1H-Benzimidazole-5,7-disulfonic acid, 2,2'-(1,4-phenylene)bis-, sodium salt (1:2)

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:

1996-03-06 to 1996-03-29

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study reliable with restrictions, because it is performed acc. to an outdated guideline version (1983) and does not totally comply with the current guideline requirements (1997), since the use of a 5th strain TA102 or E.coli WP2 is missing which should be used for detection of cross-linking mutagens. However, it does comply with the guideline of 1983. Furthermore, data on precipitation were given.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

429-750-0

EC Name:

-

Cas Number:

180898-37-7

Molecular formula:

C20H12N4O12S4.2Na

IUPAC Name:

disodium dihydrogen 2-[4-(5,7-disulfonato-1H-1,3-benzodiazol-2-yl)phenyl]-1H-1,3-benzodiazole-5,7-disulfonate

Details on test material:

- CAS name: 1H-Benzimidazole-4,6-disulfonic acid-2,2´-(1,4-diphenylene) bis, disodium salt
- Molecular formula: C20H12N4O12S4Na2
- Molecular weight: 674g/mol
- physical state: solid

Method

Target gene:

not applicable

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):

- originally obtained from Dr. Bruce N. Arnes, University of California, Berkeley, California, U.S.A.
- Type and identity of media: For preparation of the master plates Vogel-Bonner minimal medium plates enriched with histidine (260µM) and biotin (3 µM). Ampicillin (25 µg per ml medium of the plate) is added to the plates used for strains with the R-factor. The tester strain cultures used in this study were grown in Oxoid nutrient broth No.2 (2.5%). The minimal agar plates contain 25 ml of 1.5 % agar in Vogel-Bonner medium E with 2 % glucose.
- Properly maintained: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

- originally obtained from Dr. Bruce N. Arnes, University of California, Berkeley, California, U.S.A.
- Type and identity of media: For preparation of the master plates Vogel-Bonner minimal medium plates enriched with histidine (260µM) and biotin (3µM). Ampicillin (25µg per ml medium of the plate) is added to the plates used for strains with the R-factor. The tester strain cultures used in this study were grown in Oxoid nutrient broth No.2 (2.5%).The minimal agar plates contain 25 ml of 1.5 % agar in Vogel-Bonner medium E with 2 % glucose.
- Properly maintained: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate (S9 mix)

Test concentrations with justification for top dose:

Experiment I: 0, 50, 150, 500, 1500, 5000 µg/plate (with and without S9-mix)
Experiment II: 0, 50, 150, 500, 1500, 5000 µg/plate (with and without S9-mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- concentration: 10% solution in distilled water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water, 100 µL/plate

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9-mix

Migrated to IUCLID6: 0.5 µg/plate for TA1535 and TA100, dissolved in distilled water

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water, 100 µL/plate

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9-mix

Migrated to IUCLID6: 2.5 µg/plate for TA1538 and TA98, dissolved in DMSO

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water, 100 µL/plate

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9-mix

Migrated to IUCLID6: 50 µg/plate for TA1537, dissolved in distilled water

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water, 100 µL/plate

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 1.0 µg/plate for TA1538, TA98, TA100 and 3.0 µg/plate for TA1535, 1537, dissolved in DMSO

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (standard plate incorporation assay)
- each culture tube contained 2 ml of top agar, 0.1 ml of bacteria, test solution and 0.5 ml of S9-mix or phosphate buffer in the assays without metabolic activation

DURATION
- Exposure duration: 48 h at 37 °C in the dark

NUMBER OF REPLICATIONS: 3
- The experiment was repeated in full after an interval of at least 3 days.

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of reduction in the number of spontaneous revertants or clearing of the bacterial background lawn.

OTHER EXAMINATIONS:
- The plates were examined for the existence of a normal background lawn and/or precipitates and microscopically for microcolony growth
- When there was any question about the nature of colonies scored as revertants and when positive mutagenic results are obtained, the genotype of revertant colonies are spot-checked by picking and streaking on histidine free plates.

Evaluation criteria:

According to guideline. No information given in the study report.

Statistics:

A statistical analysis of the data is not required.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

In the concentration range investigated, HR 96/N00002Aq did not induce any increase in the spontaneous mutation frequency in any of the tester strains in the absence and presence of a metabolic activation system.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

At the concentrations tested HR 96/N00002Aq was not bacteriotoxic.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

In the concentration range investigated, HR 96/N00002Aq did not induce any increase in the spontaneous mutation frequency in any of the tester strains in the absence and presence of a metabolic activation system.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

At the concentrations tested HR 96/N00002Aq was not bacteriotoxic.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: The number of spontaneous revertants observed using each of the five strains was very close
to those previously established in our laboratory and was within the range obtained by Ames et al. (1975) as well as reported by De Serres and Shelby (1979).
- Similarly, the results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the full activity of the metabolizing system.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- At the concentrations tested (50 - 5000 µg/plate) HR 96/N00002Aq was not bacteriotoxic.

STATISTICS:
The estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level, using a X2-test (Mohn and Ellenberger, 1977), did not reveal a significant effect at any of the test points.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The results indicate that the test substance under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 in the presence and absence of a metabolizing system.