(isopropyl (1s,3s)-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutane-1-carboxylate)

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:

20 December 2018 - 10 January 2019

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(isopropyl (1s,3s)-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutane-1-carboxylate)

EC Number:

950-860-4

Molecular formula:

C15H20N4O2

IUPAC Name:

(isopropyl (1s,3s)-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutane-1-carboxylate)

Test material form:

solid: bulk

Details on test material:

White Crystalline Solid

Method

Target gene:

Determine the potential of PF-07094402 and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system.

Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Escherichia coli WP2uvrA strain detects base-pair substitutions.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 at 500 mg/kg body weight.

Test concentrations with justification for top dose:

Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate. The highest concentration used in the subsequent mutation assays was 5000 µg/plate or the level at which the test item exhibited limited solubility.

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: 5.4, 17, 52, 164, 512 and 2000 µg/plate. Since in testers strains TA1535, TA1537 and TA98 no dose level with precipitation and/or cytotoxicity was observed in the presence of S9-mix an additional experiment was performed with these tester strains at the dose level of 5000 µg/plate in the presence of S9-mix. This additional experiment is reported as part of the first mutation assay.

Based on the results of the first mutation assay, PF-07094402 was tested up to the dose level of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO) and Saline

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene, ICR-191

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The test system was exposed to the test article via plate incorporation and pre-incubation methods.

DURATION:
First Experiment: Direct Plate Assay:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, 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-07094402 used in the subsequent mutation assays was 5000 µg/plate or the level at which the test item exhibited limited solubility. 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.

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 (109 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. Initially, in tester strains TA1535, TA1537 and TA98 no dose level with precipitation and/or cytotoxicity was tested in the presence of S9-mix. Therefore an additional experiment was performed with these tester strains at the dose level of 5000 µg/plate in the presence of S9-mix to complete the data of the first experiment.

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 (109 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.

SELECTION AGENT (mutation assays): histidine and biotin solution

NUMBER OF REPLICATIONS: Triplicate

DETERMINATION OF CYTOTOXICITY
Evidence of test item precipitate on the plates and the condition of the bacterial background lawn will be 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

As evidenced by a reduction of the bacterial background lawn

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 6

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

As evidenced by a reduction of the bacterial background lawn

Test results 7

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Test results 8

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

As evidenced by a reduction of the bacterial background lawn

Additional information on results:

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In the direct plate test, no increase in the number of revertants was observed upon treatment with PF-07094402 under all conditions tested.
In strain TA1535, fluctuations in the number of revertant colonies above the laboratory historical control data range were observed in the absence of S9-mix. However, since the increases were not dose related and not three-fold (a maximum of 1.2-fold was reached), these increases were not considered to be relevant.

In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Any other information on results incl. tables

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. The test item was tested up to or beyond a precipitating dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in both tester strains at dose levels of 1600 and 5000 µg/plate in the absence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested in the strains TA1535, TA1537 and TA98 up to concentrations of 2000 and 5000 µg/plate in the absence and presence of S9-mix, respectively.  PF-07094402 precipitated on the plates at the dose level of 2000 µg/plate in the absence of S9-mix. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was only observed in tester strain TA1537 in the presence of S9-mix at the highest concentration tested.

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. The test item precipitated on the plates at the dose levels of 1000 and 5000 µg/plate in the absence of S9-mix. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA1537 in the presence of S9-mix. Since the test item precipitated heavily on the triplicate plates at the test item concentration of 5000 µg/plate in the absence of S9-mix in the tester strains TA1535, TA1537, TA100 and WP2uvrA, the number of revertants of this dose level could not be determined.

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.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that PF-07094402 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.