cis-N-methyl-N-(1H-pyrrolo[2,3-d]pyrimidin4-yl)cyclobutane-1,3-diamine phosphate (1:1)

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:

The experimental start date was 30 Nov 2018, and the experimental completion date was 24 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

(1s,3s)-N1-methyl-N1-{7H-pyrrolo[2,3-d]pyrimidin-4-yl}cyclobutane-1,3-diamine; phosphoric acid

EC Number:

951-920-2

Molecular formula:

C11H15N5* H3PO4

IUPAC Name:

(1s,3s)-N1-methyl-N1-{7H-pyrrolo[2,3-d]pyrimidin-4-yl}cyclobutane-1,3-diamine; phosphoric acid

Method

Target gene:

Histidine locus in several strains of Salmonella typhimurium.
Tryptophan locus of Escherichia coli.

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:

S9

Test concentrations with justification for top dose:

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.

Vehicle / solvent:

Milli-Q water

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

Positive controls:

yes

Remarks:

Without Metabolic Activation

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

Positive controls:

yes

Remarks:

With Metabolic Activation

Positive control substance:

other: 2-aminoanthracene

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 97

Metabolic activation:

without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 9

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichiacoli reverse mutation assay.

Applicant's summary and conclusion

Conclusions:

All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
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. In conclusion, based on the results of this study it is concluded that PF-03817968-09 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichiacoli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential ofPF-03817968-09 and/or its metabolites to induce reverse mutations at thehistidinelocus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichiacoli (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 GR13472 ofPF-03817968-09 was a white to off-white solid. 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 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 the second mutation experiment were cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in tester strains TA1535, TA1537, TA98 and TA100 in absence of S9-mix. 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 thatPF-03817968-09 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichiacoli reverse mutation assay.