N,N'-naphthalene-1,5-diylbis[4-[(2,3-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 December 2021 to 05 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-naphthalene-1,5-diylbis[4-[(2,3-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

EC Number:

271-178-7

EC Name:

N,N'-naphthalene-1,5-diylbis[4-[(2,3-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Cas Number:

68516-75-6

Molecular formula:

C44H26Cl4N6O4

IUPAC Name:

N,N'-naphthalene-1,5-diylbis{4-[(2,3-dichlorophenyl)diazenyl]-3-hydroxy-2-naphthamide}

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Name of Test Item : PV Fast Brown RL
Chemical Name (IUPAC) : N,N`-Naphthalene-1,5-diylbis[4-[(2,3-dichlorophenyl)azo]3-hydroxynaphthalene-2-carboxamide]
Molecular formula : C44H26Cl4N6O4
Molecular weight : 844.55 g/mol
CAS No. : 68516-75-6
Physical Appearance (with color) : Brown powder
Storage Conditions : Ambient (21 to 29°C)

Method

Target gene:

HPRT

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:

s9 Liver homogenate

Test concentrations with justification for top dose:

At a concentration of 0.125 mg/mL, the Relative Survival was greater than 10%. Therefore 0.125 mg/mL was selected as the highest concentration for testing in gene mutation test.

Vehicle / solvent:

DMSO

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

CHO AA8 Cells, a derivative of the CHO-K1, were used, CHO AA8 cells, Batch No.5000062 procured from American Type Culture Collection (ATCC) was used for the test.

Evaluation criteria:

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
 At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
 The increase is concentration-related when evaluated with an appropriate trend test.
 Any of the results are outside the distribution of the historical negative/vehicle control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
A test chemical is considered clearly negative if, in all experimental conditions examined:
 None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
 There is no concentration-related increase when evaluated with an appropriate trend test.
 All results are inside the distribution of the historical negative/vehicle control data.

Statistics:

Data of mutant frequencies were analyzed for differences among vehicle control, treatment and positive control groups by performing power transformation procedure by Snee and Irr (1981) with which, the observed mutant frequency was transformed using the formula:
Y=(X+A)B
Where,
Y = transformed mutant frequency, X = observed mutant frequency
BIO-GNT 1743 Study Report Page 19 of 36
[Where X=No. of mutant colonies per replicateACE value×100
and A, B = constants (viz. A = 1 and B = 0.15)]
Statistical analysis of the experimental data was carried out using SPSS Statistical package version 22.0. If the analysis of variance is significant at p < 0.05, Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control.
The statistical significances are designated by the superscripts as given below:
* Statistically significant (p<0.05) change than the vehicle control group.

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:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility, Precipitation and pH Test
The test item was found soluble in DMSO at 200 mg/mL. Precipitation test was conducted at 0.0625, 0.125, 0.25, 0.50, 1 and 2 mg/mL concentrations. Post 3 hours and 40 minutes of incubation, no precipitation observed at 0.125 and 0.0625 mg/mL, mild precipitation was observed at 0.25 mg/mL, moderate precipitation observed at 0.5 mg/mL and heavy precipitation observed at 1 and 2 mg/mL. No change in pH was observed at the tested concentrations up to 2 mg/mL.
Initial Cytotoxicity Test
In the initial cytotoxicity test, there was no evidence of excessive cytotoxicity (˂10% RS) at and up to 0.5 mg/mL in both presence of metabolic activation and absence of metabolic activation when compared to vehicle control. In the presence of metabolic activation, the RS values ranged from 1.23 % to 45.68 % and in the absence of metabolic activation, the RS values ranged from 1.27 % to 46.84 % at the concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL.
Gene Mutation Test
In the gene mutation test, the cells were treated with test item at the concentrations of 0.015625, 0.03125, 0.0625 and 0.125 mg/mL using DMSO as the vehicle in four plate cultures both in the presence of metabolic activation and absence of metabolic activation.
The test item resulted in mutant frequencies of 23.46 to 24.71 per 2×106 cells in the presence of metabolic activation with 24.18 per 2×106 cells in the vehicle control. In the absence of metabolic activation, mutant frequencies of 24.10 to 24.68 per 2×106 cells were observed with 23.86 per 2×106 cells in the vehicle control. There was no statistically significant increase in the mutant frequencies observed when compared with vehicle control at any of the tested concentrations.
There was no evidence of excessive cytotoxicity (˂10% RS) at any of the concentrations both in presence and absence of metabolic activation. In the presence of metabolic activation, the RS values ranged from 17.07 to 85.37 % and in the absence of metabolic activation the RS values ranged from 18.99 to 78.48 % respectively.
Application of the positive control, 3 μg/mL of Benzo (a) pyrene, resulted in a RS value of 89.02 % in the presence of metabolic activation, and a mutant frequency of 259.55 per 2×106 cells which was statistically significant when compared with the vehicle control.
The treatment with the positive control, 1 μg/mL of 4-Nitroquinoline N-oxide, resulted in a RS value 96.20 % in the absence of metabolic activation and a mutant frequency of 262.92 per 2×106 cells and was statistically significant when compared with that of vehicle control.

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Applicant's summary and conclusion

Conclusions:

Based on the results obtained, the test item is considered as non-mutagenic at and up to the concentration of 0.125 mg/mL, both in the presence and absence of metabolic activation under the tested laboratory conditions.

Executive summary:

The test item was evaluated for gene mutation test in CHO AA8 cells.
The test item was found soluble in DMSO at 200 mg/mL. Precipitation test was conducted at 0.0625, 0.125, 0.25, 0.50, 1 and 2 mg/mL concentrations. After 3 hours and 40 minutes of incubation, no precipitation was observed at 0.125 and 0.0625 mg/mL, mild precipitation was observed at 0.25 mg/mL, moderate precipitation was observed at 0.5 mg/mL and heavy precipitation was observed at 1 and 2 mg/mL. No change in pH was observed at the tested concentrations up to 2 mg/mL.
On the basis of precipitation results, 0.5 mg/mL was selected as the highest concentration for the initial cytotoxicity test. Initial cytotoxicity test was conducted at the concentrations of 0.03125, 0.0625, 0.125, 0.25 and 0.5 mg/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (3 hours and 33 minutes). Cytotoxicity was assessed by determining the Adjusted Cloning Efficiency and Relative Survival in the test.
The results of the initial cytotoxicity test indicated that the Relative Survival was greater than 10% at 0.125 mg/mL when compared with the respective vehicle control, both in the presence and absence of metabolic activation. Based on these results, 0.125 mg/mL was selected as highest concentration for gene mutation test.
The gene mutation test was conducted at the concentrations of 0.015625, 0.03125, 0.0625 and 0.125 mg/mL using DMSO as a vehicle in four plates/group in the presence and absence of metabolic activation (4 hours).
Benzo(a) pyrene and 4 Nitroquinoline N-oxide were used as Positive controls for the gene mutation test.
Cytotoxicity as Relative Survival was 17.07 to 18.99 % in presence of metabolic activation and absence of metabolic activation at the highest tested concentration of 0.125 mg/mL.
There was no statistically significant increase in mutant frequencies at any of the concentrations tested when compared with the vehicle control. Moreover, treatment with the test item resulted in mutant frequencies which were within acceptable ranges with regard to historical controls.
There was statistically significant increase in mutant frequencies for positive controls when compared with the vehicle control in both metabolic activation and absence of metabolic activation.