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Toxicological information

Genetic toxicity: in vitro

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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
Study period:
2013-01-29 - 2013-08-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Name N-((3(5)-Methyl-1H-pyrazol-1-yl)methyl)acetamide (MPA)
Batch No. P70/05/S-6
Characteristics Powder
Content 96.2%

Method

Target gene:
X-chromosome, hprt locus
Species / strain
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)
Controls
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.

Results and discussion

Test results
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'.

Applicant's summary and conclusion

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.