Nickel, 5-[2-(2-hydroxy-3,7-disulfo-1-naphthalenyl)diazenyl]-1H-1,2,4-triazole-3-carboxylate sodium complexes

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:

30 May 2003 (start of experimental phase) to 16 July 2003 (report issue)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Meets the criteria for classification as Reliable without restriction according to Klimisch et al (1997)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

The method incorporated the Prival and Mitchell modification for azo dyes.

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:

S190700

IUPAC Name:

S190700

Details on test material:

Sponsor's identification: S190700
Description: dark brown powder
Purity: 82.8%
Batch number: NBZ 3341/70
Date received: 22 October 2002
Storage conditions: room temperature in the dark

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA102, TA98 and TA100

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Uninduced hamster liver S9 for test substances. Phenobarbitone/B-naphthoflavone induced rat liver S9 for efficacy check and positive controls.

Test concentrations with justification for top dose:

Preliminary Toxicity Test: Not conducted. A previous standard ames test was conducted on the substance and although it is not stated in the report, it is assumed that the test laboratory used this data to set the doses in this study.

Test concentrations for experiment 1 and 2
Concentration range (with metabolic activation): 50, 150, 500, 1500 and 5000 µg/plate
Concentration range (without metabolic activation): 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

Solvent: Sterile distilled water

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA102

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA102

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1,8-Dihydroxyanthraquinone

Remarks:

With S9

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate preincubation)

DURATION
- Preincubation period for bacterial strains: 10 hours
- Exposure duration: 48-hours
- Selection time (incubation with a selective agent): 30 minutes at 37 degrees C

NUMBER OF REPLICATIONS: Triplicate plating

DETERMINATION OF CYTOTOXICITY
Not done. Although not stated in the report dose setting was assumed to have been based on data from a previous standard Ames study.

Evaluation criteria:

Acceptance Criteria

The reverse mutation assay may be considered valid if the following criteria are met:

All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls and are within historical control ranges.

The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkM101 plasmid R-factor etc.

There should be a minimum of four non-toxic test material dose levels.

All tester strain cultures should be in the approximate range of 1 to 9.9 X 10E9 bacteria per ml.

Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the Rat liver induced S9-mix.

There should be no evidence of excessive contamination.

Evaluation Criteria

The test material is considered positive in this test system if the following criteria are met:

The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression. Reference: Kirkland D J (Ed) (1989) Statistical Evaluation of Mutagenicity Test Data. UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report - Part III, Cambridge University Press.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA102, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. An orange/red colour was observed at and above 500 ug/plate which became darker with increasing concentration. This observation did no prevent the scoring of revertant colonies. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9- mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the rat liver S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: main test

Any other information on results incl. tables

For results of mutation tests, spontaneous mutation rates and historical control values please see attached documents in additional background material section.

Applicant's summary and conclusion

Conclusions:

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

The test substance was considerd to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction

The test substance was assessed by a method designed to meet the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Reverse Mutation Study", Method B14 of Commission Directive 92/69/EEC and the USA, EPA (TSCA) OPPTS harmonised guidelines. The method incorporated the Prival and Mitchell modification for azo dyes.

Method

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using the Ames plate pre-incubation method at five dose levels, in triplicate, both with and without the addition of a hamster liver homogenate metabolising system (30% liver S9 in modified co-factors).

Results

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion

The test material was considered to be non-mutagenic under the conditions of this test.