1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 04 SEPTEMBER 2018 TO 17 SEPTEMBER 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Bacterial Reverse Mutation Test. Adopted 21st July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

EC Number:

274-126-1

EC Name:

1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

Cas Number:

69808-32-8

Molecular formula:

C17H17ClN4O2

IUPAC Name:

1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile

Test material form:

solid: particulate/powder

Details on test material:

- State of aggregation: powder
- Particle size distribution: not determined
- Mass median aerodynamic diameter (MMAD): not determined
- Geometric standard deviation (GSD): not determined
- Shape of particles: not determined
- Surface area of particles: not determined
- Crystal structure: not determined
- Coating: none
- Surface properties: not determined
- Density: 1.3526 ± 0.0010 g/cm3 at 20.0 ± 0.2°C
- Moisture content: <0.5%
- Residual solvent: not detected
- Activation: none
- Stabilisation: none
- Other:

Method

Target gene:

All the bacterial strains used in the Ames test carry a mutant gene that prevents them from synthesizing an essential amino acid. These strains may carry additional mutations which increase their sensitivity to different types of mutagens. All S. typhimurium strains used in the test carry the rfa mutation. This mutation causes an alteration in the lipopolysaccharide (LPS) layer making the bacteria more permeable to larger molecules. The uvrB deletions eliminate the accurate excision repair mechanism resulting in an increase in the rate of mutations due to an alternative DNA repair mechanism. The plasmid pKM101 in several strains enhances the chemical mutagenesis via an increase in the error-prone recombinational DNA repair mechanism.

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other:

Remarks:

A mutant gene prevent it from synthesizing essential amino acid. This mutation causes an alteration in the lipopolysaccharide (LPS) layer making the bacteria more permeable to larger molecules. The uvrB deletions eliminate the accurate excision repair

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Remarks:

rfa mutation. This mutation causes an alteration in the lipopolysaccharide (LPS) layer making the bacteria more permeable to larger molecules. The uvrB deletions eliminate the accurate excision repair mechanism resulting in an increase in the rate of muta

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: MOLTOX MOLECULAR TOXICOLOGY, INC.
- method of preparation of S9 mix:
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): referring the QUALITY CONTROL & PRODUCTION CERTIFICATE, batch. 3970

Test concentrations with justification for top dose:

C5 - 0.02 mg/plate
C4 - 0.0067 mg/plate
C3 - 0.0022 mg/plate
C2 - 0.0007 mg/plate
C1 - 0.0002 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: the test item is soluble at concentration 5.6 mg/mL in DMSO. Nevertheless, precipitation signs were observed in the assay final mixture with PBS at concentration of 5.6, 1.85, 0.6 and 0.2 mg/mL. Therefore, the C5 selected for the cytotoxicity assay was 0.2 mg/mL (0.02 mg/plate), as recommended by the OECD guideline 471.

- Justification for percentage of solvent in the final culture medium: Therefore, the C5 selected for the cytotoxicity assay was 0.2 mg/mL (0.02 mg/plate), as recommended by the OECD guideline 471.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other:

Remarks:

Other: 2-amino-anthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min
- Exposure duration/duration of treatment: 48h
- Harvest time after the end of treatment (sampling/recovery times):

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
- Determination of polyploidy:
- Determination of endoreplication:

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
- Selection time (if incubation with a selective agent):
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:

Rationale for test conditions:

Each group was assayed with 5 concentrations of the test item (C5 to C1) and with vehicle as negative control. the concentrations were defined by solubility of the test item, with and without activation system.

Evaluation criteria:

The criteria used for determining a positive result take into account a dose-response effect in the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
A result is considered positive whenever the number of revertants of the test item-treated plates is increased when compared to the solvent-treated plates according to the following criteria:
S. typhimurium TA100 --> 2 fold

Biological elevance of the results was also considered.

The bacterial reverse mutation test for the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile was considered valid as the following criteria were met:
− The mean solvent control counts complied with Vivotecnia historical data for each strain.
− The mean reference item control counts complied with Vivotecnia historical data for each strain.

Statistics:

The number of revertant colonies (recordded by an automatic colony counter).
Average plate counts were presented with the mean and the standard deviation for each set of triplicates per test item concentration and was used to calculate the ratio of colonies per exposed plate compared to the corresponding negative control.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile does not induce point mutations or frameshifts in the genome of the bacterial strains with or without metabolic activation regardless of the procedure over the concentration range tested.

Therefore, the test item 1-butyl-5-[(4-chlorophenyl)azo]-1,2-dihydro-6-hydroxy-4-methyl-2-oxonicotinonitrile at an exposure dose range of 0.0002 – 0.02 mg/plate is considered to be NON
MUTAGENIC / NON PRO-MUTAGENIC under the experimental conditions assayed.