2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

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:

11 February to 14 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 471 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

EC Number:

815-961-9

Cas Number:

1374760-95-8

Molecular formula:

C17H17N3O2S

IUPAC Name:

2-(4-methylphenoxy)-N-(1H-pyrazol-3-yl)-N-[(thiophen-2-yl)methyl]acetamide

Test material form:

solid: particulate/powder

Details on test material:

- Physical state : White powder
- Storage condition of test material: Dry place, protected form light, (15 to 30°C)

Method

Target gene:

Histidine and tryptophan for S. typhimurium and E. coli, respectively

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 applicable

Metabolic activation:

with and without

Metabolic activation system:

10% S9: S9-mix from the livers of male rats treated with 0.1 M NaH2PO4 / Na2HPO4, pH 7.4; 5 mM Glucose-6-phosphate; 4 mM NADP; 33 mM KCl; 8 mM MgCl2

Test concentrations with justification for top dose:

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 63, 130, 250, 500, 1000 μg/plate (solubility limit), with and without S9-mix
Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 63, 130, 250, 500, 1000 μg/plate (solubility limit), with and without S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: In solubility checks performed in house the test item was noted as immiscible in sterile distilled water at 50 mg/mL but fully miscile in DMSO at the same concentration. DMSO was therefore selected as the vehicle.
- Preparation of test formulation: The test item was dissolved in DMSO at 10.0 mg/mL and serially diluted to 5.0, 2.5, 1.3 and 0.63 mg/mL. All preparations were vortexed immediately before diluting.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

Without S9-mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

cyclophosphamide

other: 2-aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM
- Bacteria were purchased as dried discs from Molecular Toxicology Inc., Boone, NC, U.S.A. and stored in a refrigerator between 2 to 8°C. Frozen permanent stocks were prepared from fresh cultures of the discs and stored at -80°C ± 10°C in the presence of 9 % dimethyl sulfoxide (DMSO)

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Exposure duration: Plates were incubated at 37 °C ± 3 °C for 48 to 72 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were examinated via the naked eye and with the aid of a microscope for evidence of thinning (toxicity).

OTHERS:
After incubation, the plates were counted. The plates were also examined for the health status of the bacterial background lawn and possible precipitates of the test article at the end of the incubation period.

Rationale for test conditions:

Maximum concentration was 1000 μg/plate. Slight precipitate was visible with the naked eye only at 1.0 mg per plate. Therefore at this concentration, the test article was evaluated at the limit of solubility in the test system

Evaluation criteria:

Positive result will be considered positive when there is a significant increase in the number of colonies per plate in comparison to the concurrent negative control and a concentration-related increase over the exposure range tested. For the cases that there is historical data available, a positive result has to present an increase in the number of revertant colonies per plate in comparison to the historical data for at least one experimental condition. Biological relevance of the results will be considered first. A statistical method may be used as an aid in evaluating the test results but it will not be the only determining factor. A positive result indicates that the test article induces point mutations in S. typhimurium or E. coli.

Negative result (no evidence of genotoxicity) is concluded if there is no substantial increase in the number of colonies per plate; i.e. the results do not exceed the upper 98 percentile limit of the historical solvent/negative control range. A negative result indicates that the test article is non-mutagenic in S. typhimurium or E. coli.

Equivocal result: If no definite judgment can be made to fit the above criteria, even after repeated experiments, then the result will be described as equivocal. An equivocal result indicates that a definitive result cannot be made by performing the bacterial reverse mutation assay under the conditions described in this protocol.

Statistics:

Statistical analysis was applied to the numbers suspected to be abnormally high or to have a dose-related increase in revertant counts. The colony counts were transformed (square root) to normalize the data prior to using the one-sided Dunnett’s test (Mahon, G.A.T., et al., 1989).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

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:

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitate was observed with the naked eye starting at 250 µg/plate and increasing in a dose-dependent manner to a level of moderate precipitate at 1000 µg/plate. Precipitate was
not observed for any of the lower concentrations.
- Other confounding effects: None

HISTORICAL CONTROL DATA
All of the spontaneous reversion controls were within the acceptable range described in the protocol for spontaneous reversion. All the concurrent positive controls for each tester strain exceeded the minimum counts of the historical positive control data indicating that the sensitivity of the experiments was at historical levels.

MUTATION TEST
There were no statistically significant (p>0.01) increases in colony counts over the concurrent negative controls. The more sensitive preincubation test was performed to confirm the results of the plate incorporation
test as negative. There were five concentrations available for evaluation for mutagenicity for all conditions (OECD, 1997). There were two slight but statistically significant (p<0.01) increases in colony counts over the concurrent negative controls (TA1535 without S9 at 0.13 and 1.0 mg/plate). The results were within the Nucro-Technics historical data range (min-max) and a dose-response was not observed for this condition. Therefore the preincubation test results were concluded as clear negative which also confirmed the plate incorporation test results as negative.

OTHERS
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Under the test condition, test item is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- with or without metabolic activation.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test item diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the Ames plate incorporation and pre‑incubation methods in Experiment 1 and Experiment 2, respectively.

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method :

63, 130, 250, 500, 1000 μg/plate, with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method :

63, 130, 250, 500, 1000 μg/plate, with and without S9-mix

Negative, vehicle (DMSO) and positive control groups were also included in mutagenicity tests.

At the end of the incubation period in the plate incorporation experiment, slight to moderate precipitate was visible between 0.25 and 1.0 mg per plate with the naked eye. Toxicity was not observed at any concentration evident by a normal background lawn and colony counts similar to the concurrent negative controls. Therefore, the test article was evaluated at the limit of solubility in the test system. For all strains and conditions, all 5 concentrations were analyzable for mutagenicity. The test item did not produced any statistically significant increases (p>0.01) in colony counts over the concurrent negative controls, with or without metabolic activation. A more sensitive preincubation test was designed to confirm the negative results of the plate incorporation test.

In the preincubation experiment, both in the presence and absence of S9, the concentrations of test item investigated were identical to the plate incorporation test. At the end of the incubation period, slight precipitate was visible with the naked eye only at 1.0 mg per plate. Therefore at this concentration, the test article was evaluated at the limit of solubility in the test system. Toxicity was similar to the plate incorporation test when compared to the concurrent negative controls with one exception. For TA1537 without S9, the colony counts were slightly reduced at the highest concentration of 1.0 mg/plate. Also, the background lawns were slightly reduced at the highest concentration of 1.0 mg/plate with and without S9. Despite this toxicity, all 5 concentrations were analyzable for mutagenicity. Therefore for these conditions, the test item was evaluated at the limit of test article toxicity. In the preincubation test with TA1535 without metabolic activation, there were two slight (1.25 and 1.44-fold), but statistically significant increases (p<0.01) in colony counts at 0.13 and 1.0 mg/plate over the concurrent negative control. Despite these increases, the results were within the historical control data range (min-max) and a dose-response was not observed. Therefore the preincubation test confirmed the negative results of the plate incorporation test.

 

For most strains and concentrations, the background lawn and the number of colony counts were observed to be normal when compared to the negative control with a few exceptions. For TA1537 with and without S9 at 1.0 mg/plate, the background lawn was slightly reduced when compared to the concurrent negative controls. Also, an obvious reduction in colony counts was observed for TA1537 without S9 at 1.0 mg/plate.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

Under the test condition, test item is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- with or without metabolic activation.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.