Pentasodium 7-(4-(4-(5-amino-4-sulfonato-2-(4-((2-(sulfonato-ethoxy)sulfonyl)phenylazo)phenylamino)-6-chloro-1,3,5-triazin-2-yl)amino-2-ureidophenylazo)naphtalene-1,3,6-trisulfonate

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:

26 Febuary 1996 to 23 May 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

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:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Notification of December 9, 1986 by EA, Environmental Agency (no. 700); MHW, Ministry of Health and Welfare (No. 1039) and MITI, Ministry of International Trade and Industry (No. 1014), Japan.

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:

FAT 40549/A

IUPAC Name:

FAT 40549/A

Test material form:

other: Solid

Details on test material:

- Batch No.: TV 1
- Colour: yellow to brown
- Stability: Pure: stable under storage conditions
In solvent: 48 hours in H2O, polyethylene glycol, and CMC
- Storage: room temperature
- Expiration Date : January 01, 2001

Specific details on test material used for the study:

Identity: FAT 40'549/A
Batch No: TV1
Expiration date: 01 Jan 2001
Stability in water: for atleast 48 hours
Solubility in water: approximately 90 mg/L at 20 degree celcius.
Aggregate state under storage conditions: solid (powder)
Colour: yellow brown.
Storage conditions: at room temperature
Safety precautions: Routine hygenic procudures are sufficient to assure personnel health and safety.

Method

Target gene:

Salmonella typhimurim histidine(his) reversion system and E-coli

Species / strain

Species / strain 1

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 Dr. B.N. Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen).

Species / strain 2

Species / strain / cell type:

E. coli, other: WP2: trp:and WP2: trp; uvrA

Details on mammalian cell type (if applicable):

The bacterial strains WP2 uvrA and WP2 were obtained from Dr. Heinz Träger, Knoll AG, D-67008 Ludwigshafen, F.R.G.

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix:
S9 (Preparation by CCR): The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
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 triple i.p. injection of 80 mg/kg b.w. phénobarbital (Desitin, D-22335 Hamburg) and ß-naphtoflavone (Aldrich, D-89555 Steinheim) orally, in corn oil 1 - 3 days previously.
After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifuged cold 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 one week 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 (7).
The protein concentration in the S9 preparation was 26.8 mg/mL (lot 301195).

S9Mix
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 concentrated co-factor solution yields the following concentrations in the S9 mix:
8mM MgC12
33 mM KCl
5mM Glucose-6-phosphate
5mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Test concentrations with justification for top dose:

33.3; 100; 333.3; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

water

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 for TA 1535, TA 100

Controls 2

Untreated negative controls:

yes

Remarks:

DMSO

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 S9 TA 1537, TA 98

Controls 3

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 for WP2 uvrA, WP2

Controls 4

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with all strains

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:
- Cell density at seeding (if applicable):
- Test substance added in medium; Plate incorporation test (experiment I) and the pre-incubation test (experiment II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 60 minutes
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY : A test article is considered mutagenic if in the strains TA 98, TA 100, WP2, and its uvrA
derivative the number of reversions will be at least twice as high and in the strains TA 1535, and TA 1537 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.

Evaluation criteria:

The criteria of mutagenicity used in this test are a doubling of the spontaneous reversion rate and a dose-effect relationship.

Statistics:

No appropriate statistical method is available.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA, WP2

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

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The plates with the test article showed normal background growth up to 5000 µg/plate in strain TA 98 and TA 100.
According to the dose selection criteria, the test article was tested at the following concentrations: 33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:

The test article showed no mutagenic effect for Salmonella typhimurium and Escheria coli at all applied test concentrations.

Executive summary:

The test substance was tested for mutagenic potential according to OECD 471 and 472 in a GLP certified laboratory.

Salmonella typhimurium (TA 98,TA 100, TA 1535, TA 1537) and Escheria coli (WP2 uvrA and WP2) strains were used at six dose levels of 33.3, 100, 333.3, 1000, 2500 and 5000 μg/plate both in the presence and absence of metabolic activation.

No toxic effects, evident as a reduction in the number of revertants, occurred in any of the test groups with and without metabolic activation. No substantial increases in revertant colony numbers of any of the six tester strains were observed following treatment with test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).

There was also no reproducible tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. All positive controls showed mutagenic effects, thereby validating the study.

Based on the findings of this study, the test substance is considered to be non-mutagenic in Salmonella typhimurium and Escherichia coli reverse mutation assay.