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EC number: 834-970-9 | CAS number: 130111-95-4
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2 018
- 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
- 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
1
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 resultsopen allclose all
- 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
- 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
- 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
- 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
- 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
- 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.
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