Registration Dossier

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:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test accoding to GLP and the study procedures described in this report were based on the most recent OECD and EC guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1,2,3-tetramethyl-1H-benzo[e]indolium 4-methylbenzenesulfonate
Cas Number:
141914-99-0
Molecular formula:
C23H25NO3S
IUPAC Name:
1,1,2,3-tetramethyl-1H-benzo[e]indolium 4-methylbenzenesulfonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: V173520
Appearance: White powder (determined by WIL Research Europe)
Batch: AN
Purity/Composition: Not indicated
Test substance storage: At room temperature
Stable under storage conditions until: 20 April 2017 (expiry date)

Purity/composition correction factor: No correction factor required
Test substance handling: No specific handling conditions required
Stability at higher temperatures: Yes, maximum temperature: 50°C(drying), maximum duration: 24 h
Chemical name (IUPAC), synonym or trade name: 1,1,2,2-tetramethyl-1H-Benz(e)indolium 4- methylbenzenesulfonate
CAS Number: 141914-99-0
Molecular formula: C23H25NO3S
Molecular weight: 395.53
Solubility in vehicle: Water: > 10% (NTU >>>)
Stability in vehicle: Water: Unknown

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The Salmonella typhimurium strains are regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
The strain is regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains, except in the tester strains TA1535 and TA98 in the presence of S9-mix.

In a follow-up experiment of the assay with additional parameters, the test substance was tested at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Toxicity was observed in all tester strains, except in the tester strains TA1535, TA98 and TA100 in the presence of S9-mix and in WP2uvrA in the absence and presence of S9-mix.
Vehicle / solvent:
V173520 was dissolved in water (sterilized by autoclaving for 20 min at 121 ± 3°C) (Millipore Corp., Bedford, MA., USA). Test substance concentrations were used within 3 hours of preparation.
Controls
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191
Details on test system and experimental conditions:
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain. The above mentioned dose range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the test substance was tested both in the absence and presence of 5% (v/v) S9- mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test substance was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.

The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in Milli-Q and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must
exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to
5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Statistics:
No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 is not greater than two (2) times the
concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or
WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 is greater than two (2) times the
concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or
WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the
tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

Since 2.4- to 13-fold, dose related increases were observed in three tester strains in the absence and presence of S9-mix (TA98, TA100 and WP2uvrA) and the results were reproducible in the repeat experiments (TA100 and WP2uvrA), these increases are biologically relevant and V173520 is considered to be mutagenic in the absence and presence of S9-mix.