1,1'-{(phenylmethanediyl)bis[(3-substituted benzene-4,1-diyl)diazene-2,1-diyl{1-[3-(dimethylamino)propyl]-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridine-5,3-diyl}]}dipyridinium chloride lactate salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 12, 2008 - January 23, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study compliant with TG guideline and conducted under GLP in recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.5100 (Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: essential amino acid requiring strains

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: essential amino acid requiring strain

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male Wistar rats treated by oral route on 3 consecutive days with Phenobarbital (80 mg/kg bw) and β-Naphtoflavone (100 mg/kg bw) for enzyme induction

Test concentrations with justification for top dose:

Pre-Experiment (Plate-incorporation Test):
3.16, 10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/plate TA 98 and TA100 without and with metabolic activation (S9).
Experiment 1 (Plate-incorporation Test, including some of the assays from the pre-experiment):
3.16, 10.0, 31.6, 100, 316, 1000, 2500 µg/plate (TA 98, TA100, TA1535, TA1537, WP2 uvrA without and with metabolic activation (S9).
5000 µg/plate (WP2 uvrA with metabolic activation (S9).
Experiment 2 (Pre-incubation Test):
3.16, 10.0, 31.6, 100, 316, 1000, 2500 µg/plate (TA 98 without and with metabolic activation (S9).
0.316, 1.00, 3.16, 10.0, 31.6, 100 µg/plate (TA100, TA1535, TA1537 without metabolic activation (S9).
3.16, 10.0, 31.6, 100, 316, 1000, 2500 µg/plate (TA100, TA1535, TA1537 with metabolic activation (S9).
10.0, 31.6, 100, 316, 1000, 2500, 5000 µg/plate (WP2 uvrA without and with metabolic activation (S9).

The factor of 1.16 was applied to all dose preparations in order to achieve dose levels equivalent to 100% active components of the test substance, i.e. all dose concentrations are expressed as amount of water- and minor impurity-free test substance per plate.

Vehicle / solvent:

Distilled water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methylmethanesulfonate; 2-aminoanthracene

Details on test system and experimental conditions:

Plate-incoroporation tests were performed in the Pre-Experiment and Experiment 1, and pre-incubation tests in Experiment 2.
No precipitation of the test substance was observed in any tester strain used in Experiments 1 and 2 (without and with metabolic activation).
Positive Control Substances:
sodium azide: positive control for TA 100 and TA 1535 without metabolic activation; vehicle = distilled water.
4-nitro-o-phenylene-diamine: positive control for TA 98 and TA 1537 without metabolic activation; vehicle = dimethylsulfoxide.
methylmethanesulfonate: positive control for WP2 uvrA without metabolic activation; vehicle = distilled water.
2-aminoanthracene: positive control for TA 98, TA 100, TA 1535, TA 1537 and WP2 uvrA with metabolic activation;vehicle = dimethylsulfoxide.

Evaluation criteria:

Degree of increase in the number of revertant colonies in any tester strain. Dose dependency of any effects. Confirmation in independent repeat experiment.

An increase in number of revertant colonies is considered to be biologically relevant:
- if in tester strains TA 100 and/or WP2 uvrA the number of revertant colonies is at least twice as high,
- if in tester strains TA 98, TA 1535 and/or TA 1537 the number of revertant colonies is at least three times higher,
than the number of revertant colonies in the solvent control.

A test substance producing neither a dose related increase in the number of revertant colonies nor a reproducible biologically relevant positive response in any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

Statistical analysis of the results attained was not considered to be necessary.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 98 and TA 100 bacteria

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in all strains and at all concentrations tested

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In TA 98 at 1000 µg/plate and higher without S9 and at 2500 µg/plate and higher with S9; in TA 100 at 100 µg/plate and higher without S9 and at 316 µg/plate and higher with S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in all strains and at all concentrations tested

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 100 µg/plate and higher without S9 and at 316 µg/plate and higher with S9; For detailed results, see Table 1 in Remarks section

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in all strains and at all concentrations tested

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 10 µg/plate and higher without S9 and at 316 µg/plate and higher with S9; For detailed results, see Table 2 in Remarks section

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Relevant data from the pre-experiment have been included in Table 1, the result table of Experiment I (Plate-incorporation Test).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: in Pre-Experiment (Plate-incorporation Test)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without and with metabolic activation (S9)

In the present study, the test substance, the test substance, was tested for mutagenicity, both at non-toxic and cytotoxic test substance concentrations, in a reverse mutation assay using four different strains of S. typhimurium and one strain of E. coli in the absence and presence of metabolic activation with S9. Mutagenicity of the test substance was not evident, as it did not induce any relevant increase in the number of revertant colonies in the five bacteria strains tested. Hence, there was no evidence of point mutations by base pair changes or frameshifts attributable to treatment with the test substance.

Executive summary:

the test substance was tested for mutagenicity, both at non-toxic and cytotoxic test substance concentrations (up to 5000 μg/plate) in an Ames test according to OECD Guideline 471 (Bacterial Reverse Mutation Assay) utilising S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and E. coli WP2 uvrA with and without metabolic activation with S9. Following a pre-experiment, two independent main experiments, the plate incorporation test (Experiment 1) and the pre-incubation test (Experiment 2), were performed. Reliability grade 1 was assigned to the study.

Precipitation of the test substance was not evident in the two main experiments in this study. Mutagenicity of the test substance was not evident in this study, as it did not induce any relevant increase in the number of revertant colonies in the five bacteria strains tested. Hence, there was no evidence of point mutations by base pair changes or frameshifts attributable to treatment with the test substance.

In the vehicle control plates receiving distilled water without the test substance, the growth rates and numbers of revertant colonies were within expected ranges. In addition, the positive control mutagens induced distinctly higher numbers of revertant colonies than the vehicle controls. Therefore, validity of the conditions and procedures adopted in the present study and of the bacterial cultures used was confirmed.