Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The ability of MACROLEX Rot B to induce mutations was investigated using Salmonella typhimurium strains TA 100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2uvrA with a pre-incubation method in the absence and presence of a metabolic activation System (S9 mix).

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

EC Number:

277-146-9

EC Name:

Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

Cas Number:

72968-71-9

Molecular formula:

C23H29N7O4S

IUPAC Name:

methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

Test material form:

solid

Specific details on test material used for the study:

Chemical name. Methyl 4-cyano-5 [[5-cyano-2,6-bis[(3-methoxy propyl)amino]-4-methyl-3-pyridyl azo]-3-methyl-2-thenoate
Other name: MACROLEX Rot B
CAS Number: 72968-71-9
Purity: >99% (w/w)

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

Negative control, 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-(2-FuryI)-3-(5-nitro-2 furyl)acrylamide, Sodium azide, 2-Aminoanthracene

Details on test system and experimental conditions:

This study was performed by the pre-incubation method without and with S9 mix. Triplicate plates were used for the negative control group, and duplicate plates were used for the positive control and the test substance treatment groups.

Procedures
After 0.1 mL of the test substance solution, vehicle or the positive control substance solution, 0.5 mL of 0.1 M sodium phosphate bufier (pH 7.4) or S9 mix and 0.1 mL of the bacterial culture were added to a test tube, the mixture as shaken at 37±0.5°C for 20 minutes. Two milliliters of the soft agar were then added to each tube and the mixture was poured onto a minimal glucose agar plate. The number of revertant colonies was counted after incubation at 37±0.5°C for 48 hours.

Confirmation of Sterility
The highest concentration of the test substance solution (0.1 mL) and S9 mix (0.5 mL) were individually mixed with 2 mL of the soft agar and were poured onto the minimal glucose agar plate in order to examine bacterial contamination.
Bacterial contamination was judged with those plates after incubation at 37±0.5°C for 48 hours.

Negative Control and Positive Controls
The vehicle was employed as a negative control. As positive controls 2-(2-FuryI)-3-(5-nitro-2 furyl)acrylamide, Sodium azide, 2-Aminoanthracene were used.

Evaluation criteria:

The test substance was judged to be positive when the number of revertant colonies increased to twice or more than that in the negative control and when the responses were dose-related and/or reproducible. The other cases were judged to be negative. No statistical methods were used.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mutagenicity of the test substance was judged to be negative because the number of the revertant colonies in the test substance treatment groups in all tester strains was less than twice that in the negative control without and with S9 mix.
The numbers of the revertant colonies in the positive controls were above twice that in the negative controls, The test results showed that the numbers of revertant colonies in the negative control and the positive controls were within the range of the historical data at the testing facility. It was also confirmed that no bacterial contamination was observed, which indicates the test results to be valid.

Applicant's summary and conclusion

Conclusions:

It was concluded that MACROLEX Rot B did not induce mutations under the present test conditions.

Executive summary:

The ability of MACROLEX Rot B to induce mutations was investigated using Salmonella typhimurium strains TA 100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2uvrA with a pre-incubation method in the absence and presence of a metabolic activation System (S9 mix).

As a result, the mutagenicity of the test substance was judged to be negative because the numbers of revertant colonies in the test substance treatment groups were less than two times that in each negative control in all testet strains with and without S9 mix.

Consequently, it was concluded that MACROLEX Rot B had no ability to induce mutations under the present test conditions.