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:
12th June 2020 to 15th July 2020
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
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
Reference substance name:
N-{[4-(2,2-DICYANO-1-HYDROXYETHENYL)PHENYL]METHYL}-5-FLUORO-2-METHOXYBENZAMIDE
IUPAC Name:
N-{[4-(2,2-DICYANO-1-HYDROXYETHENYL)PHENYL]METHYL}-5-FLUORO-2-METHOXYBENZAMIDE

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 homogenate: MolTox Inc. Boone, NC
- Lot No. 4201, (Exp. Date: 29 Jan 2022)
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. 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.

- method of preparation of S9 mix: The S9 mix was prepared on the day of use. S9-mix contained per 1 mL: MgCl2 8 mM, KCL 33 mM, β-nicotinamide-adenine dinucleotide phosphate 4 mM, glucose-6-phosphate 5mM, phosphate buffer pH 7.4 100 mM and S9 homogenate 10% v/v.

The Sham mix, containing 100 mM phosphate buffer at pH 7.4, was also prepared on the day of use.
Test concentrations with justification for top dose:
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.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Lot No: SHBL2820
Supplier: Sigma-Aldrich
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.3. x 10 9 cells/mL
- Test substance added in top agar tubes

One-half (0.5) milliliter of S9 or Sham mix, 100 µL of tester strain (cells seeded) and 50.0 µL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45±2°C. When plating the positive controls, the test article aliquot was replaced by a 50.0 µL aliquot of appropriate positive control. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.
Evaluation criteria:
All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene.

Based on historical control data (95% control limits), all tester strain cultures must exhibit
characteristic numbers of spontaneous revertants per plate with the vehicle controls. The mean
revertants per plate must be within the following ranges (inclusive).

95% Control Limits (99% Upper Limit)
TA98: -S9 6-26 (31) +S9 11-35 (41)
TA100: -S9 68-112 (123) +S9 78-130 (143)
TA1535: -S9 5-21 (25) +S9 5-21 (25)
TA1537: -S9 1-13 (16) +S9 2-14 (17)
WP2 uvrA: -S9 14-38 (44) +S9 18-42 (48)

To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3x109 cells/mL.
The mean of each positive control must exhibit at least a 3.0-fold increase in the number of revertants over the mean value of the respective vehicle control and exceed the corresponding acceptable vehicle control range cited above.
A minimum of three non-toxic dose levels is required to evaluate assay data. A dose level is
considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the
mean number of revertants per plate as compared to the mean vehicle control value. This
reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) At
least a moderate reduction in the background lawn (background code 3, 4 or 5).

Results and discussion

Test resultsopen allclose all
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
Untreated negative 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
Untreated negative 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
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Untreated negative 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:
cytotoxicity
Remarks:
Toxicity as reduction in revertant count was observed beginning at 1500 or at 5000 µg per plate with TA1537 in the presence or absence of S9 activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

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, 3532785 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, 3532785 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. No precipitate was observed. Toxicity as reduction in revertant count was observed beginning at 1500 or at 5000 µg per plate with TA1537 in the presence or absence of S9 activation. Vehicle control value for WP2 uvrA in the absence of S9 activation was outside the 95% historical control limit (HCL) but within the 99% HCL. No positive mutagenic responses were observed with any tester strain in the presence and absence of S9 activation.
These results indicate 3532785 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 of an exogenous metabolic activation system.