Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts
EC Number:
812-037-7
Cas Number:
1793011-72-9
Molecular formula:
Unknown for all components
IUPAC Name:
Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts
Test material form:
solid

Method

Species / strain
Species / strain / cell type:
other: Salmonella typhimurium TA100, TA1535, TA98 and TA1537. Escherichia coli WP2urvA
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix prepared using the livers of male rats treated with phenobarbital and benzoflavone.
Test concentrations with justification for top dose:
Preliminary test: 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate

Main test 1 and 2:
TA100, TA1535 and WP2urva (with and without S9): 313, 625, 1250, 2500 and 5000 µg/plate.
TA98 (without S9): 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/plate
TA98 (with S9): 0.0763, 0.153, 0.305, 0.610, 1.22, 2.44 and 4.88 µg/plate
TA1537 (without S9): 313, 625, 1250, 2500 and 5000 µg/plate
TA1537 (with S9): 1.22, 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In a solvent selection test the substance was dissolved at 50 mg/mL in sterilised water in DMSO. The test substance did not dissolve or suspend in acetone. The use of DMSO showed no increases in temperature, discolouration or foaming and the solvent is a standard vehicle in tests of this type.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 2-(2-Furly)-3-(5-nitro-2-furyl), 9-Aminoacridine hydrochloride monohydrate (9-AA) and 2-Aminoanthracene (2-AA)
Details on test system and experimental conditions:
Preparation of the test substance:
The test substance was weighed and dissolved in DMSO with vortex mixing to make a 50 mg/mL solution. An aliquot of this was diluted stepwise with DMSO to make the test substance solutions of each concentration. All procedures were performed under yellow light at room temperature.

Method:
The study was performed using the pre-incubation method with and without S9 mix.
For each treatment, 0.1 mL of the test substance solution, negative (solvent) control or positive control solution was added in sterilised test tube with aluminium cap. Appropriate amounts of S9 mix was included where required alongside bacterial suspensions with resultant mixtures shaken for 20 minutes at 37°C. After pre-incubation, 2 mL of the moulten top agar was added to this mixture and then poured into a minimal glucose agar plate. After the overlaid agar had solidified, the plates were incubated for 48 hours at 37°C. A preliminary sterility test was also conducted.

Observations:
Precipitation was judged by observation of the plate surface macroscopically. The background lawn of the bacterial cells that have amino-acid requirements were observed by a stereoscopic microscope and microbial growth inhibition was judged by the relationship between the test substance treated plates and the negative (solvent) control plates.

Measurements of colonies:
Revertant colonies were measured using an automatic colony counter or manual counting.

Number of replicates
Preliminary test: 1 plate/dose (2 plates for the solvent and positive controls)
Main tests: 3 plates/dose (triplicate).
Rationale for test conditions:
Standard as per OECD guidelines
Evaluation criteria:
The test substance was considered to be judged as mutagenic if it induces a dose-dependant increase in the number of revertant colonies (mean) to a level equal to or greater than 2 fold of the negative control value in any of the tester strains with or without S9 mix and when the dose dependant increase was reproducible. Other results were judged to be negative. Mutation activities (number of revertant colonies/mg) were calculated.
Statistics:
No statistical analysis was performed on the results.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None noted
- Effects of osmolality: None noted
- Evaporation from medium: None noted
- Water solubility: Soluble in DMSO
- Precipitation: Observed at 313 µg/plate and higher in all tester strains with S9 mix. in the preliminary test. In the main test precipitation of the substance was observed at 313 µg/plate or more in TA100, TA1535 and WP2urva and at 313 µg/plate in TA1537 with S9 mix.

RANGE-FINDING/SCREENING STUDIES: A preliminary test was performed at 5000 µg/plate as the maximum dose using 7 doses with a ratio of 4. The number of revertant colonies treated with the substance was greater than 2-fold when compared with the negative control in TA98 without S9 mix and in TA98 and TA1537 with S9 mix. The doses selected in the main tests were based on the results of the preliminary tests.


Any other information on results incl. tables

Results of sterility test:

Bacterial or fungus contamination was not observed in the plates used for the sterility assessments.

The number of revertant colonies was greater than 2 -fold than treated with the solvent control at the following dose levels:

Main test 1:

Without S9 mix: 625 to 5000 µg/plate (TA98)

With S9 mix: 0.610 to 4.88 µg/plate (TA98)

With S9 mix: 39.1 to 313 µg/plate (TA1537)

Main test 2:

Without S9 mix: 313 to 5000 µg/plate (TA98)

With S9 mix: 0.610 to 4.88 µg/plate (TA98)

With S9 mix: 39.1 to 313 µg/plate (TA1537)

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study the substance was deemed to be potentially mutagenic in TA98 with and without S9 mix and in TA1537 with S9 mix.