1-phenyl-3-pyrazolidone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES test

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

In vitro gene mutation assay in Mammalian cells

In vitro chromosome aberration test, Test material did not induce gene mutation in Chinese hamster lungs cells in the presence and absence of S9and hence is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data from handbook or collection of data

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

Weight of evidence prepared from various publication mention below
1,This study was performed to investigate the potential of test substance to induce gene muta¬tions in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.
2,To evaluate the mutagenic potential of test substance in bacteria.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other:

Species / strain / cell type:

S. typhimurium, other: TA 98 and TA 100

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9 metabolic activation system

Test concentrations with justification for top dose:

1,0.0 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate
2,3-5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RO water
- Justification for choice of solvent/vehicle: The test chemical was soluble in RO water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

RO water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.

Statistics:

No data

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No precipitation was noted at a dose upto 5 mg/plate in the pre-experiment
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 – 5.0 mg/plate based on the solubility and precipitation test. In TA 98 and TA 100, cyto-toxicity was observed in the treated concentrations 1.582 and 5 mg/plate (T7 to T8), moderate inhibition was observed in the treated concentrations 0.501 mg/plate (T6) and there was no reduction in colony count as well as background lawn in any of the following concentrations tested; 0.002, 0.005, 0.016, 0.050 and 0.158 ( T1 to T5) mg/plate both in absence and in the presence of metabolic activation, when compared to that of the negative control group. Based on the results of pre-experiment following doses were selected for the main study trials: 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Executive summary:

Data for the various publication was reviewed to determine the mutagenic nature of 1-phenyl-3-pyrazolidone (92-43-3). The studies are as mentioned below:

AMES test

Ames assay was performed to investigate the potential of test substance to induce gene muta­tions in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with Sodium 2-hydroxybenzoate (CAS No. 54-21-7) at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

Gene toxicity in vitro study was performed for the test compound using S typhimurium strain TA98 and TA100. Plate incorporation and preincubation assay was performed at dose levels of 3-5000 µg/plate. with and without S9 metabolic activation system. The given test substance failed to induce mutation in Salmonella typhimurium strains TA100 and TA98 with and without metabolic activation system S9 in the plate incorporation and preincubation method performed. The test material is therefore not likely to be classified as a gene toxicant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

data from handbook or collection of data

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

Weight of evidence prepared from various studies mention below
To evaluate the mutagenic potential of test chemical in Chinese hamster lung(CHL)cells by in vitro mammalian chromosome aberration test.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not specified

Species / strain / cell type:

other: Chinese hamster lung(CHL)cells

Details on mammalian cell type (if applicable):

CHL / IU cells derived from Chinese hamster lungs obtained from Dainippon Pharmaceutical Co., Ltd. (September 2003, passage: 14th generation,
frozen: 17th) were used for the test within 4 weeks after thawing succession

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

1,-S9 mix (6 hr short-term treatment); 100, 125, 150, 175, 200 μg/mL
+S9 mix (6 hr short-term treatment); 200, 225, 250, 275, 300 μg/mL
-S9 mix (24 hr continuous treatment; main test); 50, 75, 100, 125, 150 μg/mL
-S9 mix (24 hr continuous treatment; additional test); 100, 110, 120, 130, 140, 150 μg/mL
2,-S9 mix (6 hr short-term treatment); 100, 125, 150, 175, 200 μg/mL
+S9 mix (6 hr short-term treatment); 200, 225, 250, 275, 300 μg/mL
-S9 mix (24 hr continuous treatment; main test); 50, 75, 100, 125, 150 μg/mL
-S9 mix (24 hr continuous treatment; additional test); 100, 110, 120, 130, 140, 150 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Isotonic sodium chloride solution

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Isotonic sodium chloride solution

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: -S9 mix, Mitomycin C +S9 mix, Benzo [a] pyrene

Details on test system and experimental conditions:

Details on test system and conditions
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 3 days
- Exposure duration: short-time treatment method :6h
continuous treatment method:24h
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

NUMBER OF CELLS EVALUATED: 200 metaphases were scored per experimental group.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

Structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. Also analyzed 200 groups of metaphase cells per group for structural abnormalities and ploidy cells.

