PF-05201372

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:

25th August 2011 to 22nd November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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:

(2E)-N-(5-Cyano-4-{(E)-[(dimethylamino)methylene]amino}-2-methoxyphenyl)-4-(piperidin-1-yl)but-2-enamide

Molecular formula:

C20 H27 N5 O2

IUPAC Name:

(2E)-N-(5-Cyano-4-{(E)-[(dimethylamino)methylene]amino}-2-methoxyphenyl)-4-(piperidin-1-yl)but-2-enamide

Method

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:

Metabolic activation system
Rat liver microsomal enzymes (S9 homogenate) was obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that had been injected intraperitoneal with Aroclor (500 mg/kg).
Preparation of S9-mix
S9-mix was prepared immediately before use and kept on ice. 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 or 5.0 ml Milli-Q water (first or second experiment respectively) (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 in the first experiment and to 9.0 ml of S9- ix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment. The S9-batch used was no. 2732.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

Mutation assay
At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain. The highest concentration of PF-05201372 used in the mutation assays was 5000 μg/plate. In the first experiment PF-05201372 was tested both in the absence and presence of 5% (v/v) S9-mix. In an independent repeat of the assay with additional parameters, the test substance was tested both in the absence and presence of 10% (v/v) S9-mix. An additional experiment was performed with tester strain WP2uvrA in the absence of S9-mix. The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix. Top agar in top agar tubes was melted by heating to 45°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 substance in dimethyl sulfoxide 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.

Colony counting
The revertant colonies (histidine independent or tryptophan independent) were counted manually if less than 40 colonies per plate were present. If more than 40 colonies were present, these were counted automatically with a Biocount 4000 Pro-S-colony counter. Plates with abundant test substance precipitate which interfered with automated colony counting were counted manually and the evidence of test substance precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.

Rationale for test conditions:

Recommended test system in international guidelines (e.g. OECD, EC).

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle 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 independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results

Test results 1

Key result

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that PF-05201372 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of PF-05201372 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).
PF-05201372 was tested in the Salmonella typhimurium reverse mutation assay with four histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor). To obtain more information about the possible mutagenicity of PF-05201372, an additional experiment was performed with tester strain WP2uvrA in the absence of S9-mix.
The study procedures described in this report were based on the most recent OECD and EC guidelines.
Batch 902/299804/G/2/1 of PF-05201372 was an off-white powder with a purity of 98.2%. The test substance was dissolved in dimethyl sulfoxide.
In the first mutation assay, PF-05201372 was tested up to concentrations of 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the strains TA1535, TA1537, TA98 and TA100 and WP2uvrA. PF-05201372 did not precipitate on the plates at this dose level. In an independent repeat of the assay with additional parameters, PF-05201372 was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in all tester strains. PF-05201372 did not precipitate on the plates at the top dose of 5000 μg/plate. Toxicity was only observed at tester strain WP2uvrA in the absence of S9-mix (second experiment).
To verify the results obtained in tester strain WP2uvrA, in the absence of S9-mix (first experiment), a third mutation experiment was performed with this strain in the absence of S9-mix. PF-05201372 was tested up to the dose level 5000 μg/plate. No toxicity was observed. 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, except the response for WP2uvrA in the absence and presence of S9-mix (first experiment). However, since this value was just without the limit of the range, the validity of the test was considered to be not affected. 
In tester strain WP2uvrA, PF-05201372 induced an up to 1.9-fold increase in the number of revertant colonies compared to the solvent control in the absence of S9-mix (first experiment). Verification of this result was performed in an additional experiment, in which no increase in the number of revertants was observed upon treatment with PF-05201372 Since the increase was observed only in one out of three experiments and the increase was less than 2-fold, this increase is considered to be not biologically relevant and PF-05201372 is considered to be not mutagenic.
All other 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.
Based on the results of this study it is concluded that PF-05201372 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.