Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test: negative

Chromosome aberration assay: negative

HPRT test: negative

Ames-Test:

N-(3(5)-Methyl-1H-pyrazol-l-yl-methyl)-acetamid (P 70/05) was tested for a possible potential to induce gene mutation in bacteria according to OECD guideline 471.

As test organisms the Salmonella typhimuriumstrains TA 98, TA 100, TA 1535, TA 1537 and the Escherichia colistrain WP2uvrA were used. The bacteria were exposed to N-(3(5)- Methyl-1H-pyrazol-l-yl-methyI)-acetamid (P 70/05) both, in the presence and absence of rat liver S9 metabolic activation.

No biologically relevant increases in revertant numbers were obtained after treatment with N-(3(5)-Methyl-1H-pyrazol-l-yl-methyl)-acetamid (P 70/05) in any bacterial strain and at any concentration tested. This applies to the presence and absence of metabolic activation and as well as the plate incorporation test and the pre-incubation test.

Cytotoxicity was not observed at any concentration.

Therefore, N-(3(5)-Methyl-1H-pyrazol-l-yl-methyI)-acetamid (P 70/05) is considered to be non-mutagenic in the bacterial reverse mutation test.

 

Chromosome aberration assay:

N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA) tested up to a concentration of 5000 µg/mL medium (cytotoxic without S9 at a 24-hour exposure), in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage. In the same test, Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.

 

HPRT-Test:

Under the test conditions, N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA) tested up to cytotoxic concentrations in the experiments without and with metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

 

Justification for selection of genetic toxicity endpoint

Conclusion based on the following assays: Bacterial reverse mutation assay (Ames test); chromosome aberration assay, HPRT assay in mammalian cells

Link to relevant study records

Referenceopen allclose all

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
Study period:
2013-01-29 - 2013-08-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
X-chromosome, hprt locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 -induced rat S9 fraction
Test concentrations with justification for top dose:
Preliminary study : 2.5, 10, 25, 100, 250, 1000 and 2500 µg/mL
Main study: without metabolic activation: 78.13, 156.3, 312.5, 625 or 1250 µg/ N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/mL
with metabolic activation: 156.3, 312.5, 625, 1250 or 2500 µg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/mL
Vehicle / solvent:
dimethyl sulfoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Cytotoxicity experiment:
The concentrations employed were chosen based on the results of a cytotoxicity study with concentrations of 2.5, 10, 25, 100, 250, 1000 and 2500 µg/mL. In this preliminary study pronounced cytotoxicity in form of decreased plating efficiency was noted starting at concentrations of 1000 or 2500 µg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA) per mL medium in the experiment without and with metabolic activation (24-h or 4-h exposure, respectively).
Hence, a concentration of 2500 µg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/mL was employed as the top concentration for the main mutagenicity test without and with metabolic activation with a 4 h exposure and 1250 µg/mL in the second experiment without metabolic activation with a 24-h exposure.

Main study:
On day 1 of the experiment, approximately 1500000 cells were placed in 30 mL DMEM-FCS per 150 mm diameter dish. On the following day, the cells were exposed to the selected concentrations of the test item. In the absence of S9 mix, the cells were exposed in DMEM-FCS to the test item for 4 hours (1st experiment) or 24 hours (2nd experiment).
In the experiments with S9 mix, the medium was replaced by 18 mL S9 mix and the exposure limited to 4 hours. The vehicle control was treated with dimethylsulfoxide (DMSO) (the vehicle) in the same way. In addition, positive controls were employed.

Afterwards the cells were trypsinised and a relative plating efficiency (PE1) was determined for each dose to obtain an accurate measure of the toxic effect of the chemical. Three replicate plates (60 mm diameter) were used with a known number of cells. The remaining cells were replated and the culture incubation continued until day 8 with 30 mL normal DMEM-FCS with one subcultivation on day 4 or 5. This period was required for expression of the new genotype, i.e. for sufficient dilution and catabolism of the previously formed hypoxanthine guanine phosphoribosyl transferase. Afterwards the cells were harvested by trypsinisation and replated at a density of 1000000 cells per 150 mm diameter dish in DMEM-FCS containing 6-thioguanine (10 µg/mL) for selection of mutants (5 replicate plates), or at approx. 100 to 150 cells (exact number known) per 60 mm diameter dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE2), (3 replicate plates). The plates were fixed and stained after about 8 days (plating efficiency plates) or about 12 days (6-thioguanine plates).


