicity of FAT 36156/D TE (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the absence of S9 activation was 30% at 40 μg/mL, the highest test dose level evaluated for chromosome aberrations.

Toxicity of FAT 36156/D TE (cell growth inhibition relative to the vehicle control) in CHO cells when treated for 4 hours in the presence of S9 activation was 46% at 40 μg/mL, the highest test dose level evaluated for chromosome aberrations

DETERMINATION OF CYTOTOXICITY
Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at 600 μg/mL in the non-activated 4-hour exposure group and at dose levels ≥ 6 μg/mL in the non-activated 20-hour exposure group. Cytotoxicity was not observed at any dose levels in the S9-activated 4-hour exposure group.

Based on the results of the preliminary toxicity test, the dose levels selected for testing in the chromosome aberration assay were as follows:
Non-activated 4 hr: 5, 10, 20, 40, 60, 80, 100 μg/mL
Non-activated 20 hr: 0.25, 0.5, 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10 μg/mL
S9-activated 4 hr: 5, 10, 20, 40, 60, 80, 100 μg/mL

Evaluation criteria:

Criteria for Determination of a Valid Test:

Vehicle Controls:
The frequency of cells with structural chromosomal aberrations should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95% control limits, they may be acceptable as long as these data are note extreme outliers (indicative of experimental or human error).

Positive Controls:
The frequency of cells with structural chromosomal aberrations must be significantly greater than the concurrent vehicle control (p ≤ 0.05). In addition, the cytotoxicity response must not exceed the upper limit for the assay (60%).

Cell Proliferation:
The average viable cell count in the vehicle control at harvest must be ≥ 1.5-fold the average viable cell baseline value.

Test Conditions:
The test substance must be tested using a 4-hour treatment with and without S9, as well as a 20-hour treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.

Analyzable Concentrations:
At least 300 metaphases must be analyzed from at least three appropriate test substance concentrations. The number of metaphases scored may be reduced when high numbers of cells with chromosomal aberrations (≥10% metaphases) are observed as with a positive test substance or the positive control substance.

Evaluation of Test Results:
The test substance was considered to have induced a positive response if:
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and
• results were outside the 95% control limit of the historical negative control data.
The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p ≤ 0.05) for a pairwise comparison of the frequency of aberrant cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Effects of pH: The pH of the highest dose level of test substance in treatment medium was 7.4.
- Effects of osmolality: The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 120%.

COMPARISON WITH HISTORICAL CONTROL DATA:
The numerical aberrations in the 4-hr treatment were statically positive without a dose response. The induced values were in the line of the historical control range (0-5%, in the non-activated 4-hour exposure group and 0-9.5%, in the S9-activated 4-hour exposure group), therefore, it was considered biologically irreverent.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The results of the evaluation of cell growth inhibition are presented in Tables under any other information section. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at 600 μg/mL in the non-activated 4-hour exposure group and at dose levels ≥ 6 μg/mL in the non-activated 20-hour exposure group. Cytotoxicity was not observed at any dose levels in the S9-activated 4-hour exposure group.

Remarks on result:

other: strain/cell type: Chinese hamster ovary (CHO-K1) cells

Any other information on results incl. tables

Preliminary Toxicity Assay

In the preliminary toxicity assay, CHO cells were exposed to nine dose levels of FAT 36156/D TE, ranging from 0.2 to 2000 μg/mL, as well as vehicle controls, in both the absence and presence of an Aroclor-induced S9 metabolic activation system for 4 h, or continuously for 20 h in the absence of S9 activation. The test substance formed clear solutions in DMSO from 0.02 to 0.06 mg/mL and workable suspensions in DMSO from 0.2 to 200 mg/mL. Visible precipitate was observed in treatment medium at the following dose levels:

 

Treatment Condition	Treatment Time	Visible precipitate
		At the beginning of Treatment period	At the conclusion of Treatment period
Non-activated	4 h	≥ 600 µg/mL	≥ 60 µg/mL
	20 h	≥ 600 µg/mL	≥ 60 µg/mL
S9-activated	4 h	≥ 600 µg/mL	≥ 60 µg/mL

 

The osmolality in treatment medium was measured as follows:

The osmolality of the test substance dose levels in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 120%. The pH of the highest dose level of test substance in treatment medium was 7.4.