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

other: Chinese hamster lung (CHL/IU) cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: No mutagenic effect were observed

Conclusions:

In vitro chromosome aberration test, Test material did not induce gene mutation in Chinese hamster lungs cells in the presence and absence of S9and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

In vitro Mammalian cell gene mutation assay

In vitro chromosome aberration study was performed to determine the mutagenic nature of test material.SHE cells (5x105) in tertiary culture were plated into 75-cm2flasks, incubated overnight, and treatedat dose levels from 0, 0.8,3,8,30µMfor 24 h.The test chemical was dissolved in distilled, deionized water .SHE cells have the doubling time of 16 h. The cells were harvested with 0.1% trypsin for chromosome preparation. Three hours before harvest, Colcemid was administered at 0.2µg/ml and metaphase chromosomes were prepared. For determination of chromosome aberrations, 100 metaphases were scored per experimental group. Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, they were scored as breaks. Also, the chromosome aberration assay was carried out in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant (PMS), Cells (5x105) were plated on 100-mm dishes and after overnight incubation, they were treated with chemical agents for 3 h in a 5% PMS mixture.). Cells were washed twice with 5 ml PBS (-) and incubated with fresh medium for 18 h followed by chromosome preparations., Test material did not induce gene mutation inSyrian hamster embryo (SHE) cells inthe presence and absence ofmetabolic activation with rat liver post-mitochondrial supernatant (PMS)and hence is not likely to classify as a gene mutant in vitro.

In vitro chromosome aberration study was performed to determine the mutagenic nature of test material. CHL / IU cells derived from Chinese hamster lungs. 2 × 10 4 CHL / IU cells were seeded in a dish (6 cm in diameter) containing 5 mL of the culture solution and cultured in a 37 ° C. CO 2 incubator (5% CO 2) for 3 days. After the preculture, in the short-time treatment method, the test substance was treated for 6 hours in the presence and absence of S9 mix and then cultured for 18 hours in fresh culture medium. In the continuous treatment method, the test substance was continuously treated for 24 hours. The test substance was dissolved in a Physiological saline to prepare a stock solution, and then the stock solution was serially diluted with a solvent to prepare a test substance preparation solution of a 0,100,125,150,200 µg/mL concentration without S9 and 2,200,225,250,275,300 µg/mL with S9 in the short-time treatment method while 0,50,75,100,125,150 µg/mL concentration in In the continuous treatment method were used . The test substance preparation solution was added so as to be 10 % Volume of the culture solution in all tests. As a positive control group, benzo [a] pyrene was added at a concentration of 20 μg / mL in the presence of S 9 mix in a short time treatment method, and mitomycin C at a concentration of 0.1 μg / mL, and in continuous treatment, mitomycin C was set at a concentration of 0.05 μg / mL. Chromosome analysis based on structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy. The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. Also analyzed 200 groups of metaphase cells per group for structural abnormalities and ploidy cells. The appearance frequency of chromosomal structural abnormalities and polyploid cells was less than 5% in both the short treatment method S9 mix presence and in the absence of S9 mix and in the treatment group treated with continuous treatment method 24 hours. Hence test material did not induce gene mutation inChinese hamster lungs cells inthe presence and absence of S9and hence is not likely to classify as a gene mutant in vitro

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data for the various publication was reviewed to determine the mutagenic nature of 1-phenyl-3-pyrazolidone (92-43-3). The studies are as mentioned below:

AMES test

Ames assay was performed to investigate the potential of test substance to induce gene muta­tions in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with Sodium 2-hydroxybenzoate (CAS No. 54-21-7) at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

Gene toxicity in vitro study was performed for the test compound using S typhimurium strain TA98 and TA100. Plate incorporation and preincubation assay was performed at dose levels of 3-5000 µg/plate. with and without S9 metabolic activation system. The given test substance failed to induce mutation in Salmonella typhimurium strains TA100 and TA98 with and without metabolic activation system S9 in the plate incorporation and preincubation method performed. The test material is therefore not likely to be classified as a gene toxicant in vitro.

