1H-Pyrazole-3-carboxylic acid, 5-amino-1-[(2-fluorophenyl)methyl]-, ethyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 18-22, 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

non-GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

max. concentration only 2 mg/plate ; no triplicates but single plate per conc. ; no data on purity, solubility or stability of the test substance; limited scope of report

GLP compliance:

no

Remarks:

A non-GLP Study was conducted in the context of a pharmaceutical development program

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 5-amino-1-[(2-fluorophenyl)methyl]-1H-pyrazole-3-carboxylate

EC Number:

805-733-7

Cas Number:

256504-39-9

Molecular formula:

C13H14FN3 O2

IUPAC Name:

ethyl 5-amino-1-[(2-fluorophenyl)methyl]-1H-pyrazole-3-carboxylate

Test material form:

solid

Specific details on test material used for the study:

no analytical data of test compound described (purity, stability, solubility unknown)
fresh preparation of test compound in DMSO

Method

Target gene:

Histidine gene locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

0, 5, 10, 50, 100, 250, 500, 1000, 2000 µg/plate (Preincubation test, +/-S9 mix)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Remarks:

No solvent control was used since sufficient evidence was available in the literature and from testing laboratory experience, indicating that the solvents used had no influence on the spontaneous mutant counts of the used strains.

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (only TA 1535, TA 100), nitrofluorene (only TA 98), 4-nitro-1,2-phenylene diamine (TA 1537), cumene hydroperoxide (only TA 102), 2-aminoanthracene (all strains).

Remarks:

The positive controls sodium azide, nitrofluorene, 4-nitro-1,2-phenylene diamine and cumene were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix.

Details on test system and experimental conditions:

Due to limited availability of the test compound only one plate per concentraion was tested.

Evaluation criteria:

The test substance is classified as mutagenic if there is a concentration-related increase over the range tested and/or an increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system. For TA1535, TA1537, TA100 and TA98 this increase should be about twice that of negative controls, whereas for TA 102 an increase of less than 2-fold may also be judged a positive result.
In the evaluation of the test results, the magnitude of the effects, their reproducibility, bacteriotoxic effects of the test substance, the historical control data obtained in the laboratory and scientific plausibility are taken into consideration. Any positive test result should be evaluated for its biological relevance.

Statistics:

not specified

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to 2 mg/ plate

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

mutagenic potential (based on QSAR/QSPR prediction)

Any other information on results incl. tables

Table 1: Results from the Salmonella mutagenicity assay (preincubation) with Aminopyrazol (single revertant number per plate)

 

Dose (µg per plate)	Without metabolic activation
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	10	109	8	51	265
5	4	105	6	28	291
10	5	97	14	43	299
50	13	162	7	45	323
100	7	162	9	42	307
250	9	196	14	39	292
500	9	151	13	41	252
1000	8	178	7	37	251
2000	8	148	6	25	237
Positive control	622	1020	74	1044	717
Dose ( µg per plate )	With metabolic activation (liver S9 mix)
	TA 1535	TA 100	TA 1537	TA 98	TA 102
0	11	225	11	23	395
5	7	225	10	21	397
10	6	249	13	17	394
50	10	271	13	25	311
100	13	263	13	23	384
250	7	371	13	63	363
500	5	451	10	46	380
1000	10	517	13	24	369
2000	7	506	9	25	313
Positive control	118	3981	334	2798	1445

 

Applicant's summary and conclusion

Conclusions:

mutagenic potential observed in two bacterial strains with metabolic activation

Executive summary:

Viability and high cell density of the cultures of the individual bacterial strains were confirmed. The counts recorded on appropriate solvent control plates confirmed the characteristic spontaneous reversion rates of the tester strains. Furthermore, appropriate positive controls with known mutagens produced the expected numbers of revertant colonies. Precipitates in the agar were not found.

Doses up to and including 2000 μg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Evidence of mutagenic activity of Aminopyrazole was seen. A biologically relevant increase in the mutant count, in comparison with the negative controls, was observed for the Salmonella strains TA100 and TA98.