Disodium 2,4-diamino-3-{[4-(ethenylsulfonyl)phenyl]diazenyl}-5-{[4-(ethenylsulfonyl)-2-sulfonatophenyl]diazenyl}benzenesulfonate

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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD guideline 471 and in accordance with GLP

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Guidelines followed

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Method

Target gene:

Histidine auxotrophic strains of Salmonella typhimurium: TA98, TA100, TA1535 and TA1537.

Tryptophan auxotrophic strain of Escherichia coli: WP2uvrA (pKM101).

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium: TA98, TA100, TA1535 and TA1537; Escherichia coli: WP2uvrA (pKM101).

Details on mammalian cell type (if applicable):

None

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

-Initial Mutation Assay
(a) 50, (b) 158, (c) 500, (d) 1581 and (e) 5000 µg/plate

-CONFIRMATORY MUTATION ASSAY
(a) 100, (b) 266, (c) 707, (d) 1880 and (e) 5000 µg/plate.

Vehicle / solvent:

Sterile Water

Controls

Untreated negative controls:

yes

Remarks:

Sterile water

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Remarks:

2-Aminoanthracene, 2-Nitrofluorene, Sodium azide, 9-aminoacridine, 4-Nitroquinoline-1-oxide

Positive control substance:

not specified

Remarks:

None

Details on test system and experimental conditions:

Following strains of bacteria accepted under OECD for the assessment of point gene mutation were used:

Histidine auxotrophic strains of Salmonella typhimurium: TA98, TA100, TA1535 and TA1537.

Tryptophan auxotrophic strain of Escherichia coli: WP2uvrA (pKM101).

Evaluation criteria:

To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system.

The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and TA1537.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Statistics:

None

Results and discussion

Test results

Species / strain:

other: Salmonella typhimurium: TA98, TA100, TA1535 and TA1537; Escherichia coli: WP2uvrA (pKM101).

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

None

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

All criteria for a valid study were met as described in the study plan. It is concluded that the test item, FAT 40866/A TE, was not mutagenic in this bacterial reverse mutation test up to the regulatory-required top dose of 5000 µg/plate under the conditions of testing employed.

Executive summary:

The test item, FAT 40866/A TE was tested for its mutagenic potential in the bacterial reverse mutation assay. The study was conducted using TA98, TA100, TA1535 and TA1537 strains of Salmonella typhimurium and WP2uvrA (pKM101) strain of Escherichia coli in three phases, a preliminary toxicity test, an initial mutation assay and a confirmatory mutation assay. The bacterial tester strains were exposed to the test item in the presence and absence of metabolic activation system (S-9 fraction prepared from Aroclor 1254 induced rat liver).

FAT 40866/A TE is soluble in sterile water (SW) at the required concentration of 50 mg/mL and was found to be stable in SW for 24 hours at room temperature at the concentration levels of 500 µg/mL and 50000 µg/mL.

In a preliminary toxicity test for the selection of test doses for the mutation assay, FAT 40866/A TE did not precipitate the basal agar plates at any of the tested doses.FAT 40866/A TE did not cause toxicity to the tester strain up to the top dose of 5000 µg/plate either in the presence or absence of metabolic activation, as the intensity of the bacterial background lawn as well as the number of revertant colonies were comparable to the SW control plates. Based on these observations, it was decided to test up to 5000mg/plate in the initial as well as the confirmatory mutation assay both in the presence and absence of metabolic activation.

In the initial mutation assay,FAT 40866/A TE was exposed in triplicate to 50, 158, 500, 1581 and 5000mg/plate test doses in the presence and absence of metabolic activation using direct plate incorporation procedure. In the confirmatory mutation assay,FAT 40866/A TE was exposed in triplicate to 100, 266, 707, 1880 and 5000mg/plate test doses in the presence and absence of metabolic activation using pre-incubation procedure. The vehicle control (SW) and the appropriate positive controls were tested simultaneously. The mean and standard deviation of numbers of revertant colonies were calculated for each test dose and the controls for all the tester strains.

The results of the study from both the initial and confirmatory mutation assay showed that,FAT 40866/A TE did not show any positive mutagenic response at any of the tested doses either in the presence or in the absence of metabolic activation. Under identical test conditions, there was a more than 3-fold increase in the mean numbers of revertant colonies in the positive controls, demonstrating the sensitivity of the assay procedure used.

The results of the concentration analysis of the dose formulation samples of both the initial and confirmatory mutation assays confirmed that the top dose level of 5000µg/plate was achieved in both assays and the results support the validity of the study conclusion.

The study indicated that FAT 40866/A TE was not mutagenic in this Bacterial Reverse Mutation Assay up to the regulatory-required top dose of 5000mg/plate, under the conditions of testing employed.