Diammonium 5-(hexylsulfonyl)-2-{[3-methyl-2,7-dioxo-1-(3-sulfonatobenzoyl)-2,7-dihydro-3H-naphtho[1,2,3-de]heterocycle-6-yl]amino}benzenesulfonate

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:

26 November 2008 to 25 December 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Remarks:

"GLP Standards for the Test Facility on New Chemical Substances" (Notification No. 1121003 of Pharmaceutical and Food Salfety Bureau, Ministry of Health, Labour and Welfare, No.3 of Manufacturing Industries Bureau. Ministry of Economy, Trade and Industry,

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Method

Target gene:

Histidine operon was used for the S. typhimurium TA strains (Salmonella typhimurium TAl 00, Salmonella typhimurium TA 1535 (base pair substitution), Salmonella typhimurium TA98 and Salmonella typhimurium TA 1537 (frame shift)).

Tryptophan operon was used for the Escheria coli WP2 uvrA strain (base pair substitution).

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):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 2

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

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 1.2, 4.9, 20, 78, 313, 1250 and 5000 µg/plate

Main test: 5000 µg/plate

Vehicle / solvent:

- solvent used: water

- Justification for choice of solvent: Based on the information from the sponsor that the test substance was soluble in water at 20% and more.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Migrated to IUCLID6: 5 µg/plate

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of Escherichia coli.

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate.

Remarks:

With S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Migrated to IUCLID6: 5 µg/plate

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix

Migrated to IUCLID6: 5 µg/plate

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-fury)acrylamide: 0.01 µg/plate,

Remarks:

without S9 mix

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9 mix

Migrated to IUCLID6: 0.5 µg/plate

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-fury)acrylamide: 0.01 µg/plate

Remarks:

Without S9 mix

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-fury)acrylamide: 0.1 µg/plate

Remarks:

without S9 mix

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine∙2HCl: 1.0 µg/plate.

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: The culture tube was left at 4°C for 7 hours and 45 minutes.

SELECTION AGENT (mutation assays): Not applicable.

Evaluation criteria:

In the dose-finding test and the main test, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed. The test substance was to be judged positive. Statistical analysis was not done.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

In the dose-finding test and the main test, neither an increase in the number of revenant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.

The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls (mean ± 3SD) in background data, indicating that this study was performed correctly.

From these results, mutagenicity of the test substance was judged negative.

The growth inhibition of the test strains by the test substance was not observed. and the precipitate on the plates was not observed either with or without metabolic activation.

In the sterility test on the test solution and the S9 mix, no growth of bacteria was observed.

References

1) B.N. Ames, F.D. Lee, and W.E. Durston: An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens, Proc. Natl. Acad. Sci. USA Vo1.70, No.3, pp.782-786, March 1973

2) J. McCann, N. E. Spingarn, J. Kobori and B.N. Ames: Detection of Carcinogens as Mutagens: Bacterial Tester Strains with R Factor Plasmids, Proc. Natl. Acad. Sci. USA Vol.72, No.3, pp.979-983, March 1975

3) M.H.L. Green and W.J. Muriel: Mutagen Testing using Trp+ Reversion in Escherichia coli, Mutation Res. Vol.38, pp.3-32. 1976

4) T. Yahagi, M. Nagao, Y. Seino. T.Matsushima. T.Sugimura and M.Okada: Mutagenicities of N-nitrosamines on Salmonella, Mutation Res. Vol.38, pp.121-130, 1977

5) D.Maron and B.N. Ames: Revised Methods for the Salmonella Mutagenicity Test, Mutalion Res. Vol.l13, pp.173-215.1983

6) A guidebook for Mutagenicity Studies using Bacteria, New Edition, Ed. by Chemical Substances Investigation Division, Industrial Safety and Health Department, Ministry of Labor, Japan Industrial Safety and Health Association, 1986

7) Testing Methods for Environmental Mutagens: Ed. by Tajima, Kada, Kondo and Tamura, Koudan-sha Co., pp.56-68. 1980

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

From the results described above, it is concluded that H-MI is not mutagenic in the bacterial reverse mutation
assay carried out under the experimental conditions.

Executive summary:

Mutagenicity potential of H-Ml was assessed with Salmonella typhimurium TA 100, TA 1535, TA98, TA 1537 and Escherichia coli WP2 uvrA. In this study, neither an increase in the number of revertant colonies more than twice in comparison with that of the negative control nor a dose-related response was observed in any strains of base-pair substitution type or frame-shift type, with or without metabolic activalion.

From the above, it is judged H-MI has no mutagenicity to bacteria under the described study conditions.