5,5'-{ethane-1,2-diylbis[thio-1,3,4-thiadiazole-5,2-diyldiazene-2,1-diyl(5-amino-3-tert-butyl-1H-pyrazole-4,1-diyl)]}diisophthalic acid

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:

The experimental phase of this study was performed between 08 June 2011 and 14 July 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

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:

Tetra potassium 5,5'-[ethane-1,2-diylbis[thio-1,3,4-thiadiazole-5,2-diyldiazene-2,1-diyl(5-amino-3-tert-buyl-1H-pyrazole-4,1-diyl)]}diisophthalate

EC Number:

700-616-6

Cas Number:

849608-59-9

Molecular formula:

C36H32K4N14O8S4

IUPAC Name:

Tetra potassium 5,5'-[ethane-1,2-diylbis[thio-1,3,4-thiadiazole-5,2-diyldiazene-2,1-diyl(5-amino-3-tert-buyl-1H-pyrazole-4,1-diyl)]}diisophthalate

Test material form:

solid

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

Vehicle: sterile distilled water

Justification for choice of solvent/vehicle:
The test item was found to be soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house. Therefore, this solvent was selected as the vehicle of choice.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA102

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates 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 mix

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates 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 mix

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates 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 mix

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates 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 mix

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA102

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Details on test system and experimental conditions:

METHODS OF APPLICATION: in agar (pre-incubation) – Experiment 1 and 2
- Pre-incubation period for bacterial strains: 10hrs
- Exposure duration: 48-72hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (in incubation with a selective agent): 25 minutes at 37 degrees C


NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
-Method: the test item must have caused a reduction in the number of spontaneous revertants (below a factor of 0.5 fold under the concurrent solvent control value) and/or the bacterial lawn should have exhibited evidence of thinning when viewed microscopically.

Evaluation criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per ml.
All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination

Evaluation Criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation
Dunnetts Regression Analysis

Results and discussion

Test results

Test results 1

Key result

Species / strain:

bacteria, other: TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

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

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECFIC CONFOUNDING FACTORS:
- Colouration: A blue/black test item colouration was observed at and above 150 µg/plate. This observation did not prevent the scoring of revertant colonies.

STERILITY, VEHICLE AND POSITIVE CONTROL DATA:
- The test material formulation and S9-mix used in this experiment were both shown to be effectively sterile. Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The S9 mix and top agar used in both experiments was shown to be sterile.
Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test

Any other information on results incl. tables

Table1          Spontaneous Mutation Rates (Concurrent Negative Controls)

 

EXPERIMENT1

 

Number of revertants(mean number of colonies per plate)
Base-pairsubstitution type					Frameshifttype
TA100		TA1535	TA102		TA98			TA1537
90		15	218		20			7
132	(112)	27        (21)	198	(202)	13		(14)	14         (12)
115		20	190		10			14

 

EXPERIMENT 2

 

 

 

Numberofrevertants(meannumberofcoloniesperplate)
Base-pairsubstitution type					Frameshifttype
TA100		TA1535	TA102		TA98			TA1537
97		25	207		8			11
98	(98)	22           (5)	191	(229)	15		(15)	4	(7)
98		27	289		21			5
					19
					14        (16)t
					16

 

Table1          SpontaneousMutationRates(ConcurrentNegativeControls)

 

EXPERIMENT1

 

Numberofrevertants(mean number of colonies per plate)
Base-pair substitution type					Frame shift type
TA100		TA1535	TA102		TA98			TA1537
90		15	218		20			7
132	(112)	27          (21)	198	(202)	13		(14)	14         (12)
115		20	190		10			14

 

EXPERIMENT 2

 

 

 

Numberofrevertants(mean number of colonies per plate)
Base-pair substitution type					Frameshift type
TA100		TA1535	TA102		TA98			TA1537
97		25	207		8			11
98	(98)	22          (25)	191	(229)	15		(15)	4	(7)
98		27	289		21			5
					19
					14        (16)t
					16

 

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

Introduction.  

The method incorporated the Prival and Mitchell modification for azo dyes. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines. The method incorporated the Prival and Mitchell modification for azo dyes.

Methods.

Salmonella Typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test item using the Ames 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). The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations.oo The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the induced rat liver S9-mix and the uninduced hamster liver S9-mix were validated.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test item was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate.  A yellow item colouration was observed at and above 150 µg/plate. This observation did not prevent the scoring of revertant colonies.

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

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