2-Propenamide, N-[5-[[4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]-2-pyrimidinyl]amino]-4-methoxy-2-(4-morpholinyl)phenyl]

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:

From 2016-03-17 to 2016-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation Harmonised Tripartite Guideline, Safety Guidelines, Genotoxicity Studies S2(R1) Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Version / remarks:

09 November 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Ministry of Food and Drug Safety Notification No. 2015-82 Test Guidelines for Safety Evaluation of Drugs Annex 4 Genetic Toxicity Study-Bacterial Reverse Mutation Test

Version / remarks:

11 November 2015

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-{5-[(4-{4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl}pyrimidin-2-yl)amino]-4-methoxy-2-(morpholin-4-yl)phenyl}prop-2-enamide

EC Number:

841-500-6

Cas Number:

1903008-80-9

Molecular formula:

C30H34N8O3

IUPAC Name:

N-{5-[(4-{4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl}pyrimidin-2-yl)amino]-4-methoxy-2-(morpholin-4-yl)phenyl}prop-2-enamide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- KIT Code No.: K-5523
- Batch/Lot No.: 5219-10
- Purity: 99.6% (by HPLC)
- Purity test date : 2016-01-07
- Expiry date: 2016-07-07 (retest date)
- Appearance: Light yellow solid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated, protected from light and tightly closed
-Stability under storage conditions: not indicated
- Stability under test conditions: not indicated
- Solubility and stability of the test item in the solvent/vehicle: not indicated

OTHER SPECIFICS: As per the sponsor’s request, the correction for purity was not conducted.

Method

Target gene:

Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not relevant

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Experiment 1: Sprague-Dawley rat (male) liver tissue; Experiment 2: human (male) liver tissue (pooled)
- Method of preparation of S9 mix: The final concentration for the composition of cofactor mix per 1.0 mL of metabolic activation system was as follows:
- MgCl2 8 μmol,
- KCl 33 μmol,
- Glucose 6, phosphate (G-6-P) 5 μmol,
- Nicotinamide adenine dinucleotide phosphate reduced (NADPH) 4 μmol,
- Nicotinamide adenine dinucleotide reduced (NADH) 4 μmol,
- Sodium phosphate buffer (pH 7.4) 100 μmol
- Concentration or volume of S9 mix and S9 in the final culture medium: The metabolic activation system containing 5% v/v of S9 fraction consisted of the following: S9 fraction 0.5 mL, Cofactor mix 9.5 mL.
- Quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 fraction was checked by the manufacturer and the quality control statement, for each batch and type of S9 fraction used, was included in the raw data
- Use of metabolic activation system: The metabolic activation system was prepared immediately prior to use and placed in an ice bucket during experiment. The remaining metabolic activation system was discarded after the treatment. Each plate was treated with 0.5 mL of the prepared metabolic activation system, which was held in an ice batch for use. Same volume of 0.2 M sodium phosphate buffer solution (pH 7.4) was used for treatment in the absence of metabolic activation system. The activity of metabolic activation system was confirmed by the induction of revertant colonies in the plates treated with 2-AA and BP in S. typhimurium TA1535 and TA98, respectively.

Test concentrations with justification for top dose:

DRF: 50, 100, 500, 750, 1000 and 2000 μg/plate
Experiment 1: 0, 2.5, 8, 23, 70 and 200 µg/plate with and without metabolic activation (rat liver S9 fraction)
Experiment 2: 0, 2.5, 8, 23, 70 and 200 µg/plate with and without metabolic activation (human liver S9 fraction)

See section Any other information on materials and methods incl. tables for the justification

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: DMSO was selected as vehicle according to the information provided by sponsor and was used to dilute for dose formulation.

- Justification for percentage of solvent in the final culture medium: not indicated

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9; S. typhimurium TA100 and TA1535 (0.5 μg/plate per strain)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9; S. typhimurium TA98 (2 μg/plate)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9; S. typhimurium TA1537 (50 μg/plate)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9; E. coliWP2uvrA (1 μg/plate)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

With S9; S. typhimurium TA1535 (2 μg/plate) and E. coliWP2uvrA (4 μg/plate) in exp 1. S. typhimurium TA1535 (0.5 and 1 μg/plate), TA98 (0.1 and 0.5 μg/plate), TA1537 (0.5 and 1 μg/plate) and E. coliWP2uvrA (4 and 6 μg/plate) in exp 2.

