3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

substrain K3

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from rats induced with phenobarbital i.p. and β-naphthoflavone orally

Test concentrations with justification for top dose:

250 - 2000 µg/mL in all experiments (2000 µg = ca. 11.4 mM, limit dose)

Vehicle / solvent:

DMSO, 1%

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without metabolic activation: 300 mg EMS/mL; with metabolic activation: 10 µg MCA/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Attachment period: 20 - 24 hours
- Exposure duration: 4 hours both with and without metabolic activation and 24 hours without metabolic activation (see “Experimental setup” below)
- Expression time (cells in growth medium): about 7-9 days
- Selection time (if incubation with a selection agent): about 6-7 week
- Fixation: with methanol

SELECTION AGENT: 6-thioguanine
STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
Cloning efficiency 1 (CE1; survival)
For the determination of the influence of the test substance directly after the exposure period, per dose group about 200 cells were seeded in 25 cm² flasks in duplicate using 5 mL Ham's F12 medium incl. 10% (v/v) FCS. After an attachment period of 20 – 24 hours, the cells were treated with the vehicle, test substance or positive control for 4 hours or 24 hours. The exposure periods were completed by rinsing several times with HBSS. Then the flasks were topped up with 5 mL Ham's F12 medium incl. 10% (v/v) FCS.
Cloning efficiency 2 (CE2; viability)
The mutation rate after the expression period was determined in parallel to the selection of mutants. For each dose group about 200 cells were taken in duplicate, seeded in 25 cm2 flasks using 5 mL Ham's F12 medium incl. 10% (v/v) FCS.
In all cases, after seeding of the cells the flasks were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted. The absolute and relative cloning efficiencies (%) were calculated for each test group according to the formulas given below.

Cytotoxicity (CE, CE1, CE2)
The cloning efficiency (CE, %) was calculated for each test group as follows:
CEabsolute = total number of colonies in the test group/ total number of seeded cells in the test group x 100
CErelative = CEabsolute of the test group/ CEabsolute of the vehicle/negative control x 100
The number of colonies in every flask was counted and recorded. Using the formula above the values of absolute cloning efficiencies (CEabsolute, CE1 absolute and/or CE2 absolute) were calculated. Based on these values the relative cloning efficiencies (CErelative, CE1 relative and/or CE2 relative) of the test groups were calculated and given in percentage compared with the respective CEabsolute value of the corresponding vehicle/negative control (vehicle/negative control = 100%).

Mutant frequency
The number of colonies in every flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized to 10e6 cells seeded.
The uncorrected mutant frequency (MFuncorr.) per 10e6 cells was calculated for each test group as follows:
MFuncorr. = total number of mutant colonies/ number of seeded cells x 10e6
The uncorrected mutant frequency was corrected with the absolute cloning efficiency 2 for each test group to get the corrected mutant frequency (MFcorr.):
MFcorr. = MFuncorr./ CE2 absolute x 100

EXPERIMENTAL SETUP
1st Experiment
without S9 mix (4-hour exposure period)
0; 250; 500; 1 000; 2 000 μg/mL
with S9 mix (4-hour exposure period)
0; 250; 500; 1 000; 2 000 μg/mL
2nd Experiment
without S9 mix (24-hour exposure period)
0; 250; 500; 1 000; 2 000 μg/mL
with S9 mix (4-hour exposure period)
0; 250; 500; 1 000; 2 000 μg/mL

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data.
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.

Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10e6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

Due to the clearly negative findings, a statistical evaluation was not carried out.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of results                                                                        

Exp.	Exposure period	Test groups	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10e6 cells]	Cytotoxicity***
						CE1 [%]	CE2 [%]
1	4 hrs
	Vehicle	control1	-	-	2.83	100	100
	250	μg/mL	-	-	3.12	94.5	99.6
	500	μg/mL	-	-	4.39	92.2	105
	1000	μg/mL	-	-	0.64	101.7	101.1
	2000	μg/mL	-	-	2.85	95.5	96.8
	Positive	control2	-	-	77.13	96.3	80.7
2	24 hrs
	Vehicle	control1	-	-	2.82	100	100
	250	μg/mL	-	-	3.3	100.5	110.6
	500	μg/mL	-	-	1.52	98.4	111.3
	1000	μg/mL	-	-	6.71	98.4	122.6
	2000	μg/mL	-	-	0.89	66.2	122.3
	Positive	control2	-	-	367.17	80.6	82.9
1	4 hrs
	Vehicle	control1	+	-	6.44	100	100
	250	μg/mL	+	-	2.63	92.5	97.8
	500	μg/mL	+	-	1.76	103.7	92.1
	1000	μg/mL	+	-	2.27	109.8	99.4
	2000	μg/mL	+	-	2.34	111.6	95.1
	Positive	control3	+	-	75.49	94.6	87
2	4 hrs
	Vehicle	control1	+	-	1.94	100	100
	250	μg/mL	+	-	4.92	98.2	86.8
	500	μg/mL	+	-	1.51	104.2	102
	1000	μg/mL	+	-	1.73	104.4	104.7
	2000	μg/mL	+	-	4.51	101.4	99.7
	Positive	control3	+	-	47.14	101.1	100.3

* Precipitation in culture medium at the end of exposure period            

** Mutant frequency MFcorr.: number of mutant colonies per 106 cells corrected with the CE2 value              

*** Cloning efficiency related to the respective vehicle control             

1 DMSO 1% (v/v)                 

2300 μg/mL                 

3 MCA 10 μg/mL