isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

EC Number:

950-484-0

Molecular formula:

C9H17NO2*C4H6O4

IUPAC Name:

isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

Test material form:

solid

Details on test material:

Batch (Lot) Number: GR13335
Expiry date: 31 January 2020 (expiry date)
Physical Description: White solid
Purity/Composition: 97.1%
Storage Conditions: At room temperature

Method

Target gene:

Histidine (Salmonella typhimurium spp)
Tryptophan (Escherichia coli spp)

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:

Metabolic Activation System
S9-Fraction
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).
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and
2-aminoanthracene, which require metabolic activation, in tester strain TA100 at
concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per
10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg
glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water
(Millipore Corp., Bedford, MA., USA); 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.

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose-range finding test with the
strains TA100 and WP2
uvrA, both with and without 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 PF-07097547-24 used in the subsequent mutation assays was
5000 µg/plate. At least five different doses (increasing with approximately half-log steps) of
the test item were tested in triplicate in each strain in the absence and presence of S9-mix.
The first experiment was a direct plate assay and the second experiment was a pre-incubation
assay.
The negative control (vehicle) and relevant positive controls were concurrently tested in each
strain in the presence and absence of S9-mix.

Vehicle / solvent:

The vehicle of the test item was Milli-Q water

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

methylmethanesulfonate

other: ICR-191 TA1537 2.5µg/plate direct plate assay without S9; 2-nitrofluorene: TA98 10µg/plate wihtut S9, TA1537 15µg/plate preincubation without S9; 2-aminoanthracene: All strains, 1-15µg/plate with S9.

Details on test system and experimental conditions:

Cell Culture
Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth
(Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C,
150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10
Freshly grown cultures of each strain were used for a test.

Agar plates
Agar plates (ø 9 cm) contained 25 mL glucose agar medium. Glucose agar medium contained
per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose
(Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella
typhimurium strains also contained 12.5 µg/plate biotin (Merck) and 15 µg/plate histidine
(Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate
tryptophan (Sigma).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v)
sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 mL top agar were
transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for
20 min at 121 ± 3°C.

Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C
(actual range 35.4 – 37.6°C). The temperature was continuously monitored throughout the
experiment. Due to addition of plates (which were at room temperature) to the incubator or
due to opening and closing the incubator door, temporary deviations from the temperature
may occur. Based on laboratory historical data these deviations are considered not to affect
the study integrity.

Experimental Design
4.7.1. Dose-range Finding Test
Selection of an adequate range of doses was based on a dose-range finding test with the
strains TA100 and WP2
uvrA, both with and without 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 PF-07097547-24 used in the subsequent mutation assays was
5000 µg/plate. At least five different doses (increasing with approximately half-log steps) of
the test item were tested in triplicate in each strain in the absence and presence of S9-mix.
The first experiment was a direct plate assay and the second experiment was a pre-incubation
assay.
The negative control (vehicle) and relevant positive controls were concurrently tested in each
strain in the presence and absence of S9-mix.

First Experiment: Direct Plate Assay
The above mentioned dose-range finding study with two tester strains is reported as a part of
the direct plate assay. In the second part of this experiment, the test item was tested both in
the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. 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
(10e9 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item in Milli-Q water
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 were mixed on a Vortex and the content of
the top agar tube was poured onto a selective agar plate. 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) were counted.

Second Experiment: Pre-Incubation Assay
The test item was tested both in the absence and presence of S9-mix in all tester strains. Top
agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were
pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C, 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),
0.1 mL of a fresh bacterial culture (10e9 cells/mL) of one of the tester strains, 0.1 mL of a
dilution of the test item in Milli-Q water. After the pre-incubation period the solutions were
added to 3 mL molten top agar. The ingredients were mixed on a Vortex and the content of
the top agar tube was poured onto a selective agar plate. 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) were counted. Initially tester strain
TA100 (absence of S9-mix) was rejected due to infections, this part of the study was
repeated. An additional pre-incubation experiment was performed with tester strain WP2
uvrA in the absence of S9-mix.

Colony Counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates
with sufficient test item precipitate to interfere with automated colony counting were counted
manually. Evidence of test item precipitate on the plates and the condition of the bacterial
background lawn were evaluated when considered necessary, macroscopically and/or
microscopically by using a dissecting microscope.

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation
assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without
S9-mix) must exhibit a characteristic number of revertant colonies when compared
against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit
limited solubility as demonstrated by the preliminary toxicity range-finding test or should
extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen
event. If the results are considered invalid due to contamination, the experiment will be
repeated.
All results presented in the tables of the report are calculated using values as per the raw data
rounding procedure and may not be exactly reproduced from the individual data presented.

Statistics:

No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants should
be evaluated for its biological relevance including a comparison of the results with the
historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2
uvrA is not greater than two
times the concurrent control, and the total number of revertants in tester strains TA1535,
TA1537 or TA98 is not greater than three times the concurrent 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 tester strain TA100 or WP2
uvrA is greater than two
times the concurrent control, or the total number of revertants in tester strains TA1535,
TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat 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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

First Experiment: Direct Plate Assay

PF-07097547-24 was initially tested in the tester strains TA100 and WP2

uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the

absence and presence of S9-mix. Based on the results of the dose-range finding test, the

following dose-range was selected for the mutation assay with the tester strains, TA1535,

TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and

5000 µg/plate. The results are shown in Table 1 and Table 2. The individual data are

presented in Appendix 3.

