hexasodium 5,5'-{[2,7-disulfonato-9-(2-sulfonatophenyl)-9H-xanthene-9-ylium-3,6-diyl]bis(azanediyl)bis[(2,4,6-trimethyl-3,1-phenylene)aminosulfonyl]}diisophthalate

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:

Date of experimental starting : May 26, 2016 experimental completed June 3, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

hexasodium 5,5'-{[2,7-disulfonato-9-(2-sulfonatophenyl)-9H-xanthene-9-ylium-3,6-diyl]bis(azanediyl)bis[(2,4,6-trimethyl-3,1-phenylene)aminosulfonyl]}diisophthalate

EC Number:

854-560-3

Cas Number:

1891094-25-9

Molecular formula:

C53H38N4Na6O22S5

IUPAC Name:

hexasodium 5,5'-{[2,7-disulfonato-9-(2-sulfonatophenyl)-9H-xanthene-9-ylium-3,6-diyl]bis(azanediyl)bis[(2,4,6-trimethyl-3,1-phenylene)aminosulfonyl]}diisophthalate

Test material form:

solid: particulate/powder

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver,phenobarbital (PB) injection of 5,6-benzoflavone, S9 mix

Test concentrations with justification for top dose:

The tests were conducted in the presence (+S9mix) and absence (−S9mix) of metabolic
activation. Prior to the main study, a dose-finding test for each strain using the test
item at concentrations of 62, 185, 556, 1667, and 5000 µg/plate was conducted. The results showed that the test
item did not increase the number of revertant colonies more than 2-fold compared to the negative
control, in the presence or absence of metabolic activation, at all concentrations tested and for all
the strains used.
In addition, water was used , as a negative control . 5 doses of 5,000 µg/plate (highest dose) with a common ratio of 2.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100 ,0.01 µg/plate

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

without S9mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA 1535 ,0.5 µg/plate

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9mix

Controls 3

Untreated negative controls:

yes

Remarks:

spontaneous mutation WPuvRA ,0.01µg/plate

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

without S9

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98, 0.1 µg/plate

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

without S9

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537, 80 µg/plate

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100, 5µg/plate

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535 2µg/plate

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

with S9

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WPuvRA, 10µg/plate

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

with S9

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98, 5µg/plate

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537,2µg/plate

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

with S9

Details on test system and experimental conditions:

Concentration of test substances and number of tests:
・Dose-finding test : 5 doses of 5,000 µg/plate (highest dose) with a common ratio of 3.
・Main study : 5 doses of 5,000 µg/plate (highest dose) with a common ratio of 2. Bacterial strains tested TA100, TA1535, WP2uvrA, TA98, and TA1537 (5 strains). Metabolic system: Induced rat S9 (concentration of S9 in S9mix: 10% v/v)
Test method: Pre-incubation method.
Pre-incubation : 37°C for 20 min.
Incubation : 37°C for 48 h.

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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

upto 5000 μg/plate (highest dose)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

upto 5000 μg/plate (highest dose)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

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

Remarks:

upto 5000 μg/plate (highest dose)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

upto 5000 μg/plate (highest dose)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

upto 5000 μg/plate (highest dose)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test item was deemed non-mutagenic for the five bacterial strains listed above.

Executive summary:

Introduction

The study, a reverse mutation test of the test item in bacteria (mutagenicity test in microorganisms),

was carried out to determine whether the test item has the mutagenicity, and if it does, how potent the mutagenicity is, following the guideline of “OECD GUIDELINE FOR TESTING CHEMICALS 471” (July 21, 1997)

Results and conclusion

The mutagenicity of the test item was tested in four strains of Salmonella typhimurium (TA100, TA1535, TA98, and TA1537) and Escherichia coli (WP2uvrA). The results showed that the test item did not increase the number of revertant colonies more than 2-fold compared to the negative control, in the presence or absence of metabolic activation, at all concentrations tested and for all strains used.

The test item was deemed non-mutagenic for the five bacterial strains listed above.