Disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-amino-4-hydroxynaphthalene-2-sulphonate]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 06 February 1995; Experiment start date - 08 March 1995; Experiment end date - 03 April 1995; Study completion date - 26 April 1995.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-amino-4-hydroxynaphthalene-2-sulphonate]

EC Number:

257-854-4

EC Name:

Disodium 5,5'-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-amino-4-hydroxynaphthalene-2-sulphonate]

Cas Number:

52333-30-9

Molecular formula:

C47H38N6O14S4.2Na

IUPAC Name:

disodium 5,5'-[propane-2,2-diylbis(4,1-phenyleneoxysulfonyl-2,1-phenylenediazene-2,1-diyl)]bis(6-amino-4-hydroxynaphthalene-2-sulfonate)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Specific details on test material used for the study:

Code number: FAT 21021/D
Batch-Nr.: 9
Purity: ca. 70 %
Appearance: solid
Solubility: 30 g/l
Storage: room temperature
Expiration: 12/99

Method

Target gene:

histidine auxotrophy for S. typhimurium.

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The bacterial strains TA 1535, TA 98 and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.).The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen).
Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in CCR according to Ames et al. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
Storage: The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

S9 (Preparation by C C R)
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats, strain Hanlbm (BRL, CH-4414 Füllinsdorf weight approx. 220 - 320 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Ettlingen, F.R.G.) in olive oil 5 days previously. After cervical dislocation the livers of the animals were re-moved, washed in 150 mM KCI and homogenised. The homogenate was diluted 1 + 3 in KCI and centrifuged at 9,000 g for 10 minutes at 4° C. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80° C. Small numbers of the ampoules are kept at -20° C for up to several weeks before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 München: Bio-Rad protein assay, Catalogue 500 000 6 (6). The protein concentration in the S9 preparation was 33.0 mg/ml (lot 310894) in the pre-experiment and in experiment I, and 37.2 mg/ml (lot 230195) in experiment I and II.

S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v. The composition of the co-factor solution was concentrated to yield the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix will be stored in an ice bath. The S9 mix preparation will be performed according to Ames et al.

Test concentrations with justification for top dose:

With and without microsomal activation: 33.3; 100; 333; 1000; 2500; and 5000 µg/plate

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation - TA 1535 and TA 100

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Without metabolic activation - TA 1537, TA 98

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

With metabolic activation - TA 1535, TA 1537, TA98, TA 100

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition.

METHODS FOR MEASUREMENTS OF GENOTOXICIY : Concentration dependent increases in revertant colony numbers.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the second experiment without metabolic activation at a concentration of 2500.0 ug/plate and higher.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium 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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the first experiment without metabolic activation at a concentration of 1000.0 ug/plate and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Toxic effects, evidenced by a reduction in the number of revertants, occurred in the strain TA 98 in the first experiment without metabolic activation at a concentration of 1000.0 µg/plate and higher and in strain TA 1537 in the second experiment without metabolic activation at a concentration of 2500.0 µg /plate and higher. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used. Substantial and concentration dependent increases in revertant colony numbers were observed in strain TA 98 with metabolic activation following treatment with FAT 21021/D in both experiments. This increase started at 1000.0 µg/plate in both experiments. The factor of 3 required as described under "evaluation of results" was exceeded at 5000 µg/plate in experiment I and 2500 µg/plate in experiment 2. The other strains showed no relevant increases either with or without metabolic activation in both experiments. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Applicant's summary and conclusion

Conclusions:

FAT 21021/D induced gene mutations by frameshifts in the genome of the strain TA 98. Therefore, is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

A key study was performed to investigate the potential of FAT 21021/D according to the plate incorporation test (experiment I and II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in two independent experiments, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test substance was tested at the following concentrations: 33.3, 100, 333.3, 1000, 2500, and 5000 µg/plate (active ingredient). Toxic effects, evidenced by a reduction in the number of revertants, occurred in the strains TA 98 and TA 1537 without metabolic activation. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used substantial and concentration dependent increases in revertant colony numbers were observed in strain TA 98 with metabolic activation following treatment with FAT 21021/D in both experiments. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Under the experimental conditions reported, the test article did induce gene mutations by frameshifts in the genome of the strain TA 98. Therefore, FAT 21021/D is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.