Precipitate

Precipitation of  PF-07097547-24 on the plates was not observed at the start or at the end of

the incubation period in any tester strain.

Toxicity

To determine the toxicity of the test item, the reduction of the bacterial background lawn, the

increase in the size of the microcolonies and the reduction of the revertant colonies were

observed. The definitions are stated in Appendix 2.

No reduction of the bacterial background lawn and no biologically relevant decrease in the

number of revertants were observed.

Mutagenicity

In the direct plate test, no increase in the number of revertants was observed upon treatment

with  PF-07097547-24 under all conditions tested.

Second Experiment: Pre-Incubation Assay

To obtain more information about the possible mutagenicity of the test item, a pre-incubation

experiment was performed in the absence and presence of S9-mix. Based on the results of the

first mutation assay,  PF-07097547-24 was tested up to the dose level of 5000 µg/plate in the

tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The results are shown in

Table 3, the individual data are presented in Appendix 3.

Precipitate

Precipitation of  PF-07097547-24 on the plates was not observed at the start or at the end of

the incubation period.

Toxicity

There was no reduction in 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.

In tester strain WP2 uvrA in the absence of S9-mix, the positive control item showed toxic

responses, as evidenced by a reduction in the bacterial background lawn.

In strain TA1537 (presence of S9-mix), a fluctuation in the number of revertant colonies

below the laboratory historical control data range was observed at the dose level of

5000 µg/plate. However, since no toxicity was observed in the first mutation experiment and

at any of the other tester strains, this reduction is not considered to be caused by toxicity of

the test item. It is more likely this reduction is caused by an incidental fluctuation in the

number of revertant colonies and is also coupled to a low number of the solvent control.

Mutagenicity

In the pre-incubation test, no increase in the number of revertants was observed upon

treatment with the test item under all conditions tested.

Additional Second Experiment: Pre-Incubation Assay

An additional experiment was performed with this tester strain to verify the results obtained

in the positive control. In the additional experiment, the test item was tested up to the

concentration of 5000 µg/plate in tester strain WP2uvrA in the absence of S9-mix in the preincubation

assay.

The results are shown in Table 4, the individual data are presented in Appendix 3.

Precipitate

Precipitation of  PF-07097547-24 on the plates was not observed at the start or at the end of

the incubation period.

Toxicity

There was no reduction in the bacterial background lawn and no biologically relevant

decrease in the number of revertants at any of the concentrations tested.

The positive control item showed toxic responses, as evidenced by a reduction in the bacterial

background lawn.

Mutagenicity

In the pre-incubation test, no increase in the number of revertants was observed upon

treatment with the test item under all conditions tested.

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this study it is concluded that PF-07097547-24 is not
mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli
reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of  PF-07097547-24 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 or absence of an exogenous

mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was

performed and secondly a pre-incubation assay. An addition pre-incubation experiment was

performed with tester strain WP2uvrA in the absence of S9-mix.

The study procedures described in this report were based on the most recent OECD and EC

guidelines.

Batch GR13335 of  PF-07097547-24 was a white solid with a purity of 97.1%. The vehicle of

the test item was Milli-Q water.

In the dose-range finding study, the test item was initially tested up to concentrations of

uvrA in the direct plate assay. In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the

strains TA1535, TA1537 and TA98. In the second mutation experiment, the test item was

tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98,

TA100 and WP2 uvrA in the pre-incubation assay. In all three experiments the test item did

not precipitate on the plates at this dose level. The bacterial background lawn was not

reduced at any of the concentrations tested and no biologically relevant decrease in the

number of revertants was observed.

In tester strain WP2uvrA in the absence of S9-mix, the positive control item showed toxic

responses in the second mutation experiment, as evidenced by a reduction in the bacterial

background lawn. An additional experiment was performed with this tester strain to verify the

results obtained in the positive control. In the additional experiment, the test item was tested

up to the concentration of 5000 µg/plate in tester strain WP2uvrA in the absence of S9-mix in

the pre-incubation assay. The test item did not precipitate on the plates at this dose level.

The bacterial background lawn was not reduced at any of the concentrations tested and no

biologically relevant decrease in the number of revertants was observed. The positive control

item showed toxic responses in the additional mutation experiment, as evidenced by a

reduction in the bacterial background lawn.

The negative and strain-specific positive control values were within the laboratory historical

control data ranges indicating that the test conditions were adequate and that the metabolic

activation system functioned properly.

Although, the positive control item in the pre-incubation assay in tester strain WP2

uvrA showed toxic responses in two experiments, the mean number of revertants were within the

historical data range and therefore the reduction of the bacterial background lawn has no

effect on the study integrity.

The test item did not induce a significant dose-related increase in the number of revertant

(His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in

the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and

presence of S9-metabolic activation. These results were confirmed in a follow-up

experiment.

In conclusion, based on the results of this study it is concluded that  PF-07097547-24 is not

mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia  coli

reverse mutation assay.