Evaluation criteria:
So far no satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments such as those performed here (see UKEMS guidelines for discussion). Experience has shown that the following predetermined descriptive criteria are the most useful for interpretation of the results:
- If in both independent experiments solvent and positive controls show results within the norm and if the test item does not increase the mutation frequency 2-fold above the mean of the solvent controls under any condition, or if the mutation frequency is always lower than 40 x 10-6 and if at least 1000000 cells per condition have been evaluated, the item is considered as negative in the test.
- In case of a dose-dependent increase of the mutation frequency in both independent experiments (at similar concentrations) to at least 2-fold solvent control and at least 40 x 10-6 both in the presence and/or absence of S9 mix, the item is considered as positive in the test.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative

Under the test conditions, N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA) tested up to cytotoxic concentrations in the experiments without and with metabolic activation was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.
Executive summary:

N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA) was tested for mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) according to OECD guideline 476 both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced animals. The duration of the exposure with the test item was 4 hours or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix.

The concentrations employed were chosen based on the results of a cytotoxicity study with concentrations of 2.5, 10, 25, 100, 250, 1000 and 2500 µg/mL.In this preliminary study pronounced cytotoxicityin form of decreased plating efficiency was noted starting at concentrations of 1000 or 2500 µg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA) per mL medium in the experiment without and with metabolic activation (24-h or 4-h exposure, respectively).

Hence, a concentration of 2500 µg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/mL was employed as the top concentration for the main mutagenicity test without and with metabolic activation with a 4‑h exposure and 1250 µg/mL in the second experiment without metabolic activation with a 24-h exposure.

 

Main study:

Five concentrations ranging from 156.3 to 2500 µgN-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/mL were selected for the experiments without and with metabolic activationwith a 4‑h exposure,andrangingfrom 78.13 to 1250 µg/mL in the second experiment without metabolic activation with a 24-h exposure.

 

Cytotoxicity:

In the main study, cytotoxicityin form of decreased plating efficiency (PE1) wasnoted in the experiments without and with metabolic activation at the top concentration of 2500µgIPETC (DANAFLOAT™ 262)/mL medium (4‑h exposure) or at 1250 µg/mL in the second experiment without metabolic activation with a 24-h exposure.

 

Experiments without metabolic activation:

The mutation frequency of the vehicle controldimethylsulfoxide (DMSO) was 20.84and 17.17 x 10-6cloneable cells. Hence, the vehicle controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 156.3, 312.5,625, 1250 or 2500 µg/mL or 78.13, 156.3,312.5,625 or 1250 µg/ N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/mL culture medium ranged from 10.67 to 20.00 x 10‑6cloneable cells. These results are within the normal range of the vehicle controls.

Experiments with metabolic activation:

The mutation frequency of the vehicle controldimethylsulfoxide (DMSO) was 25.54 and 17.14 x 10-6cloneable cells. Hence, the vehicle controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 156.3,312.5,625, 1250 or 2500 µg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/mL culture medium ranged from11.90 to 20.28x 10‑6cloneable cells. These results are within the normal range of the vehicle controls.

The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 706.67 to 1545.71 10-6cloneable cells in the case of EMS and ranging from 707.83 to 1610.67 x 10-6cloneable cells in the case of DMBA, indicating the validity of this test system.

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:
2007-05-02 - 2007-07-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
All used bacterial strains are defective in DNA-repair (AuvrB and AuvrA respectively). The Salmonella strains also have a defective lipopolysaccharide barrier on the cell wall (rfa). These properties confer extra sensitivity to DNA damage and also greater permeability to large molecules. The strains TA 98 and TA 100 also contain a plasmid (pKMlOl) which enhances the error prone repair and confers ampicillin resistance.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
DNA polymerase A deficient
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: defective lipopolysaccharide barrier on the cell wall (rfa)
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
According to the standard plate incorporation method at concentrations of 5000, 1000, 500, 100, 50, 10 and 5 μg/plate and according to the preincubation method at concentrations of 5000, 1000, 500, 100, 50 and 10 μg/plate.
Vehicle / solvent:
dimethyl sulfoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Bacterial

tester

strain

MA

Exp.

