Disodium 5-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]azo]-4-hydroxy-6-(methylamino)naphthalene-2-sulphonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date: 27 August 2022; Experimental starting date: 01 September 2022; Study completion date - 23 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 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted on 29th July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 5-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]azo]-4-hydroxy-6-(methylamino)naphthalene-2-sulphonate

EC Number:

274-436-7

EC Name:

Disodium 5-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]azo]-4-hydroxy-6-(methylamino)naphthalene-2-sulphonate

Cas Number:

70210-39-8

Molecular formula:

C20H17BrN4O8S2.2Na

IUPAC Name:

disodium 5-({4-[(2-bromoacryloyl)amino]-2-sulfonatophenyl}diazenyl)-4-hydroxy-6-(methylamino)naphthalene-2-sulfonate

Test material form:

solid

Remarks:

powder

Details on test material:

Name of Test Item : FAT 92354
Physical Appearance (with colour) : Dark brown powder
Batch No. : C/TE (MC-PD-R5B-03-21)
Purity (as per certificate of analysis) : 67.6 %
Batch Produced by (Name and address) : Third party (confidential)
Date of Manufacture : July 2021
Date of Expiry : 02 August 2026
Date of Retest : NA
Storage Conditions : Cool and dry (+2 to +8 ºC)

Specific details on test material used for the study:

Name of Test Item: FAT 92354
CAS No: 70210-39-8
Physical Appearance (with colour): Dark brown powder
Batch No: C/TE (MC-PD-R5B-03-21)
Purity (as per certificate of analysis): 67.6 %
Date of Manufacture: July 2021
Date of Expiry: 02 August 2026
Storage Conditions: Cool and dry (+2 to +8 ºC)

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.
Source of supply: In house bred animals

Sex:

male

Details on test animals or test system and environmental conditions:

Animal species: Rat (Rattus norvegicus)
Strain: Wistar
Justification for selection of species: The rat is one of the recommended species by regulatory agencies for conducting in vivo comet assay among rodents.
Source of supply: In house bred animals
No. of groups: Main Study: 6 groups (1 vehicle control, 2 positive control and 3 treatment groups)
No. of animals / group and sex: 6 males/group (36 males)
Age at treatment: 9 weeks
Body weight range at receipt (g): 181.05 to 229.99 g
Animal identification: Acclimatization period: All the animals were identified by tail marking using a black marker pen. Additionally, a cage card was displayed which included study no., cage no., sex, animal no. (temporary), start date and end date of acclimatization period.
Treatment period: All the animals were identified by body marking using turmeric solution, potassium permanganate and additionally, a cage card was displayed which included at least study no., cage no., sex, animal no. (permanent), treatment date and date of necropsy.

Husbandry:

Environmental Conditions: Animals were housed under standard laboratory conditions, in an environmentally monitored air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.3 to 22.5 ºC and relative humidity 49 % to 64 % in main study, with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.

Housing: Maximum of three animals of same sex and group were housed in a standard polysulphonate rat cage (L 430 × B 280 × H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Sterilized corn cob was used as a bedding material.
Feed: Altromin Maintenance Diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.

Water: Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on 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.

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:

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.

Frequency of treatment:

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.

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

Remarks:

High dose group

No. of animals per sex per dose:

6 males/group (36 males)

Control animals:

yes

Positive control(s):

Cyclophosphamide Monohydrate

Examinations

Details of tissue and slide preparation:

Minimum of three slides were prepared. The smears before staining were fixed by immersing the slides in methanol for 5 minutes. The air dried slides were stained with May-Gruenwald and Giemsa stain for evaluation.

All the slides including those of positive and vehicle controls were coded before microscopic evaluation to avoid group bias during evaluation.
For each animal, a minimum of 500 erythrocytes (which included mature and immature erythrocytes) were scored from first slide of the animal to determine PCEs: total RBC ratio along with the incidence of micronucleus. The subsequent slides were scored only for the number of PCEs and incidence of micronucleated PCEs. For each animal, a minimum of 4000 Polychromatic Erythrocytes (PCEs) were scored for the incidence of micronucleated immature erythrocytes (MNPCEs).

