2-{4-[4-[4-fluoro-6-(2-(2-vinylsulfonylethoxy)ethylamino)-1,3,5-triazin-2-ylamino]phenylazo]phenylazo}naphthalene-4,6,8-trisulfonate, trisodium salt

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:

09 August 2001 to 25 October 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Kanpoan No. 287 - Environment Protection Agency

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: Japanese Guideline: Eisei No. 127 - Ministry of Health & Welfare

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Japanese Guideline: Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade & Industry

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

442-230-8

EC Name:

-

Cas Number:

321679-52-1

Molecular formula:

Hill formula: C31 H25 F N9 Na3 O12 S4 CAS formula: C31 H28 F N9 O12 S4 · 3 Na

IUPAC Name:

trisodium 7-(2-{4-[2-(4-{[4-({2-[2-(ethenesulfonyl)ethoxy]ethyl}amino)-6-fluoro-1,3,5-triazin-2-yl]amino}phenyl)diazen-1-yl]phenyl}diazen-1-yl)naphthalene-1,3,5-trisulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 40'800/A
- Lot/batch No.: WP 2/01
- Purity: approx. 70%
- Stability in solvent: 24 hours in water, saline, polyethylene glycol, CMC, vaseline, and FCA
- Expiry date: 14 February 2007
- Storage: room temperature

Specific details on test material used for the study:

Identification: FAT 40800/B
Description: dark orange powder
Batch number: REM1
Purity: approx. 70 %
Stability of test item: Stable under storage conditions;
Expiration date: February 14, 2007
Stability in solvent: 24 hours in water, saline, polyethylene glycol, CMC, Vaseline, and FCA
Storage conditions: at room temperature

Method

Target gene:

Histidine for the S. typhimurium strains and Tryptophan for the E.coli strain

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/beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

water

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:

Strain TA 1535, TA 100, without metabolic activation

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

Remarks:

Strain TA 1537, TA 98, without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Strain WP2, without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains, with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

DURATION
- Preincubation period: 60 minutes at 37 °C
- After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

DETERMINATION OF CYTOTOXICITY: Toxic effects were evidenced by 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 item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required.

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:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No relevant toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
No substantial and reproducible increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 40800/A at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). A moderate but not strictly dose dependent increase of the number of revertant colonies was observed in strain TA 98 in the first experiment with metabolic activation. The colony count exceeded the historical range of negative and solvent controls somewhat at 33 - 333 µg/plate. At 333 µg/plate the threshold of twice the colony count of the corresponding solvent control was just exceeded (factor of 2.1 versus a threshold of 2.0) However, compared to the corresponding negative control a factor of only 1.5 was calculated. Furthermore this effect was not reproduced in the second assay. Therefore, this irreproducible increase was judged to be based upon fluctuations rather than indicating a possible mutagenic potential. There was also no reproducible 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. They showed a distinct increase of induced revertant colonies. The positive control somewhat exceeded the historical range of positive controls in strains TA 1535 and TA 100 of the first experiment without metabolic activation. However, this effect indicates the sensitivity of the strains rather than compromising the validity of the assay. The positive control of strain TA 1535 in the second experiment with metabolic activation fell just short of the lower border of the historical range (61 colonies versus 68-511). Since the threshold of thrice the corresponding solvent control was clearly exceeded this minor deviation was also judged as biologically irrelevant.

Applicant's summary and conclusion

Conclusions:

FAT 40800/A did not show any mutagenic activity in any strain.

Executive summary:

In a GLP-compliant Ames test, performed according to OECD guideline 471, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and one E. Coli strain (WP2 uvrA) were used to test the mutagenic potential of the test substance (33, 100, 333, 1000, 2500, and 5000 µg per plate), in two independent experiments (each in triplicate), both with and without metabolic activation. The test substance did not show any mutagenic activity in any strain and no toxic effects were observed. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.