Mean numbers of revertants per plate

Evaluation

Controls

μg test item per plate

UTC

VC

PC

5000

1000

500

100

50

10

5

Salmonella

typhimurium

TA1535

no

1st

15.0

19.3

2309.3

21.0

20.0

27.3

19.7

13.7

16.3

22.7

-

no

2nd

11.0

14.0

2149.3

13.3

12.3

13.0

11.7

13.0

11.0

n.e.

-

yes

2st

14.0

14.7

832.0

13.3

14.3

14.0

15.0

19.3

14.7

20.0

-

yes

2nd

16.0

17.3

904.0

22.3

11.3

14.0

13.7

18.0

19.3

n.e.

 

TA1537

no

1st

14.0

13.0

4672.0

10.3

12.3

14.0

13.0

10.0

10.3

14.7

-

no

2nd

16.0

17.3

982.0

16.3

17.0

15.0

17.7

12.3

13.3

n.e.

-

yes

1st

10.0

10.3

173.7

10.3

9.0

6.7

11.7

10.7

9.3

12.3

_

yes

2nd

25.7

19.3

313.3

24.7

17.0

18.0

18.7

14.7

19.0

n.e.

 

TA98

no

1st

24.0

29.7

952.0

32.0

23.7

23.7

28.0

20.7

25.0

31.3

-

no

2nd

26.7

31.7

784.7

28.3

26.0

34.0

26.0

22.0

25.3

n.e.

-

yes

1st

23.7

30.7

3133.3

23.7

25.0

23.7

27.7

22.3

27.7

27.0

-

yes

2nd

39.7

31.3

2821.3

29.3

36.3

37.3

37.7

41.3

37.3

n.e.

-

TA100

no

1st

228.0

223.0

2282.7

253.7

233.0

225.0

230.0

208.3

224.7

240.3

-

no

2nd

201.7

182.3

1528.0

171.0

181.0

196.0

179.7

199.0

193.3

n.e.

-

yes

1st

230.3

244.0

5730.7

205.7

215.7

239.7

245.0

208.5

203.0

209.7

-

yes

2nd

235.7

186.3

5648.0

214.0

201.0

192.7

205.7

225.0

181.7

n.e.

-

Escherichiacoli

WP2 uvrA

no

1st

21.7

20.0

225.0

19.0

21.7

25.0

24.0

22.0

20.7

23.7

-

no

2nd

26.0

22.7

729.3

28.0

29.7

24.7

24.7

32.3

31.0

n.e.

_

yes

1st

29.7

39.3

621.7

32.7

33.7

38.0

35.7

40.7

34.7

31.7

-

yes

2nd

36.7

34.7

576.0

37.0

31.7

29.7

44.7

46.7

29.3

n.e.

-

Conclusions:
Interpretation of results
negative

Based on the results of the reported study it is concluded that N-(3(5)-Methyl-1H-pyrazol-1- yl-methyl)-acetamid (P 70/05) does not induce gene mutation in Salmonella typhimurium and Escherichia coli under the experimental conditions described.
Executive summary:

N-(3(5)-Methyl-1H-pyrazol-l-yl-methyl)-acetamid (P 70/05) was tested for a possible potential to induce gene mutation in bacteria according to OECD guideline 471.

As test organisms the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and the Escherichia colistrain WP2uvrA were used. The bacteria were exposed to N-(3(5)- Methyl-1H-pyrazol-l-yl-methyI)-acetamid (P 70/05) both, in the presence and absence of rat liver S9 metabolic activation. Two independent experiments were performed according to the standard plate incorporation method (experiment I) or the pre-incubation method (experiment II), respectively.

The test item was tested according to the standard plate incorporation method at concentrations of 5000, 1000, 500, 100, 50, 10 and 5 µg/plate and according to the pre­incubation method at concentrations of 5000, 1000, 500, 100, 50 and 10 µg/plate in the experiments without and with metabolic activation in all five bacterial strains.

