main component 1 (isomer 1): 2-{6-fluoro-4-[3-(2,5-disulfo-phenylazo)-4-hydroxy-2-sulfonapht-7-ylamino]-1,3,5-triazin-2-ylamino}-3-{6-fluoro-4-[3-(1,5-disulfonaphth-2-ylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-propane sodium salt;main component 1 (isomer 2): 2-{6-fluoro-4-[3-(2,5-disulfo-phenylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-3-{6-fluoro-4-[3-(2,5-disulfo-phenylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-propane sodium salt;main component 2: 2,3-bis-{6-fluoro-4-[3-(2,5-disulfo-phenylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-propane sodium salt;main component 3: 2,3-bis-{6-fluoro-4-[3-(1,5-disulfonaphth-2-ylazo)-4-hydroxy-2-sulfonaphth-7-ylamino]-1,3,5-triazin-2-ylamino}-propane sodium salt

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:

23 October 1996 to 03 December 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:

yes

Remarks:

See overall remarks attachment section

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Revised Chemical Substance Law (1987) according to the 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.

GLP compliance:

yes (incl. QA statement)

Remarks:

CCR, Cytotest Cell Research GmbH & Co. KG, In den Leppsteinswiesen 19, D-64380 Roßdorf

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

422-610-1

EC Name:

-

Molecular formula:

multi constituent

IUPAC Name:

tetracosasodium 2-[(1E)-2-[6-({4-[(1-{[4-({6-[(1E)-2-(2,5-disulfonatophenyl)diazen-1-yl]-5-hydroxy-7-sulfonatonaphthalen-2-yl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}propan-2-yl)amino]-6-fluoro-1,3,5-triazin-2-yl}amino)-1-hydroxy-3-sulfonatonaphthalen-2-yl]diazen-1-yl]benzene-1,4-disulfonate 2-[(1E)-2-[6-({4-[(2-{[4-({6-[(1E)-2-(2,5-disulfonatophenyl)diazen-1-yl]-5-hydroxy-7-sulfonatonaphthalen-2-yl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}propyl)amino]-6-fluoro-1,3,5-triazin-2-yl}amino)-1-hydroxy-3-sulfonatonaphthalen-2-yl]diazen-1-yl]naphthalene-1,5-disulfonate 3-[(1E)-2-[6-({4-[(1-{[4-({6-[(1E)-2-(4,8-disulfonatonaphthalen-2-yl)diazen-1-yl]-5-hydroxy-7-sulfonatonaphthalen-2-yl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}propan-2-yl)amino]-6-fluoro-1,3,5-triazin-2-yl}amino)-1-hydroxy-3-sulfonatonaphthalen-2-yl]diazen-1-yl]naphthalene-1,5-disulfonate 5-[(1E)-2-[6-({4-[(2-{[4-({6-[(1E)-2-(2,5-disulfonatophenyl)diazen-1-yl]-5-hydroxy-7-sulfonatonaphthalen-2-yl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}propyl)amino]-6-fluoro-1,3,5-triazin-2-yl}amino)-1-hydroxy-3-sulfonatonaphthalen-2-yl]diazen-1-yl]naphthalene-1,6-disulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

Identity: FAT 40557/A
Batch-no.: PV1
Purity: 65 %
Appearance: Solid powder, red-brown
Solubility (g/l): > 100
Stability: Pure: stable under storage conditions; In solvent: 48 hours in H2O, saline, polyethylene glycol, and CMC
Expiration date: 30 September, 2002
Storage conditions: At room temperature

Method

Target gene:

Histidine and tryptophan.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

33.3, 100, 333.3, 1000, 2500 and 5000 µg/plate.

Vehicle / solvent:

Water. The solvent was chosen because of its solubility properties.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA, with metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Strains: TA 1535, TA 100, without metabolic activation

Controls 3

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

Remarks:

Strains: TA 1537, TA 98, without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Strain: WP2 uvrA, without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

DURATION: After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.

DETERMINATION OF CYTOTOXICITY: Toxicity of the test article results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

NUMBER OF REPLICATIONS: Two independent experiments performed in triplicate.

Evaluation criteria:

- A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a 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 reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
- A mutagenic response is described as follows: A test article is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA 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.

Statistics:

No appropriate statistical method is available.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
- To evaluate the toxicity of the test article a pre-experiment was performed with strains TA 98 and TA 100 exposed to 3.3, 10, 33.3, 100, 333.3, 1000, 2500, or 5000 µg/plate. The plates with the test article showed normal background growth up to 5000 µg/plate in strain TA 98 and TA 100.

ADDITIONAL INFORMATION ON GENOTOXICITY:
- No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40557/A at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects evident as a reduction in the number of revertants occurred up to the highest investigated dose in both experiments.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Pre-Experiment for Toxicity:

8 concentrations were tested for toxicity and mutation induction with each 3 plates.

The plates with the test article showed normal background growth up to 5000.0 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; 100.0; 333.3; 1000.0, 2500.0; and 5000.0 µg/plate.

Applicant's summary and conclusion

Conclusions:

FAT 40557/A is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

In a GLP-compliant Ames test, performed according to OECD guideline 471 and 472, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and the Escherichia coli strain WP2 uvrA were used to the test the mutagenic potential of the test substance (33.3, 100, 333.3, 1000, 2500, 5000 µg per plate), both with and without metabolic activation.

No toxic effects evident as a reduction in the number of revertants occurred up to the highest investigated dose in both experiments. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40557/A at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used and therefore, FAT 40557/A is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.