reaction mass of: pentasodium 4-amino-5-hydroxy-3-{(E)-4-[2-(sulfonatooxy)ethylsulfonyl]phenylazo}-6-{(E)-2-sulfonato-4-[2-(sulfonatooxy)ethylsulfonyl]phenylazo}naphthalene-2,7-disulfonate;tetrasodium 4-amino-5-hydroxy-3-{(E)-4-[2-(sulfonatooxy)ethylsulfonyl]phenylazo}-6-[(E)-2-sulfonato-4-(vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate;tetrasodium 4-amino-5-hydroxy-6-[(E)-2-sulfonato-4-[2-(sulfonatooxy)ethylsulfonyl]phenylazo}-3-[(E)-4-(vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate;trisodium 4-amino-5-hydroxy-3-[(2-hydroxyethylsulfonyl)-phenylazo]-6-[(E)-2-sulfonato-4-(vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate;trisodium 4-amino-5-hydroxy-3-[(E)-4-(vinylsulfonyl)phenylazo]-6-[(E)-2-sulfonato-4-(vinylsulfonyl)phenylazo]naphthalene-2,7-disulfonate;trisodium 4-amino-5-hydroxy-3-[(E)-4-(vinylsulfonyl)phenylazo]-6-[-2-sulfonato-4-(2-hydroxyethylsulfonyl)phenylazo]naphthalene-2,7-disulfonate

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:

From February 05 to March 14, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

445-280-9

EC Name:

-

Cas Number:

371921-40-3

Molecular formula:

No molecular formula possible, it is a reaction mass.

IUPAC Name:

docosasodium 4-amino-3-[(1E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-5-hydroxy-6-[(1E)-2-[4-(2-hydroxyethanesulfonyl)-2-sulfonatophenyl]diazen-1-yl]naphthalene-2,7-disulfonate 4-amino-3-[(1E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-5-hydroxy-6-[(1E)-2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate 4-amino-5-hydroxy-6-[(1E)-2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl]-3-[(1E)-2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate 4-amino-6-[(1E)-2-[4-(ethenesulfonyl)-2-sulfonatophenyl]diazen-1-yl]-3-[(1E)-2-[4-(ethenesulfonyl)phenyl]diazen-1-yl]-5-hydroxynaphthalene-2,7-disulfonate 4-amino-6-[(1E)-2-[4-(ethenesulfonyl)-2-sulfonatophenyl]diazen-1-yl]-5-hydroxy-3-[(1E)-2-[2-(2-hydroxyethanesulfonyl)phenyl]diazen-1-yl]naphthalene-2,7-disulfonate 4-amino-6-[(1E)-2-[4-(ethenesulfonyl)-2-sulfonatophenyl]diazen-1-yl]-5-hydroxy-3-[(1E)-2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl]naphthalene-2,7-disulfonate

Method

Target gene:

Histidine locus for Salmonella and tryprophan for E. Coli

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: from male rats (HanBrl:WIST SPF), delivered by RCC Ltd, Animal Breeding and Biotechnology, Füllinsdorf, Switzerland.
- method of preparation of S9 mix: the animals were treated with Aroclor 1254 (Analabs lnc., delivered from Antechnika, Karslruhe, Germany), 500 mg/kg, i.p. 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl. The homogenate was centrifuged for 15 minutes at 9000x g and the resulting supernatant (S9 fraction) was stored at approximately -80°C for no longer than one year. The protein content of the 59 fraction was 31.31 mg/ml.
- concentration or volume of S9 mix and S9 in the final culture medium: on day of experiment an appropr¡ate quantity of 59 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10 % v/v in the mixture

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

bidistilled water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; plate incorporation test (first experiment with and without metabolic activation, second experiment without activation) and the pre-incubation test (second experiment with
metabolic activation). For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the minimal agar plates:
- 100 µl Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control):
- 500 µl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 µl Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µl Overlay agar
ln the pre-incubation assay 100 µl test solution, 500 µl S9 mix and 100 µl bacterial suspension were mixed in a test tube and shaken at about 37°C for 30 minutes. After pre-incubation 2.0 ml overlay agar (about 45 °C) was added to each tube and well mixed. The mixture was poured on minimal agar plates. ln addition, the respective controls (solvent) and positive controls were run together with each strain.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C ± 2 °C in the dark.

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 controls such an increase is not considered biologically relevant.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 4

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 and 5000 µg/plate

Test results 6

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 µg/plate

Test results 7

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 8

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 ans 5000 µg/plate

Applicant's summary and conclusion

Conclusions:

The substance induces increased number of revertant colonies in E. Coli WP2 uvrA, with metabolic activation

Executive summary:

The test item was tested for mutagenic effects in vitro in histidine-requir¡ng strains of Salmonella typhimurium
and in a tryptophan-requiring strain of Escherichia coli, according to the OECD guideline 471. The following strains were used: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA.
The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. Each test item concentration, the negative and positive and controls were tested in triplicates. The test item was dissolved in bidistilled water and tested at five concentrations: 312.5, 625, 1250, 2500 and 5000 µg/plate.
ln order to confirm the results, the experiment was repeated with and without metabolic activation at the same concentrations used in the first experiment. The test with metabolic activation was carried out as pre-incubation assay.
Since a weak increase in the number of revertant colonies was observed in strain WP2 uvrA in the experiment with metabolic activation (pre-incubation assay), the experiment with this strain was performed in addition with the same concentrations tested before.
Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.
Previously, a pre-experiment for toxicity (range finding test) was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiment was performed with and without metabolic activation with the concentrations of 20.6, 61 .7, 185.2, 555.6, 1666.7 and 5000 µg/plate.
Normal background growth was observed with both strains. The number of revertant colonies was not reduced at any concentrations tested. The test item did not precipitate on the surface of the agar plates.
From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 5000.0 µg/plate with and without metabolic activation.
ln the first mutagenicity test performed with and without metabolic activation, no substantial increase in the number of revertant colonies was observed after treatment with Navy MGi 1571 at any concentrations.
ln the second mutagenicity test carried out with metabolic activation, treatment of strain E. coli WP2 uvrA with the test item led to an increase in the number of revertant colonies at the concentrations of 1250 and 5000 µg/plate. No similar effects were observed with strains of S. typhimurium.
ln the supplement experiment carried out as pre-incubation assay with metabolic activation with E. coli WP2 uvrA again an increase in the number of revertant colonies occurred at the concentrations of 625 to 5000 µg/plate.
ln case of the increased number of revertant colonies reached the threshold for a positive response colonies from one plate of each respective concentration group were tested on selective agar plates for their mutant properties, in order to avoid false positive or false negative results.

Toxic effects, evident as a reduction in the number of revertants, were visible in the first experiment with activation in strain TA 1535 at the concentrations of 2500 and 5000 µg/plate and in strain TA 1537 at the concentration of 5000 µg/plate. ln the experiment without activation in strain TA 1537 a reduction in the number of reveftants occurred at the concentrations of 2500 and 5000 µg/plate.
ln both mutagenicity tests normal background growth was observed with all strains at all concentrations. The test item did not precipitate on the surface of the agar plates.
ln the experiments negative (solvent) and positive control treatments were included for all strains. The mean numbers of revertant colonies on negative control plates were found to be within acceptable ranges. The positive controls induced appropriate increases in the number of revertant colonies in all experiments, thus demonstrating the correct strain functioning and the activity of the S9-mix.

ln conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item induced gene mutations by base-pair changes and others in the genome of strain E. coliWP2 uvrA.
Therefore, Navy MGi 1571 is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.