An untreated, a vehicle control and appropriate positive controls were included into the experimental design. Triplicate plates were scored for each experimental point.

No biologically relevant increases in revertant numbers were obtained after treatment with N-(3(5)-Methyl-1H-pyrazol-l-yl-methyl)-acetamid (P 70/05) in any bacterial strain and at any concentration tested. This applies to the presence and absence of metabolic activation and as well as the plate incorporation test and the pre-incubation test.

The revertant frequencies of the negative controls were within the expected range and the positive control chemicals induced marked increases in revertant colonies.

Cytotoxicity was not observed at any concentration.

Based on the results of the reported study it is concluded that N-(3(5)-Methyl-1H-pyrazol-l- yl-methyl)-acetamid (P 70/05) does not induce gene mutation inSalmonella typhimurium and Escherichia coli under the experimental conditions described.

Therefore, N-(3(5)-Methyl-1H-pyrazol-l-yl-methyI)-acetamid (P 70/05) is considered to be non-mutagenic in the bacterial reverse mutation test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-01-29 - 2013-06-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human peripheral lymphocyte
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat S9 fraction
Test concentrations with justification for top dose:
Preliminary cytotoxicity study: 10, 25, 100, 250, 1000, 2500 and 5000 µg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA) per mL medium
Main Study: 5000 µg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA) per mL medium
Vehicle / solvent:
dimethyl sulfoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Culture establishment:
Human peripheral blood was obtained by venipuncture from healthy donors known to be without any medication and collected in heparinised vessels. Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of complete culture medium. The tubes were sealed and incubated at 37°C with occasional shaking to prevent clumping.

Preparation of the test item solution:
The test item was completely dissolved in dimethylsulfoxide (DMSO) . A correction factor of 1.04 was used as the content of the test item was 96.2% only.
In order to determine the exposure concentration of the test item in the mixture, one sample of 1 concentration level in the high range and one sample of 1 concentration level in the medium range of one independent experiment were retained and made available to the Sponsor for analysis.
In line with normal practice in this type of in vitro study, no analysis of the dose form is required.

Preliminary toxicity test / Exposure concentrations:
Concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 µg test item per mL medium were employed in an experiment without and with metabolic activation. Each treatment was tested in the absence and in the presence of S9 mix.
Samples (50 µL) of the test item solutions were added 48 hours after culture establishment. After the 4-hour exposure period the cultures were washed with fresh medium and incubated for further 4 or 24 hours at 37°C..
Cultures were harvested and 1 slide per culture was prepared. 1000 lymphocytes per culture were examined at a magnification of x 400; the mitotic index was calculated as the percentage of lymphocytes examined which were in mitosis (metaphase). Slides were coded before analysis.

Main study:
After 48 hours of culture in complete medium the tubes were centrifuged, and the cell pellet resuspended to 4.5 mL (for S9 mix addition) or 5.0 mL with treatment medium (see Appendix 1) including the test item at the final concentrations. Treatments have been added at a volume of 50 µL.
S9 mix (0.5 mL) was added to the appropriate cultures. During treatment the tubes were incubated for 4 hours in a shaking water bath at 37°C.
After this period, the tubes were centrifuged and the cells were washed with 5 mL treatment medium to remove the test item and S9 mix. After a further centrifugation the cell pellet was resuspended in 5 mL of complete medium and returned to the incubator for further 20 hours.
The incubation procedure took place in the dark.
In a second set of the experiment a continuous treatment of 24 hours without metabolic activation was carried out and the 4-hour treatment with metabolic activation was repeated.
Evaluation criteria:
Observed aberrations were noted and scored according to J. R. K. SAVAGE (1975). In addition, the total number of gaps was recorded in 100 metaphases for each culture, if possible.
Metaphases which differed from the normal diploid complement (46) were excluded from evaluation. However, test item-related variations of the normal chromosome number were noted (polyploidy / endoreduplication).
Metaphases were assigned to one of the following 5 categories:

1 normal metaphases
2 metaphases with 1 - 2 aberrations
3 metaphases with multiple aberrations
4 pulverised metaphases: extreme (≥ 50%) fragmentation of chromosomal material
5 polyploidy / endoreduplication

To examine the toxicity of the test item, 1000 cells were scored and the mitotic index was calculated as the percentage of cells in metaphase.
When all examinations had been completed, the slides were decoded and the results collated.

