Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19th Nov 2018 to 13th Dec 2018
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
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

Constituent 1
Chemical structure
Reference substance name:
cis-N-hydroxy-3-[methyl(1H-pyrrolo[2,3-d]pyrimidin- 4-yl)amino]cyclobutanecarboxamide
EC Number:
951-622-2
Molecular formula:
C12H15N5O2
IUPAC Name:
cis-N-hydroxy-3-[methyl(1H-pyrrolo[2,3-d]pyrimidin- 4-yl)amino]cyclobutanecarboxamide

Method

Target gene:
Histidine locus in 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
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
PF-07085579 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.
Vehicle / solvent:
dimethyl sulfoxide
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
mutagenic potential (based on QSAR/QSPR prediction)
Remarks:
PF-07085579 is mutagenic in the Salmonella typhimurium reverse mutation assay and that PF-07085579 is not mutagenic in the Escherichia coli reverse mutation assay.

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this study it is concluded that PF-07085579 is mutagenic in the Salmonella typhimurium reverse mutation assay and that PF-07085579 is not
mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations including a pre-incubation step.
Executive summary:

The objective of this study was to determine the potential of  PF-07085579 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 GR13449 of  PF-07085579 was a white solid with a purity of 100%. 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 tester strains TA100 and WP2uvrA in the direct plate assay. The test item precipitated on the plates at the dose level of 5000 µg/plate. 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. 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 up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the dose level of 5000 µg/plate. 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 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 was tested up to or beyond a precipitating 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 in tester strain WP2uvrA showed toxic responses, 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 WP2uvrA showed toxic responses, 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.

In the absence of S9-mix,  PF-07085579 induced dose related increases in three tester strains (TA1537, TA98 and TA100) after the pre-incubation step only. The increases observed in the tester strains TA1537 and TA100 were above the laboratory historical control data range and 3.4 to 3.5-fold the concurrent control. The increase observed in tester strain TA98 was not above the laboratory historical control data range and just above three times the concurrent

control. In the presence of S9-mix,  PF-07085579 induced dose related increases in three tester strains (TA1537, TA98 and TA100) after the pre-incubation step only. The increases observed in the tester strains TA1537 and TA100 were above the laboratory historical control data range and 2.1 to 9.8-fold the concurrent control. The increase observed in tester strain TA98 was not above the laboratory historical control data range and only two times the concurrent control. Since 2.1- to 9.8-fold, dose related increases were observed in three tester strains, both in the absence and presence of S9-mix, these increases are considered biologically relevant, PF-07085579 is mutagenic in the absence and presence of S9-mix. In the other tester strains,  PF-07085579 did not induce a dose-related, two-fold increase in the number of revertant colonies. In conclusion, based on the results of this study it is concluded that  PF-07085579 is

mutagenic in the Salmonella typhimurium reverse mutation assay and that  PF-07085579 is not mutagenic in the Escherichia  coli  reverse mutation assay. The mutagenicity was confined only to incubations including a pre-incubation step.