Propan-2-yl-oxocyclobutane carboxylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12-11-2018 to 29-11-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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

1

Reference substance name:

propan-2-yl 3-oxocyclobutane-1-carboxylate

EC Number:

834-970-9

Cas Number:

130111-95-4

Molecular formula:

C8H12O3

IUPAC Name:

propan-2-yl 3-oxocyclobutane-1-carboxylate

Test material form:

liquid

Details on test material:

Batch (Lot) Number: GR13224
Expiry date: 31 January 2020 (expiry date)
Physical Description: Colourless to pale yellow liquid
Purity/Composition: 99.6%
Storage Conditions: At room temperature

Method

Target gene:

S.typhimurium - Histidine
E. Coli- Tryptophan

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:

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-06238566 used in the subsequent mutation assays was 5000
µg/plate.

Vehicle / solvent:

DMSO

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 TA1537 2.5µg/plate 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 35.2 - 38.3°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.

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-06238566 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 DMSO 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 DMSO. 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.

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:

ACCEPTABILITY 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

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

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

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

Positive controls validity:

valid

Test results 6

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

Positive controls validity:

valid

Any other information on results incl. tables

First Experiment: Direct Plate Assay

PF-06238566 was initially tested in the tester strains TA100 and WP2uvrA 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-06238566 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, except in tester strain TA98 in the absence of S9-mix

where cytotoxicity, as evidenced by a decrease in the number of revertants was observed at

the top dose level tested.

Mutagenicity

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

with  PF-06238566 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-06238566 was tested up to the dose level of 5000 µg/plate in the

tester strains TA1535, TA1537, TA98, TA100 and WP2

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

Precipitate

Precipitation of  PF-06238566 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, except in tester strain TA98 in the absence of S9-mix

where cytotoxicity, as evidenced by a decrease in the number of revertants was observed at

the top dose level tested.

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

below the laboratory historical control data range was observed. However, since no dose relationship

was observed, 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.

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.

In strain TA1535, a fluctuation in the number of revertant colonies above the laboratory

historical control data range was observed in the presence of S9-mix at the dose level of

5000 µg/plate. However, since the increase was not three-fold (a maximum of 2.3-fold was

reached), this increase was not considered to be relevant.

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this study it is concluded that PF-06238566 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-06238566 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.

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

guidelines.

Batch GR13224 of  PF-06238566 was a colourless to pale yellow liquid with a purity of

99.6%. The vehicle of the test item was dimethyl sulfoxide.

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

5000 µg/plate in the strains TA100 and WP2uvrA 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 WP2uvrA 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, except in tester strain TA98 in the absence of S9-mix in

the first and second experiment where cytotoxicity, as evidenced by a decrease in the number

of revertants was observed at the top dose level tested.

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.

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-06238566 is not

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

reverse mutation assay.