Statistics:
The assessment was carried out by a comparison of the number of chromosome aberrations of the samples with those of the solvent control, using the exact test of R. A. FISHER (p ≤ 0.05) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, Statistical evaluation of mutagenicity test data, 1989).
The test item is judged to have mutagenic properties with respect to chromosomal or chromatid change, if the following criteria are fulfilled:
o the number of chromosomal aberrations is significantly (at p  0.05) increased compared with the solvent control
o the increase observed is concentration-dependent
o both duplicate cultures lead to similar results
o the increase should not occur in the severely cytotoxic range (mitotic index <0.25), as it is known that high cytotoxicity causes artefacts in the form of aberrations in in vitro chromosomal aberration tests
o a reproducible increase in the number of cells with chromosomal aberrations.

Key result
Species / strain:
lymphocytes: human peripheral lymphocyte
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only at the top concentration of 5000 µg/mL medium
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative

N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA) tested up to a concentration of 5000 µg/mL medium (cytotoxic without S9 at a 24-hour exposure), in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.
In the same test, Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.
Executive summary:

Test samples ofN-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)were assayed in an in vitro cytogenetic study according to OECD guideline 473 using human lymphocyte cultures both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals.

The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The experiment with S9 mix was carried out twice. The harvesting time was 24 hours after starting of exposure. The incubation procedure took place in the dark. The study was conducted in duplicate.

The N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA) was completely dissolved in dimethyl sulfoxide (DMSO). A correction factor of 1.04 was used as the content of the test item was 96.2% only.The vehicle DMSO served as the negative control.

A preliminary cytotoxicity study was conducted to establish the top concentration for the main cytogenetic test.Concentrations of 10, 25, 100, 250, 1000, 2500 and 5000 µg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA) per mL medium were employed in an experiment without and with metabolic activation(24 h or 4‑h exposure).In this preliminary experiment no signs of cytotoxicity were noted in the experiment with metabolic activation (4-h exposure) up to the top concentration of 5000 µg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/mL. Pronounced cytotoxicity was noted in the experiment without metabolic activation (24-h exposure) at the top concentration of 5000 µg/mL medium.

Hence, the highest concentration employed in the main study was 5000 µg N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)/mL medium.

In the main study no signs of cytotoxicity were noted in the experiments with a 4-hour exposure without and with metabolic activation up to the top concentration of 5000 µg/mL medium. Pronounced to complete cytotoxicity was noted in the second experiment without metabolic activation (24-h exposure) at the top concentration of 5000 µg/mL medium.

Mitomycin C and cyclophosphamide were employed as positive controls in the absence and presence of metabolic activation, respectively.

Tests without metabolic activation (4- and 24-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with the N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA) at concentrations from 625 to 5000 or 2500 µg/mL medium (4 h or 24‑h exposure, respectively), in the absence of metabolic activation ranged from 1.0% to 3.5%.These results were within the range of the historical control data (0 - 4%).

The result for the vehicle control cultures was a mean of 1.0 or 0.5% cells with aberrations (excluding gaps), respectively, which is within the historical control range. The positive control cultures had a significantly increased frequency of cells with aberrations, which was in line with the historical control range. Therefore, the test is considered to be valid.

Test with metabolic activation(4-hour exposure)

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with the N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA) at concentrations from 625 to 5000 µg/mL medium in the presence of metabolic activation in the first and second experiment ranged from 1.0% to 3.5%. These results were within the range of the historical control data (0 - 4%).

The result for the vehicle control cultures was a mean of 1.0 or 0.5% cells with aberrations (excluding gaps), respectively, which is within the historical control range. The positive control cultures had a significantly increased frequency of cells with aberrations, which was in line with the historical control range. Therefore, the test is considered to be valid.

No test item-related polyploidy or endoreduplication were noted in the experiments without or with metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

According to the CLP Regulation (EU GHS Regulation (EC) No 1272/2008) classification and labelling is not required for genetic toxicity of N-(3(5)-Methyl-1 H-pyrazol-1 -yl-methyl)-acetamid, based on reliable data from 3 in-vitro tests.