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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 30 July 2022; Experiment start date - 03 August 2022; Experiment completion date - 16 November 2023; Study completion date - 10 January 2023.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted on 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

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

Details on test material:

- Identification: FAT 36156/D TE
- Appearance: Dark powder
- Lot: Lot.20140804 (China)

Specific details on test material used for the study:

Name of Test Item: FAT 36156
Chemical Name (IUPAC): N-{2-[-(2-cyano-4,6-dinitrophenyl)diazenyl]-5-(diethylamino) phenyl}acetamide
CAS No. : 24170-60-3
Physical Appearance (with colour): green powder
Batch No.: FAT 36156 D/TE (20140804 (China))
Purity (as per certificate of analysis): 96.9 %
Batch Produced by (Name and address) : Third party (confidential)
Date of Manufacture: August 2014
Date of Expiry : April 28th, 2023
Date of Retest: May 3rd, 2021
Storage Conditions: Ambient (21 to 29 °C)

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is one of the recommended species by regulatory agencies for conducting in vivo comet assay among rodents.

Sex:

male

Details on test animals or test system and environmental conditions:

Name of Test Item: FAT 36156
CAS No : 24170-60-3
Physical Appearance (with colour): green powder
Batch No: D/TE (20140804 (China))
Purity (as per certificate of analysis): 96.9 %
Date of Manufacture: August 2014
Date of Expiry: April 28th, 2023
Date of Retest: May 3rd, 2021
Storage Conditions: Ambient (21 to 29 °C)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

0.5 % carboxymethyl cellulose

Details on exposure:

The test item was administered through oral route. The oral route is one of the probable routes of human exposure. The test item was administered through oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula. All the doses were administered in an equal volume of 10 mL/kg bw/day the dose of 500 (G2), 1000 (G3) and 2000 (G4) mg/kg bw/day as low, mid and high dose, respectively. Vehicle control group (G1) animals were administered with vehicle. Positive control group G5 and G6 animals were administered with ethyl methanesulfonate and cyclophosphamide monohydrate, respectively, at the dose volume of 10 mL/kg bw/day. Ethyl methanesulfonate and cyclophosphamide monohydrate were dissolved in distilled water and administered at a dose of 250 and 100 mg/kg bw/day, respectively.

Duration of treatment / exposure:

oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula.

Frequency of treatment:

oral route once a day for 3 consecutive days (0 day, 24 hours and 45 hours), using gavage cannula

Post exposure period:

Post 3 hours of the last day dosing, terminal sacrifice was done for all animals and all the animals were subjected to gross pathological examination.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Low dose group

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Mid dose group

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

High dose group

No. of animals per sex per dose:

6 males per group

Control animals:

yes

Positive control(s):

Ethyl methanesulfonate

Examinations

Tissues and cell types examined:

The animals were subjected to gross pathological examination.

Details of tissue and slide preparation:

Animals were euthanized by cervical dislocation 3 hours after the third treatment and subjected to gross pathological examination. Bone marrow was collected for micronucleus test. Liver, glandular stomach, duodenum and testicles were collected for the comet assay.

The liver, glandular stomach and duodenum for the comet assay was placed into ice-cold mincing and/or homogenization buffer (DPBS) and stored on ice. Tissues were rinsed sufficiently with cold mincing buffer to remove residual blood and stored in ice-cold mincing buffer until processed. A portion of the liver, glandular stomach and duodenum were collected and preserved in 10 % Neutral Buffered Formalin fixative and testes were preserved in modified Davidson’s fixative for 24 to 48 hours and then transferred to 10 % NBF for histopathology.

All the slides were coded before evaluation to avoid group bias during evaluation. Before scoring, slides were rehydrated with chilled distilled water for 30 minutes and stain with ethidium bromide, covered with a fresh coverslip and cells were scored under 400 X magnification.

Evaluation criteria:

At least 150 cells were analysed per sample. The open comet software was used for analysis. The comet endpoints collected was % tail DNA, tail length in microns measured from the estimated edge of the head region closest to the anode. The frequency of hedgehogs was determined for at least 150 cells per sample.

Statistics:

Body weight of day 1, 2 and 3 was analysed by SPSS, at a 95 % level (p ≤0.05) of significance. Inter group comparison of body weight of Day 1, 2 and 3 were done.

The clinical chemistry examination was analysed by SPSS at a 95% level (p ≤0.05) of significance. Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Gamma-glutamyl transpeptidase (GGT) were subjected to statistical analysis.
The percentage of tail DNA was analysed by SPSS at a 95 % level of confidence (p <0.05) of significance*.
The data of positive control and the treatment groups were compared with that of the vehicle control for the incidence of MNPCEs and the proportion of PCEs among total RBCs by SPSS at a 95 % level (p ≤0.05) of significance. All analysis and comparisons were evaluated at the 95 % level of confidence (p <0.05). Statistically significant changes obtained were designated by the superscripts in the summary table throughout the report as stated below:
*: Statistically significant (p <0.05).

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The dose formulation samples were analysed for dose concentration by HPLC and the results are found within the range of ± 15 % (i.e. 85 % to 115 %) recovery to the nominal concentration.

There was no statistically significant variation in body weight for treated animals.
No test item related gross or histopathological changes were seen in any of the treated animals.
No adverse treatment-related changes were observed in clinical chemistry parameters.

Any other information on results incl. tables

Clinical Signs of Toxicity and Mortality
The administration with the test item resulted in no clinical signs or mortality in any of the treated animals.