 

 

Dose tested	Dose levels (µg/mL)	Osmolality (mmol/kg)
Vehicle	0	430
Highest soluble	200	438
Lowest precipitating	600	440
Highest	2000	417

 

In the chromosome aberration assay, the test substance formed clear solutions in DMSO from 0.025 to 0.05 mg/mL and workable suspensions in DMSO from 0.1 to 10 mg/mL. Visible precipitate was observed in treatment medium at the following dose levels:

Treatment Condition	Treatment Time	Visible precipitate
		At the beginning of Treatment period	At the conclusion of Treatment period
Non-activated	4 h	None	≥ 40 µg/mL
	20 h	None	None
S9-activated	4 h	None	≥ 40 µg/mL

 

Applicant's summary and conclusion

Conclusions:

FAT 36156/D TE was concluded to be positive for the induction of structural chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Executive summary:

FAT 36156/D TE, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells following OECD guideline 473 in compliance to GLP in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system. A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, CHO cells were treated for 4 and 20 h in the non-activated test system and for 4 h in the S9-activated test system. All cells were harvested 20 hours after treatment initiation. Dimethylsulfoxide (DMSO) was used as the vehicle.

 

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL. Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at 600 μg/mL in the non-activated 4-h exposure group and at dose levels ≥ 6 μg/mL in the non-activated 20-hour exposure group. Cytotoxicity was not observed at any dose levels in the S9-activated 4-h exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 60 μg/mL in all three treatment conditions. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 5 to 100 µg/mL for the non-activated and S9-activated 4-h exposure groups, and from 0.25 to 10 µg/mL for the non-activated 20-h exposure group.

 

In the chromosome aberration assay, 55 ± 5% cytotoxicity (reduction in cell growth index relative to the vehicle control) was observed at dose levels ≥ 3.5 μg/mL in the non-activated 20-hour exposure group. Cytotoxicity was not observed at any dose levels in the non-activated and S9-activated 4-h exposure groups. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 40 μg/mL in the non-activated and S9-activated 4-h exposure groups. The dose levels selected for microscopic analysis were 10, 20, and 40 µg/mL for the non-activated and S9-activated 4-hour exposure groups, and 1, 2, and 4.5 µg/mL for the non-activated 20-h exposure group.

 

The percentage of cells with structural and numerical aberrations in the non-activated 4-h exposure group was statistically significantly increased relative to vehicle control at dose levels 10, 20, and 40 μg/mL (p ≤ 0.05 or p ≤ 0.01, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose-response (p > 0.05).

 

The percentage of cells with structural aberrations in the S9-activated 4-h exposure group was statistically significantly increased relative to vehicle control at 40 μg/mL (p ≤ 0.01, Fisher's Exact test). The Cochran-Armitage test was also positive for a dose-response (p ≤ 0.05).

 

The percentage of cells with numerical aberrations in the S9-activated 4-h exposure group was statistically significantly increased relative to vehicle control at dose levels 10 and 20 μg/mL (p ≤ 0.01, Fisher's Exact test). However, the Cochran-Armitage test was negative for a dose-response (p > 0.05). The percentage of cells with structural aberrations in the non-activated 20-h exposure group was statistically significantly increased relative to vehicle control at dose levels 2 and 4.5 μg/mL (p ≤ 0.01, Fisher's Exact test). The Cochran-Armitage test was also positive for a dose-response (p ≤ 0.05). The percentage of cells with numerical aberrations in the test substance-treated group was not significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher's Exact test).

 

The numerical aberrations in the 4-h treatment were statically positive without a dose response. The induced values were in the line of the historical control range (0-5%, in the non-activated 4-h exposure group and 0-9.5%, in the S9-activated 4-h exposure group), therefore, it was considered biologically irreverent. All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤ 0.01). Thus, all criteria for a valid study were met.

 

Under the conditions of the assay described in this report, FAT 36156/D TE was concluded to be positive for the induction of structural chromosome aberrations in both non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells. Although there was a significant induction in numerical aberrations, it was considered biologically irrelevant.