In vitro gene mutation assay in Mammalian cells

In vitro chromosome aberration study was performed to determine the mutagenic nature of test material. CHL / IU cells derived from Chinese hamster lungs. 2 × 10 4 CHL / IU cells were seeded in a dish (6 cm in diameter) containing 5 mL of the culture solution and cultured in a 37 ° C. CO 2 incubator (5% CO 2) for 3 days. After the preculture, in the short-time treatment method, the test substance was treated for 6 hours in the presence and absence of S9 mix and then cultured for 18 hours in fresh culture medium. In the continuous treatment method, the test substance was continuously treated for 24 hours. The test substance was dissolved in a Physiological saline to prepare a stock solution, and then the stock solution was serially diluted with a solvent to prepare a test substance preparation solution of a 0,100,125,150,200 µg/mL concentration without S9 and 2,200,225,250,275,300 µg/mL with S9 in the short-time treatment method while 0,50,75,100,125,150 µg/mL concentration in In the continuous treatment method were used . The test substance preparation solution was added so as to be 10 % Volume of the culture solution in all tests. As a positive control group, benzo [a] pyrene was added at a concentration of 20 μg / mL in the presence of S 9 mix in a short time treatment method, and mitomycin C at a concentration of 0.1 μg / mL, and in continuous treatment, mitomycin C was set at a concentration of 0.05 μg / mL. Chromosome analysis based on structural abnormalities such as breakage and exchange of chromosome type or chromosome type, presence and absence of gap, and ploidy. The presence or absence of cells (polyploid) was observed. The gap was not included in structural abnormality. Also analyzed 200 groups of metaphase cells per group for structural abnormalities and ploidy cells. The appearance frequency of chromosomal structural abnormalities and polyploid cells was less than 5% in both the short treatment method S9 mix presence and in the absence of S9 mix and in the treatment group treated with continuous treatment method 24 hours. Hence test material did not induce gene mutation in Chinese hamster lungs cells inthe presence and absence of S9and hence is not likely to classify as a gene mutant in vitro.

In vitro chromosome aberration study was performed to determine the mutagenic nature of test material. SHE cells (5x105) in tertiary culture were plated into 75-cm2flasks, incubated overnight, and treatedat dose levels from 0, 0.8,3,8,30µMfor 24 h.The test chemical was dissolved in distilled, deionized water .SHE cells have the doubling time of 16 h. The cells were harvested with 0.1% trypsin for chromosome preparation. Three hours before harvest, Colcemid was administered at 0.2µg/ml and metaphase chromosomes were prepared. For determination of chromosome aberrations, 100 metaphases were scored per experimental group. Achromatic lesions greater than the width of the chromatid were scored as gaps unless there was displacement of the broken piece of chromatid. If there was displacement, they were scored as breaks. Also, the chromosome aberration assay was carried out in the presence of exogenous metabolic activation with rat liver post-mitochondrial supernatant (PMS), Cells (5x105) were plated on 100-mm dishes and after overnight incubation, they were treated with chemical agents for 3 h in a 5% PMS mixture.). Cells were washed twice with 5 ml PBS (-) and incubated with fresh medium for 18 h followed by chromosome preparations., Test material did not induce gene mutation inSyrian hamster embryo (SHE) cells inthe presence and absence ofmetabolic activation with rat liver post-mitochondrial supernatant (PMS)and hence is not likely to classify as a gene mutant in vitro.

Based on the data summarized, 1-phenyl-3-pyrazolidone (92-43-3)did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.

Justification for classification or non-classification

Thus based on the above annotation and CLP criteria the test chemical 1-phenyl-3-pyrazolidone (92-43-3) not likly to nduce gene mutation .Hence it is not likely to be mutagenic in vitro.