[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 22 Feb to 15 Mar 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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:

(S)-tert-butyl 5'-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-3'H-spiro[azetidine-3,1'-isobenzofuran]-1-carboxylate

Cas Number:

1398610-06-4

Molecular formula:

C25H22Cl2F4N2O4

IUPAC Name:

(S)-tert-butyl 5'-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydroisoxazol-3-yl)-3'H-spiro[azetidine-3,1'-isobenzofuran]-1-carboxylate

Specific details on test material used for the study:

Batch No.: 465884
Purity: 100%

Method

Target gene:

histidine locus of Salmonella typhimurium and tryptophan locus of Escherichia coli

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:
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
- method of preparation of S9 mix:
S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per
10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL or 5.0 mL Milli-Q
water (first or second experiment respectively); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized. To 9.5 mL of S9-mix components, 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 mL of S9-mix components, 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Test concentrations with justification for top dose:

Dose-range Finding Test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Experiment 1: 21, 67, 210, 655, 1288 and 2500 μg/plate
Experiment 2: 86, 154, 275, 492, 878 and 1568 μg/plate

Vehicle / solvent:

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

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:

methylmethanesulfonate

Remarks:

For TA100 without S9 Activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For TA1535 without S9 Activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

For TA1537 without S9 Activation

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

For WP2 uvrA (pKM101) without S9 Activation

Details on test system and experimental conditions:

Dose-range Finding Test
Selection of an adequate range of concentrations was based on a dose-range finding test with
the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight
concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate.
The highest concentration of the test item used in the subsequent mutation assays was the
level at which the test item exhibited limited solubility.

Mutation Assay
At least five different concentrations (increasing with approximately half-log steps) of the test
item 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 item 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 item was tested both in the absence
and presence of 10% (v/v) S9-mix in all tester strains.
The negative control 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 item in DMSO or a
control solution, 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 of each solution were
mixed on a Vortex and the content of the top agar tubes were poured onto selective agar
plates. 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 bacteria were counted.

Colony Counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates
with sufficient test item precipitate that interfered with automated colony counting were
counted manually.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strains TA100 or WP2uvrA is less than two
times the concurrent negative control, and the total number of revertants in tester strains
TA1535, TA1537 or TA98 is less than three times the concurrent negative control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strains TA100 or WP2uvrA is greater than or
equal to two times the concurrent negative control, or the total number of revertants in
tester strains TA1535, TA1537, TA98 is greater than or equal to three times the
concurrent negative 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 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:

Dose-range Finding Test/First Mutation Experiment
No increase in the number of revertants was observed upon treatment with the test item under
all conditions tested.

Second Mutation Experiment
Precipitation of the test item on the plates was observed at concentrations of 878 and 1568 μg/plate.
There was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix, except in tester strain TA1537 where, in the absence of S9-mix, a decrease in the number of revertants was observed.
In tester strain TA100 in the presence of S9-mix, a decrease in the number of revertants was observed in both test item concentrations and negative control and is, therefore, not considered a biologically relevant decrease.
In the second mutation assay, no increase in the number of revertants was observed upon
treatment with test item under all conditions tested.
In tester strain TA1537 in the absence of S9-mix, the test item induced a 3-fold increase in the
number of revertant colonies compared to the negative control. However, this increase was
not dose-related. Furthermore, this increase was linked to a low number of revertant colonies
in the negative control and was within the historical control data range. Therefore, this
increase was considered to be not biologically relevant.

Applicant's summary and conclusion

Conclusions:

The test item is not mutagenic in the Salmonella typhimurium reverse mutation assay or in the Escherichia coli reverse mutation assay.

Executive summary:

This study was to determine the potential of test item and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence and absence of an exogenous mammalian metabolic activation system (S9-mix).

The test item is not mutagenic in the Salmonella typhimurium reverse mutation assay or in the Escherichia coli reverse mutation assay.