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9; S. typhimurium TA100, TA98 and TA1537 (2 μg/plate per strain)

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

With S9; S. typhimurium TA100 (0.5 and 1 μg/plate) in the second experiment

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)
- Preincubation period, if applicable: approximately 10 hours in a shaking incubator (120 to 200 rpm) at 37±1°C
- Treatment of test and control items: Three plates for each concentration were used in the absence and presence of metabolic activation system, respectively. Autoclaved top agar (2.0 mL) was dispensed into each 12×75 mm tube maintained at approximately 45°C in a heating block. Then 0.1 mL of test item solution, 0.5 mL of metabolic activation system (or 0.2 M sodium phosphate buffer, pH 7.4), and 0.1 mL of bacterial culture were added to each tube. This mixture (2.7 mL) was stirred for 2 to 3 seconds using a vortex mixer and overlaid onto the surface of a minimal glucose agar plate.
Negative control (vehicle) group was treated with 0.1 mL of vehicle instead of test item solution.
Positive control group was treated with 0.1mL of specific positive control for each tester strain.
Sterility checks were performed on test item solution (maximum concentration only, 0.1 mL) and metabolic activation system (0.5 mL), without bacterial culture, using additional plates. After the overlay solidified, the plates were incubated upside down for approximately 48 hours at 37±1°C in the dark.
- Harvest time after the end of treatment (sampling/recovery times): Cultures were harvested when the tester strain culture titers were more than 1×10^9 cells per milliliter.
- Counting of revertant colony: Upon completion of the incubation, the plates were scored immediately. The numbers of revertant colonies were counted using a hand counter. Absence or diminution of background lawn, formation of microcolonies, and the presence of turbidity and /or precipitation resulting from test item for each plate were evaluated with the unaided eye and recorded.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: a concentration-dependent decrease in the revertant counts, or absence or diminution of background lawn or formation of microcolonies

Rationale for test conditions:

In the DRF study, YH25448 did not cause a positive response in all strains in the absence and presence of metabolic activation system. However, antibacterial effect (toxicity), turbidity and/or precipitation were observed at some concentrations regardless of the metabolic activation system application. Therefore, to include the concentration with a visible precipitate on the basis of these results, 200 μg/plate was selected as the highest concentration in the definitive study. Treatment concentrations of the definitive study were 0, 2.5, 8, 23, 70 and 200 μg/plate.

Evaluation criteria:

The assay must be considered valid in accordance with the assay acceptance criteria (see additional information on material and methods)

Antibacterial effect (toxicity): A ≥50% reduction in the number of revertants per plate relative to the negative control (vehicle) values and exhibiting a concentration-dependent decrease in the revertant counts, or absence or diminution of background lawn or formation of microcolonies (Richard et al., 2002).

The assay was considered positive if there is a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one tester strain with or without the metabolic activation system, ≥ two times (for S. typhimurium TA98, TA100 tester strains, and E. coli WP2uvrA tester strain) or ≥ three times (for S. typhimurium TA1535 and TA1537 test strains) that of the mean concurrent negative control (vehicle) value (Richard et al., 2002).

An equivocal response was a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited.

The assay was considered negative: if none of the above criteria are met.

Statistics:

No statistical analyses were performed. The ‘fold-increase’ relative to the negative control
(vehicle) value was calculated in order to compare the mean values for all treatment groups
with those obtained for negative control (vehicle) groups.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

rat S9-mix

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at the highest dose level 200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

rat S9-mix

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

200 μg/plate was the highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

rat S9-mix

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at the highest dose level 200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

rat S9-mix

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

200 μg/plate was the highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

rat S9-mix

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

200 μg/plate was the highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

rat S9-mix

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at the highest dose level 200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

rat S9 mix

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

200 μg/plate was the highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

human S9-mix

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

200 μg/plate was the highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

human S9-mix

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at the highest dose level 200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

human S9-mix

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

200 μg/plate was the highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 11

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

human S9-mix

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed at the highest dose level 200 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 12

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

human S9-mix

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

200 μg/plate was the highest concentration tested

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The turbidity of test item was observed at 200 μg/plate concentration in all strains in the absence and presence of metabolic activation system when the test item solutions were mixed with top agar in the first and second experiments.
When the test item solutions were incubated for 48 hours in the first experiment, the precipitation of test item was observed at 200 μg/plate concentration in all strains except for S. typhimurium TA1537 strain in the
absence of metabolic activation system. The precipitation of test item was observed at 70 μg/plate and 200 μg/plate concentrations in S. typhimurium TA1537 strain in the absence of metabolic activation system.
In the second experiment, the precipitation of test item was observed at 200 μg/plate concentration in S. typhimurium TA1535, TA98 and E. coli WP2uvrA strains, and at 70 μg/plate and 200 μg/plate concentrations in S. typhimurium TA100 and TA1537 strains in the absence of metabolic activation system. The precipitation of test item was observed at 200 μg/plate concentration in S. typhimurium TA100 and E. coli WP2uvrA strains, and at 70 μg/plate and 200 μg/plate concentrations in S. typhimurium TA1535, TA98 and TA1537 strains in the presence of metabolic activation system.


