2,4,6-trifluorobenzamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April 2020 to 26 May 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

Test material information

Constituent 1

Reference substance name:

2,4,6-trifluorobenzamide

Cas Number:

82019-50-9

IUPAC Name:

2,4,6-trifluorobenzamide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Test Article
Identification: 3459540
Lot No. Vial: ALL-H81956-016
Lot No. Characterization Form: CPr123012-01-02-01-28-01
Potency: 99.71%
Molecular Weight: 175.109 g/mol
Description: White powder
Storage Conditions: Room temperature, protected from light

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. 4173, Exp. Date: 04 Dec 2021) was purchased commercially from MolTox (Boone, NC).
Upon arrival at BioReliance, 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 TA10

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 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 0.3x109 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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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. Neither toxicity nor precipitate was observed. A 1.8-fold, maximum increase was observed with WP2 uvrA in the presence of S9 activation with the increase outside of the 99% Historical Control Limit (HCL). However, this increase was not dose responsive and there was variability between plate count within any given dose level and from dose to dose. Therefore, this increase is not indicative of mutagenic activity. A 1.9- and 1.6-fold, maximum increase was observed with tester strain TA98 in the absence and presence of S9 activation, respectively. However, the counts and mean at the response were within the respective HCL. 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:

These results indicate the substance 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

Executive summary:

The test article 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. Neither toxicity nor precipitate was observed. A 1.8-fold, maximum increase was observed with WP2 uvrA in the presence of S9 activation with the increase outside of the 99% Historical Control Limit (HCL). However, this increase was not dose responsive and there was variability between plate count within any given dose level and from dose to dose. Therefore, this increase is not indicative of mutagenic activity. A 1.9- and 1.6-fold, maximum increase was observed with tester strain TA98 in the absence and presence of S9 activation, respectively. However, the counts and mean at the response were within the respective HCL. No positive mutagenic responses were observed with any tester strain in the presence and absence of S9 activation. These results indicate the substance 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.