m,m'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[2,4,6-trimethylbenzenesulphonic] acid, compound with hexane-1,6-diamine (1:1)

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:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July, 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May, 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August, 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

m,m'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[2,4,6-trimethylbenzenesulphonic] acid, compound with hexane-1,6-diamine (1:1)

EC Number:

279-213-8

EC Name:

m,m'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[2,4,6-trimethylbenzenesulphonic] acid, compound with hexane-1,6-diamine (1:1)

Cas Number:

79665-26-2

Molecular formula:

C32 H30 N2 O8 S2 . C6 H16 N2

IUPAC Name:

m,m'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[2,4,6-trimethylbenzenesulphonic] acid, compound with hexane-1,6-diamine (1:1)

Test material form:

solid: particulate/powder

Method

Target gene:

- Salmonella typhirium: histidine
- Escherichia coli: tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA100, TA1537, TA98

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and ß-napthoflavone-induced rat liver S9-mix

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: due to the limited solubility of the test substance in water, DMSO will be used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2-AA), 4-nitro-o-phenylenediamine (NOPD), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- 1st Experiment (standard plate test with and without S-9 mix)
- 2nd Experiment: (preincubation test with and without S-9 mix)
The 2nd experiment was performed as no mutagenicity was observed in the standard plate test.

DURATION
1st experiment: incubation for 48-72 hours.
2nd experiment: preincubation period: 20 min. Then, incubation for 48-72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: decrease in the number of revertants (factor ≤ 0.6), clearing or diminution of the background lawn (= reduced his- or trp- background growth)

Evaluation criteria:

Generally, the experiment was considered valid if the following criteria were met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- Fresh bacterial culture containing approximately 1E9 cells per mL were used.

The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if the number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 1535, TA100, TA1537, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found with and without S9 mix.

HISTORICAL CONTROL DATA
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 2500 μg/plate onward. In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions from about 2500 μg/plate onward.

Applicant's summary and conclusion

Conclusions:

The test substance did not induce mutagenicity in this reverse mutation assay, in the absence and presence of metabolic activation.

Executive summary:

In this GLP-compliant reverse mutation assay performed according to OECD 471, EC No 440/2008; B.13/B.14 and US EPA OPPTS 870.5100, the test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

 

- STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

- DOSE RANGE: 33 μg - 5000 μg/plate (SPT)

33 μg - 5000 μg/plate (PIT)

- TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

- SOLUBILITY: No precipitation of the test substance was found.

- TOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 2500 μg/plate onward.

 

MUTAGENICITY:

A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

 

CONCLUSION:

Thus, under the experimental conditions of this study, the test substance Oracet Blue 700 FA is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.