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Toxicological information

Genetic toxicity: in vitro

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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 to an outdated guideline version (1983) and does not totally comply with the current guideline requirements (1997), since the use of a 5th strain TA102 or E.coli WP2 is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:
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

Reference
Name:
Unnamed
Type:
Constituent
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 / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (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 characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
liver homogenate (S9 mix)
Species / strain:
S. typhimurium TA 1538
Details on mammalian cell lines (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 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:
- 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.
Controlsopen allclose all
Negative controls:
yes
Solvent 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
Negative controls:
yes
Solvent 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
Negative controls:
yes
Solvent 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
Negative controls:
yes
Solvent 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 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 resultsopen allclose all
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:
no
Remarks:
At the concentrations tested HR 96/N00002Aq was not bacteriotoxic.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
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:
no
Remarks:
At the concentrations tested HR 96/N00002Aq was not bacteriotoxic.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
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.

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.