N'-(2-Cyano-5-methoxy-4-nitrophenyl)-N,N-dimethylmethanimid amide

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 9th 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

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:

N'-(2-Cyano-5-methoxy-4-nitrophenyl)-N,N-dimethylimidoformamide

EC Number:

813-618-8

Cas Number:

1269400-04-5

Molecular formula:

C11H12N4O3

IUPAC Name:

N'-(2-Cyano-5-methoxy-4-nitrophenyl)-N,N-dimethylimidoformamide

Test material form:

solid

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 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. The S9-batch used was no. 2732.

Vehicle / solvent:

The test substance was dissolved in dimethyl sulfoxide (SeccoSolv, Merck, Darmstadt, Germany).
Test substance concentrations were used within 3.5 hours after preparation.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

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. In the mutation experiment PF-05188294 was tested both in the absence and presence of S9-mix in tester strains TA1535, TA1537, TA98 and TA100.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Since PF-05188294 showed clear positive responses, no verification in an independent repeat experiment was needed. Omission of the second test, under the conditions that a clear positive response is observed in the first test, is acceptable according to OECD Guideline no. 471. 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- ix (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.

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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Observed at 1000 ug/ml in the absence of S9. Observed at all dose levels in he presence of S9

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at dose levels of 3330 & 5000 μg/plate in the absence of S9-mix and at 1000 μg/plate and above in the presence of S9-mix.

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In this study, 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 the absence of S9-mix, PF-05188294 induced dose related increases in three tester strains (TA1537, TA98 and TA100). The increases observed in these strains were above the laboratory historical control data range and were 2.7 to 36-fold the concurrent vehicle control. In the presence of S9-mix, PF-05188294 induced dose related increases in four tester strains (TA1535, TA1537, TA98 and TA100). The increase observed in these strains were above the laboratory historical control data range and were 7.3 to 53-fold the concurrent vehicle control.
Based on the results of this study it is concluded that PF-05188294 is mutagenic in the Salmonella typhimurium reverse mutation assay and is not mutagenic in the Escherichia coli reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of PF-05188294 in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent repeat).
PF-05188294 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 the presence and absence of S9-mix (rat liver S9-mix induced by a combination of Aroclor).
The study procedures described in this report were based on the most recent OECD and EC guidelines.
Batch E010011212 of PF-05188294 was an orange powder with a purity of 99.7%. The test substance was dissolved in dimethyl sulfoxide.
In the dose range finding test, PF-05188294 was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. PF-05188294 precipitated on the plates at dose levels of 3330 and 5000 μg/plate. In tester strain TA100, toxicity was observed at dose levels of 1000 μg/plate and above in the absence of S9-mix and at all dose levels tested in the presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 3330 and 5000 μg/plate in the absence of S9-mix and at 1000 μg/plate and above in the presence of S9-mix.
Based on the results of the dose range finding test, PF-05188294 was tested in the mutation assay at a concentration range of 3 to 1000 μg/plate in the absence of S9-mix and at a range of 0.3 to 33 μg/plate in the presence of S9-mix in tester strains TA1535, TA1537, TA98 and TA100. Toxicity was observed in tester strains TA1535, TA1537 and TA100
In this study, 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 the absence of S9-mix, PF-05188294 induced dose related increases in three tester strains (TA1537, TA98 and TA100). The increases observed in these strains were above the laboratory historical control data range and were 2.7 to 36-fold the concurrent vehicle control.
In the presence of S9-mix, PF-05188294 induced dose related increases in four tester strains (TA1535, TA1537, TA98 and TA100). The increase observed in these strains were above the laboratory historical control data range and were 7.3 to 53-fold the concurrent vehicle control.
Based on the results of this study it is concluded that PF-05188294 is mutagenic in the Salmonella typhimurium reverse mutation assay and is not mutagenic in the Escherichia coli reverse mutation assay.