Reaction mass of 5-[{4-[Benzyl(methyl)amino]phenyl}diazenyl]-1,4-dimethyl-4H-1,2,4-triazol-1-ium bromide and 3-[{4-[Benzyl(methyl)amino]phenyl}diazenyl]-1,4-dimethyl-4H-1,2,4-triazol-1-ium bromide

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:

Study completion date - 25 July 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3(or5)-[[4-[benzylmethylamino]phenyl]azo]-1,2(or1,4)-dimethyl-1H-1,2,4-triazolium bromide

EC Number:

289-660-0

EC Name:

3(or5)-[[4-[benzylmethylamino]phenyl]azo]-1,2(or1,4)-dimethyl-1H-1,2,4-triazolium bromide

Cas Number:

89959-98-8

Molecular formula:

C18H21N6.Br

IUPAC Name:

Reaction mass of 3-[{4-[Benzyl(methyl)amino]phenyl}diazenyl]-1,4-dimethyl-4H-1,2,4-triazol-1-ium bromide and 5-[{4-[Benzyl(methyl)amino]phenyl}diazenyl]-1,4-dimethyl-4H-1,2,4-triazol-1-ium bromide

Test material form:

solid

Specific details on test material used for the study:

Name: FAT 31016/C
Purity: 85 %

Method

Target gene:

The experiments were performed to detect any properties of the test material or its metabolites to induce gene mutations in histidine-requiring strains of Salmonella typhimurium.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 fraction

Test concentrations with justification for top dose:

25, 75, 225, 675 and 2025 µg/0.1 ml

Vehicle / solvent:

Dimethylsulfoxyde

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunorubicin-HCl for TA 98; 2-nitrofluorene for TA 1538

Remarks:

Without microsomal activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With microsomal activation

Details on test system and experimental conditions:

Bacterial cultures were prepared from frozen stocks or from colonies on plates, and on the following days the Standard Plate Test was carried out with and without the addition of activation mixture (rat liver microsomes and co-factors) .
The test was performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. The substance was dissolved in DMSO. DMSO alone was used for the negative controls.

Each Petri dish contained:
1) approx. 20 ml of minimum agar (Difco agar noble, Difco Laboratories, Detroit, Michigan, U.S.A., Art.No.0142-01, plus salts (Vogel-Bonner Medium E) and glucose),
2) 0.1 ml of the solution of the test substance or the vehicle and 0.1 ml of a bacterial culture (in nutrient broth: Bacto Nutrient Broth dehydrated, Difco Laboratories, Detroit, Michigan, U.S.A., Art.No.0003 0.8 % plus 0.5 % NaCl) in 2.0 ml of soft agar. The soft agar was composed of: 100 ml of 0.6 % agar solution (Difco agar noble) with 0.6 % NaCl and 10 ml of a solution of 1-histidine, 0.5 mM (Fluka, Buchs, Switzerland, Art.No.14400) and +biotin 0.5 mM (Fluka, Buchs, Switzerland, Art .No .53320). In the experiments in which the substance was metabolically activated, 0.5 ml of an activation mixture was added also. 1 ml activation mixture contains: 0.3 ml S9 fraction of liver from rats induced with Aroclor 125 4 (Analabs, Inc., North Haven, Connecticut, U.S.A.,, No.RCS-088), 8 ymoles MgCl , 33 µmoles KCl, 5 µmoles glucose-6-phosphate, 4 µmoles NADP and 100 µmoles phosphate buffer, pH 7.4.
Positive control experiments were carried out simultaneously with the following substances: 1) for Strain TA 98: daunorubicin-HCl (DAUNOBLASTIN , Farmitalia, Montedison Farmaceutica GmbH, Freiburgi.Br., Germany), 5 and 10 µg/0.1 ml phosphate buffer; 2) for Strain TA 1538: 2-nitrofluorene (Fluka, Buchs, Switzerland, Art. No.73330), 5 and 10 µg/0.1 ml DMSO. The activation mixture was tested with Strain TA 15 35 and cyclophosphamide (ENDOXAN-ASTA , Asta-Werke, Bielefeld, Germany), 250 µg/0.1 ml phosphate buffer.
In the experiments with and without the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). In the positive control experiments two Petri dishes were used per strain and per group.

The plates were incubated for about 48 hours at 37 °C in darkness.

Evaluation criteria:

The test substance was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled at any concentration.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

other: TA 98 and TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

In the experiments performed without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with FAT 31016/C revealed no marked differences.

In the experiments with microsomal activation, on the other hand, treatment with FAT 31 016/C led to an increase in the number of back-mutant colonies of Strains TA 98 and TA 1538. This effect was observed at the concentrations of 75 µg/0.1 ml and above. At the highest concentration there was again a reduction in the number of back-mutant colonies, due to a growth-inhibiting effect of the substance on the bacteria.

Applicant's summary and conclusion

Conclusions:

FAT 31016/C was found to exert a mutagenic effect on strains TA 98 and TA 1538 in the presence of metabolic activation.

Executive summary:

FAT 31016/C was tested for mutagenic effects on histidine auxotrophic mutants of Salmonella typhimurium. This study was conducted according to method similar to OECD test guideline 471. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 ml. In the experiments performed without microsomal activation, comparison of the numbers of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 31016/C revealed no marked deviations. In the experiments in which activation mixture was added to the cultures, the number of back-mutant colonies of Strains TA 98 and TA 1538 was distinctly greater after treatment with FAT 31016/C than in the controls. Hence, it can be concluded that FAT 31016/C was found to exert a mutagenic effect on strains TA 98 and TA 1538 in the presence of metabolic activation.