Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 July 2020 to 21 Aug 2020
Reliability:
1 (reliable without restriction)

Data source

Reference
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
N-[(4-{5-AMINO-4-CYANO-1-[(2S)-1,1,1-TRIFLUOROPROPAN-2-YL]-1H-PYRAZOL-3-YL}PHENYL)METHYL]-5-FLUORO-2-METHOXYBENZAMIDE
EC Number:
953-182-7
Cas Number:
2101703-40-4
Molecular formula:
C22H19F4N5O2
IUPAC Name:
N-[(4-{5-AMINO-4-CYANO-1-[(2S)-1,1,1-TRIFLUOROPROPAN-2-YL]-1H-PYRAZOL-3-YL}PHENYL)METHYL]-5-FLUORO-2-METHOXYBENZAMIDE
Specific details on test material used for the study:
Test Article
Identification: 3532789

Method

Target gene:
TA98, TA100, TA1535, TA1537 and WP2 uvrA
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™
1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice. The S9 (Lot No. 4203, Exp. Date: 04 Feb 2022) was purchased commercially.
Upon arrival, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize benzo(a)pyrene and
2-aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.
Test concentrations with justification for top dose:
1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate
Vehicle / solvent:
DMSO
Controls
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976).
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations

Preparation of Tester Strain
Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel, containing 50 to 65 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each
culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x10^9 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.
Evaluation criteria:
Evaluation of Test Results
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:
Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.

Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
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
Positive controls validity:
valid
Additional information on results:
The results of the mutagenicity assay conducted at dose levels of 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate in DMSO. The test article in DMSO formed clear solutions at concentrations from 0.030 to 100 mg/mL. Precipitate was observed beginning at 500 µg per plate in the absence of S9 activation and beginning at 1500 µg per plate in the presence of S9 activation. Toxicity as reduction in revertant count was observed beginning at 50.0 µg per plate with TA1537 in the absence of S9 activation. No positive mutagenic responses were observed with any tester strain in the presence and absence of S9 activation.
Remarks on result:
other:
Remarks:
substance was negative for the ability to induce reverse mutations

Applicant's summary and conclusion

Conclusions:
All criteria for a valid study were met as described in the protocol. The results of the Bacterial
Reverse Mutation Assay indicate that, under the conditions of this study, 3532789 did not cause
a positive mutagenic response with any tester strain in the presence and absence of
Aroclor-induced rat liver S9.
Executive summary:

The test article, 3532789 was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle. In the mutagenicity assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and
5000 µg per plate. The test article in DMSO formed clear solutions at concentrations from 0.0300 to 100 mg/mL. Precipitate was observed beginning at 500 µg per plate in the absence of S9 activation and beginning at 1500 µg per plate in the presence of S9 activation. Toxicity as reduction in revertant count was observed beginning at 50.0 µg per plate with TA1537 in the absence of S9 activation. No positive mutagenic responses were observed with any tester strain
in the presence and absence of S9 activation. These results indicate 3532789 was negative for the ability to induce reverse mutations at selected loci of a several strain of Salmonella typhimurium and at the tryptophan locus of Escherichia coli WP2 uvrA in the presence and absence of an exogenous metabolic activation
system.

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