RANGE-FINDING/SCREENING STUDIES: Increase in the number of colonies compared to the negative control (vehicle) in S. typhimurium TA1535, TA98 and TA1537 strains in the absence of metabolic activation system, and in S. typhimurium TA98 and TA1537 strains in the presence of metabolic activation system. To confirm the results of these strains, the verification test of colony was conducted using sixteen randomly selected colonies per plate that were removed and streaked onto minimal glucose agar plates supplemented with biotin but without histidine or the test item. No growth was observed at 500 to 2000 μg/plate concentrations in S. typhimurium TA1535 and TA1537 strains confirming that the colonies were not revertants, but growth was observed at 500 μg/plate concentration and at 500 to 2000 μg/plate concentrations in S. typhimurium TA98 strain in the absence and presence of metabolic activation system, respectively. However, the criterion for considering test item as positive (≥ two times for S. typhimurium TA98 strain) was not met in S. typhimurium TA98 strain regardless of metabolic activation system application. Antibacterial effect (toxicity) defined as a diminution of background lawn, formation of microcolonies was observed at ≥500
μg/plate concentrations in all strains in the absence of metabolic activation system, and at ≥500 μg/plate concentrations in S. typhimurium TA98 and TA1537 strains and at ≥750 μg/plate concentrations in S.
typhimurium TA100, TA1535 and E. coli WP2uvrA strains in the presence of metabolic activation system. Antibacterial effect (toxicity) defined as the ≥50% reduction in the number of revertants relative to the negative control (vehicle) values were also observed at ≥500 μg/plate concentrations in S. typhimurium TA100 strain in the absence of metabolic activation system, and at 500, 750 and 2000 μg/plate concentrations in S. typhimurium TA100 and TA1535 strains in the presence of metabolic activation system. In addition, turbidity of test item was observed at ≥500 μg/plate concentrations, and precipitation of test item was observed at ≥100 μg/plate concentrations in all strains when the test item solutions were mixed with top agar. After 48 hours incubation, precipitation of test item was observed at ≥100 μg/plate concentrations in all strains regardless of the metabolic activation system application. In particular, precipitation of test item was observed at 50 μg/plate concentration in S. typhimurium TA100 strain in the absence of metabolic activation system.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The activity of the metabolic activation system was verified using 2-AA and BP, both of which produced significant mutagenicity in the presence of metabolic activation system. In the absence of metabolic activation system, 2-AA and BP produced no mutagenicity in the S. typhimurium TA1535 or TA98 stains. The number of revertant colonies in the negative control (vehicle) group was within the range of historical control data. The positive control groups showed significant increases (≥ two times) in mutagen-induced revertant colonies compared to that of the concurrent negative control (vehicle) group. These data indicated that the present study was performed under acceptable experimental conditions and the results were valid.
- Signs of toxicity: In both experiment, antibacterial effect (toxicity) defined as a diminution of background lawn, formation of microcolonies and/or the ≥50% reduction in the mean number of revertants per plate relative to the mean negative control (vehicle) values was observed at the highest concentration (200 μg/plate) in some strains in the absence and presence of metabolic activation system.
- There was no increase in the number of revertant colonies compared to the negative control (vehicle) at any concentrations of YH25448 in the absence and presence of metabolic activation system in any strain tested in the first experiment. Therefore, to confirm the results of the first experiment, the second experiment was performed by changing the type of S9 fraction of metabolic activation system. The results of S. typhimurium TA1535 strain were rejected according to the criteria for assay acceptance in the first experiment, since the contamination was observed at all plates. So, the retest for S. typhimurium TA1535 strain was additionally carried out when performing the second experiment. The number of revertant colonies was not increased at any concentrations of YH25448 in the absence and presence of metabolic activation system in any strain tested in the second experiment.
- In case of S. typhimurium TA98 strain, there was a slight discrepancy between the results of DRF study and the definitive study. The primary difference between DRF and definitive studies is the preparation method of 10-fold stock solutions of each target concentration for treatment. Since the concentration of the highest stock solution in the definitive study was 10 times lower than in the DRF study, the stock solution was clearly dissolved by using the unaided eye. The results of the DRF study was obtained with only one plate per each concentration and did not meet the criteria for considering test item as positive (≥ two times
for S. typhimurium TA98 strain). In addition, the negative results were obtained from two independent experimental conditions of the definitive study. Therefore, it is considered that a biologically relevant increase in the number of revertant colonies was not observed following treatment with YH25448 at any concentrations in the absence and presence of metabolic activation system.

Applicant's summary and conclusion

Conclusions:

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Study of YH25448 in the Salmonella typhimurium/Escherichia coli Plate Incorporation Assay indicated that, under the conditions of this study, YH25448 did not cause a positive response up to 200 μg/plate in the absence and presence of Aroclor 1254-induced rat liver S9 homogenate. The second experiment with human liver S9 did not show mutagenic potential in all tester strains in the absence and presence of metabolic activation system.