Evaluation criteria:

For each animal, a minimum of 500 erythrocytes (which included mature and immature erythrocytes) were scored from first slide of the animal to determine PCEs: total RBC ratio along with the incidence of micronucleus. The subsequent slides were scored only for the number of PCEs and incidence of micronucleated PCEs. For each animal, a minimum of 4000 Polychromatic Erythrocytes (PCEs) were scored for the incidence of micronucleated immature erythrocytes (MNPCEs).
The average numbers of micronucleated polychromatic erythrocytes (MNPCEs) were observed for a minimum of 4000 polychromatic erythrocytes (PCEs). The average percentage of MNPCEs in animals dosed with vehicle was 0.07. In the animals dosed with the test item at 500, 1000 and 2000 mg/kg bw/day, the average percentage of MNPCEs were 0.07, 0.09 and 0.10. There was no statistically significant increase in the percentage of MNPCEs (per 4000 PCEs scored) at any of the doses, in comparison with vehicle control.

Statistics:

Body weight of day 1, 2 and 3 was analysed by SPSS, at a 95 % level (p≤0.05) of significance. Intergroup comparison of body weight of Day 1, 2 and 3 were done. The slides from main study were decoded after analysis, the number of PCEs (Polychromatic erythrocytes), RBC (Red blood corpuscles), MNPCEs (Micronucleated Polychromatic erythrocytes) and PCEs/ total erythrocytes ratio (Polychromatic erythrocytes/ total erythrocytes) and frequency of MNPCEs was calculated. 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). 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.

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 average percentage of MNPCEs in animals dosed with vehicle was 0.07. In the animals dosed with the test item at 500, 1000 and 2000 mg/kg bw/day, the average percentage of MNPCEs were 0.07, 0.09 and 0.10. There was no statistically significant increase in the percentage of MNPCEs (per 4000 PCEs scored) at any of the doses, in comparison with vehicle control.

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

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
Grossly, pinkish discoloration of the stomach contents was observed in animals of G2, G3 and G4 group and this was attributable to the physical nature of the test item.
However, the mucosa of stomach was found to be within normal limits. There were no other gross pathological observations in the study.

Histopathology Findings
There was no test item-related histopathological findings in the study. The minimal, multifocal increased apoptosis in duodenal crypt epithelium in G5 group of animals (2/6), is considered to be related to administration of ethyl methanesulfonate which is used as positive control in the study.
Few microscopic findings observed in the study such as infiltration of mononuclear cells in liver and other findings were considered incidental as they occurred randomly across the groups including vehicle control group 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.

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 92354, is neither clastogenic nor aneugenic at and up to 2000 mg/kg bw/day.

Executive summary:

A study was conducted according to OECD test guideline 474 to determine the genotoxic potential of FAT 92354 (test item) in the micronucleus test using bone marrow cells of Wistar rat.

The test item, vehicle control and positive control were dosed for three consecutive days by oral route using oral gavage cannula. After the last dosing, animals were sacrificed and bone marrow cells were collected. The slides of bone marrow cells were stained with May-Gruenwald and Giemsa stain and observed for incidences of micronucleated polychromatic erythrocytes (MNPCEs). 

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 corn oil as vehicle. The animals designated as groups G2, G3 and G4 were administered 500, 1000 and 2000 mg/kg bw/day of FAT 92354, respectively.

The animals in group G6 were administered 100 mg/kg bw/day of the positive control cyclophosphamide monohydrate (for micronucleus test).

Bone marrow was collected for G6 group.

In the micronucleus test, the average percentage of MNPCEs was 0.07 in males dosed with vehicle. For the animals dosed with the test item at 500, 1000 and 2000 mg/kg bw/day, the average percentage of MNPCEs were 0.07, 0.09 and 0.10, respectively. There was no statistically significant increase in the percentage of MNPCEs (per 4000 PCEs scored) at the doses of 500, 1000 and 2000 mg/kg bw/day of test item, in comparison with the vehicle control.

The positive control group (G6), cyclophosphamide monohydrate at 100 mg/kg bw/day exhibited statistically significant increase in the numbers of MNPCEs when compared to vehicle control and the average percentage of MNPCEs (per 4000 PCEs scored) in positive control was 0.79. This demonstrated the sensitivity of the test system towards positive controls and confirmed that the test conditions were 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 92354, is neither clastogenic nor aneugenic at and up to 2000 mg/kg bw/day.