Body Weight
No statistically significant changes in body weight were observed in any of the treated animals when compared to vehicle control group.

Gross Pathology
No gross pathological findings were observed in any of the animals dosed with the test item at the doses of 500, 1000 and 2000 mg/kg bw/day.

HISTOPATHOLOGY FINDINGS
There were no test item-related histopathological findings observed in the study. Few microscopic findings were observed in the study such as infiltration of mononuclear cells in liver and all other findings were considered incidental as they occurred randomly across the groups and/or were expected for laboratory rats.

Clinical Chemistry
No adverse treatment related variation was noted in clinical chemistry parameters.
There were no statistically significant variations noted.

EVALUATION OF DNA DAMAGE
All the slides were coded before evaluation. Before scoring, slides were rehydrated with chilled distilled water for 30 minutes, stained with ethidium bromide, covered with a fresh coverslip and cells were then scored under 400X magnification.
At least 150 cells were analyzed per sample. The comet endpoint collected was % tail DNA, tail length in microns measured from the estimated edge of the head region closest to the anode. The frequency of hedgehogs was determined in at least 150 cells per sample bw/day. The average % tailing for DNA from liver cells was 3.66, 3.74, 3.69 and 3.76 at 0, 500, 1000 and 2000 mg/kg bw/day, respectively. The average % tailing for DNA from glandular stomach cells were 3.28, 3.35, 3.22 and 3.13 respectively and in duodenum, the % tailing observed were 3.77, 3.74, 3.81, and 3.65, at 0, 500, 1000 and 2000 mg/kg bw/day, respectively. There was no dose-dependent or statistically significant increase in the % tail DNA in any of the test item dosed animals, in comparison with the vehicle control group. The average % tail DNA observed in the liver, glandular stomach and duodenum of the positive control (at 250 mg/kg bw/day of ethyl methyl sulfonate) dosed animals were 8.25, 8.90 and 8.03, respectively.

Applicant's summary and conclusion

Conclusions:

Based on the results obtained under the conditions employed during this experiment, it is concluded that the test item, FAT 36156 not lead to increased DNA damage at and up to 2000 mg/kg bw/day.

Executive summary:

The test item, FAT 36156, was evaluated in the “In vivo Mammalian Alkaline Comet Assay” as per OECD Guideline No. 489, adopted on 29 July 2016. This study was conducted to determine if the test item, FAT 36156, can cause an increase in DNA damage in cells from specific organs and also to detect damage. The comet assay detects single and double stranded breaks when DNA is analyzed under alkaline conditions (pH >13). These strand breaks, when they occur in vivo, may be repaired, resulting in no persistent effect, or may be lethal to the cell, or may be fixed into a mutation resulting in a permanent viable change. This study used 6 groups of rats and each group consisted of 6 males. The animals received designated treatment for 3 consecutive days by oral route using gavage canula. The animals designated as group G1 animals were administered with 0.5 % carboxymethyl cellulose as vehicle. The animals designated as groups G2, G3 and G4 were administered 500, 1000 and 2000 mg/kg bw/day of FAT 36156, respectively. The animals in group G5 were administered 250 mg/kg bw/day of the positive control ethyl methanesulfonate (for comet assay). Approximately 3 hours after the last dosing, all rats were sacrificed by cervical dislocation and the designated organs (liver, duodenum, glandular stomach and testis) were collected for G1 to G5 groups. The collected tissues were processed, single cells were isolated, and slides were prepared. Slides were run through submarine-type electrophoresis and drained. Drained slides were stained with ethidium bromide and evaluated for % tailing of DNA, i.e. tail length in microns measured from the estimated edge of the head region closest to the anode. In the comet assay, the average % tailing for DNA from male liver cells was 3.66, 3.74, 3.69 and 3.76 at 0, 500, 1000 and 2000 mg/kg bw/day respectively. The average % tailing for DNA from glandular stomach cells were 3.28, 3.35, 3.22 and 3.13 and in duodenum, the % tailing observed were 3.77, 3.74, 3.81 and 3.65 at 0, 500, 1000 and 2000 mg/kg bw/day, respectively. There was no dose-related or statistically significant increase in the % tailing of DNA from cells of any of the organs for any of the FAT 36156 treated groups when compared to the vehicle control group. The average % tail DNA observed in the liver, glandular stomach and duodenum of the positive control (at 250 mg/kg bw/day of ethyl methyl sulfonate) dosed animals were 8.25, 8.90 and 8.03 respectively. The positive control [G5], ethyl methanesulfonate at a dose of 250 mg/kg bw/day produced a statistically significant increase in % tailing of DNA in cells from all the organs which were assessed (liver, duodenum and glandular stomach) when compared to the equivalent cells from organs of vehicle control animals [G1]. These data support the conclusion that the test conditions and sensitivity of the comet assay for this test of FAT 36156 were fully adequate. There was no statistically significant variation in body weight for treated animals. No test item related gross or histopathological changes were seen in any of the treated animals. No adverse treatment-related changes were observed in clinical chemistry parameters. The dose formulation samples were analyzed for dose concentration by HPLC and the results are found within the range of ±15 % (i.e. 85 % to 115 %) recovery to the nominal concentration. Based on the results obtained under the conditions employed during this experiment, it is concluded that the test item, FAT 36156 not lead to increased DNA damage at and up to 2000 mg/kg bw/day.