[No public or meaningful name is available]

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:

From 2012-10-19 to 2012-11-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-(5'-((5S)-5-(3,5-Dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-3'-H-spiro(azetidine-3,1'-(2)benzofuran)-1-yl)-2-(methylsulfonyl)ethanone

Cas Number:

1398609-39-6

Molecular formula:

C23H18Cl2F4N2O5S

IUPAC Name:

1-(5'-((5S)-5-(3,5-Dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-3'-H-spiro(azetidine-3,1'-(2)benzofuran)-1-yl)-2-(methylsulfonyl)ethanone

Specific details on test material used for the study:

Batch No.: 1000712
Purity: 95.7%

Method

Target gene:

selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA (pKM101)

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain 2

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: The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, 5 days prior to sacrifice. The S9 was prepared by and purchased from Moltox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used.
- method of preparation of S9 mix: The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate, 4 mM β-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2, and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use.
- concentration or volume of S9 mix and S9 in the final culture medium: One-half mL of S9 or Sham mix was added to 2.0 mL of molten selective top agar
- quality controls of S9: No contaminant colonies were observed on the sterility plates for the S9 and Sham mixes.

Test concentrations with justification for top dose:

Initial Toxicity-Mutation Assay/Experiment B1: 1.5, 5.0, 15, 50, 150, 500, 1500, and 5000 μg per plate
Confirmatory Mutagenicity Assay: Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.
- Experiments B2: The dose levels tested were 15, 50, 150, 500, 1500 and 5000 μg per plate with tester strains TA1537 and WP2 uvrA (pKM101) in the absence of S9 activation and tester strains TA98, TA100 and TA1535 in the presence of S9 activation and 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate with the remaining test conditions.
- Experiments B3: The dose levels tested were 15, 50, 150, 500, 1500 and 5000 μg per plate in the absence of S9 activation and 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate in the presence of S9 activation for TA1537.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test article and compatibility with the target cells. After sonication at 29.2ºC for 5 minutes in the solubility test, the test article formed a clear solution in DMSO at a maximum concentration of approximately 500 mg/mL.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

For TA98, TA100, TA1535, and TA1537, WP2 uvrA (pKM101) with S9 Activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

For TA98 without S9 Activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For TA100, TA1535 without S9 Activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

For TA1537 without S9 Activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

For WP2 uvrA (pKM101) without S9 Activation

Details on test system and experimental conditions:

- Initial Toxicity-Mutation Assay:
The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. Vehicle control, positive controls and 8 dose levels of the test article were plated, 2 plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537, and WP2 uvrA (pKM101) on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9.

- Confirmatory Mutagenicity Assay:
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article. Six or seven dose levels of test article along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537, and WP2 uvrA (pKM101) on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test article, vehicle control, and positive controls were plated in triplicate.
- Plating and Scoring Procedures:
The test system was exposed to the test article via the plate incorporation methodology.
On the day of its use, minimal top agar, containing 0.8% agar (w/v) and 0.5% NaCl (w/v), was melted and supplemented with L-histidine, D-biotin, and L-tryptophan solution to a final concentration of 50 μM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water. Bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (w/v) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (w/v) agar and supplemented with 2.5 % (w/v) Oxoid Nutrient Broth Number 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (w/v) Oxoid Nutrient Broth Number 2 (dry powder).
One-half mL of S9 or Sham mix, 100 μL of tester strain (cells seeded), and 50 μL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45 ± 2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 μL aliquot of appropriate positive control. 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.
The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification.

Evaluation criteria:

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 1 tester strain over a minimum of 2 increasing concentrations of test article. Non-dose related increases that were reproducibly greater than the thresholds indicated below for each tester strain were also evaluated as positive.
Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100, and WP2 uvrA (pKM101) were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.
An equivocal response was a biologically relevant increase in a revertant count that partially met 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

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Initial Toxicity-Mutation Assay:
In Experiment B1 (Initial Toxicity-Mutation Assay), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 500 or 1500 μg per plate. No toxicity was observed.

- Confirmatory Mutagenicity Assay:
In Experiment B2 (Confirmatory Mutagenicity Assay), no positive mutagenic responses were observed with tester strains TA98, TA100, TA1535 and WP2 uvrA (pKM101) in either the presence or absence of S9 activation. Precipitate was observed beginning at 150, 500, 1500 or at 5000 μg per plate. No toxicity was observed. Due to tester strain contamination, tester strain TA1537 was not evaluated for mutagenicity but was retested in Experiment B3 based on the precipitate and toxicity profile observed.
In Experiment B3 (Retest of the Confirmatory Mutagenicity Assay), no positive mutagenic responses were observed with tester strain TA1537 in either the presence or absence of S9 activation. Precipitate was observed beginning at 1500 μg per plate. No toxicity was observed.

Applicant's summary and conclusion

Conclusions:

The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, the test item did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

Executive summary:

The test article was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA (pKM101) in the presence and absence of Aroclor-induced rat liver S9 in accordance with OECD 471. The assay was performed in 2 phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 500 or 1500 μg per plate. No toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with tester strains TA98, TA100, TA1535 and WP2 uvrA (pKM101) in either the presence or absence of S9 activation. Precipitate was observed beginning at 150, 500, 1500 or at 5000 μg per plate. No toxicity was observed. Due to tester strain contamination, tester strain TA1537 was not evaluated for mutagenicity but was retested based on the precipitate and toxicity profile observed.

In the retest of the confirmatory mutagenicity assay, no positive mutagenic responses were observed with tester strain TA1537 in either the presence or absence of S9 activation. Precipitate was observed beginning at 1500 μg per plate. No toxicity was observed.

Under the conditions of this study, test article was concluded to be negative in the Bacterial Reverse Mutation Assay.