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EC number: 210-483-1 | CAS number: 616-45-5
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
1. Gene mutation in bacteria: Jagannath,
1987. Mutagenicity Test on 2-Pyrrolidone in the
Ames-Salmonella/Microsome Reverse Mutation Assay. GLP, comparable to the
OECD guideline 471, S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and
TA 100, with and without metabolic activation (Ames test), negative
2. Cytogenicity in mammalian cells in vitro: BASF AG, 1987. In Vitro
Cytogenetic Investigations of 2-Pyrrolidon in Human Lymphocytes. Report
No. 30M0286/8617. According to the OECD guideline 473, Chromosome
Aberration Assay, with and without metabolic activation, negative
3. Gene mutation in mammalian cells: Wells, 2019. 2-pyrrolidone in vitro
gene mutation test in CHO cells (HPRT locus assay), OECD 476, GLP, up to
851 µg/mL, negative
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Comparable to guideline study (GLP) with acceptable restrictions (strain combination not suitable for the detection of cross links)
- Principles of method if other than guideline:
- According to Ames et al. Mut. Res., 31, 347-364, 1975
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his-
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced rat liver
- Test concentrations with justification for top dose:
- 0, 110, 1100, 5500, 11000, 27500, 55000, 110000 and 165000 µg/plate (= 0, 0.1, 1.0, 5.0, 10, 25, 50, 100 and 150 µL/plate)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- sodium azide
- other: without metabolic activation: Quinacrine mustard and with metabolic activation: 2-Anthramine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: plate incorporation
NUMBER OF REPLICATIONS: 3 - Evaluation criteria:
- Evaluations considered if a dose-response was observed and strain-specific evaluation criteria. For strains TA-1535, TA-1537 and TA-1538, the data set is evaluated as positive if a dose-response is observed over a minimum of three test concentrations and the increase in revertants is equal to or greater than three times the solvent control value at the peak of the dose-response. The solvent control value should be within the normal range for evaluating the results. For strains TA-98 and TA-100, the data set is evaluated as positive if a dose-response is observed over a minimum of three test concentrations and the increase in revertants achieves a doubling of the solvent control value at the peak of the dose-response. The solvent control value should be within the normal range for evaluating the results.
- Statistics:
- Formal statistical methods were not used to evaluate the data.
- Species / strain:
- other: Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 165000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results: negative
2-Pyrrolidone is negative in this Ames Test - Executive summary:
The test item was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver).
The experiments were carried out as plate incorporation tests. The test item was completely dissolved in distilled water. The solvent served as the negative control.
Preliminary test
Concentrations of test substance were selected based on a preliminary toxicity assay at 14 concentration levels using two-fold dilutions from a high concentration of 150 microliter per plate (for liquids). As no significant toxicity was observed, 150 microliters per plate was used as the top concentration in the studies.
Main study
Eight concentrations ranging from 0.1 to 150 μL test substance/plate were employed in the plate incorporation test (in detail the following concentrations were employed: 0, 0.1, 1.0, 5.0, 10, 25, 50, 100 and 150 microliters per plate for all strains in both of the two independent repeats: with and without metabolic activation).
Cytotoxicity was noted in the plate incorporation test, carried out without and with metabolic activation at 165000 µg/plate..
No increase in revertant colony numbers as compared with control counts was observed for the test substance, tested up to a concentration of 150 μL/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively. The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.
In conclusion, under the present test conditions the test substance 2 -pyrrolidone tested up to a concentration of 150 µL/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in the plate incorporation test carried out without and with metabolic activation.
- 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
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (GLP)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- his-
- Species / strain / cell type:
- lymphocytes: human
- Metabolic activation:
- with and without
- Test concentrations with justification for top dose:
- 1250, 2500 und 3500 µg/mlL(without S9-Mix), 2500, 5000 und 6000 µg/mL (with S9-Mix)
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- 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:
- NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 (positive controls: 50) per replicate - Statistics:
- The Fisher exact test was applied to determine significant differences between the relative frequencies of a characteristic of two groups.
- Species / strain:
- lymphocytes: human
- 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:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results: negative
2-pyrrolidone is not genotoxic in human lymphocyte chromosome aberation assay. - Executive summary:
2 -pyrrolidone was tested in the Human Lymphocytes Chromosome Aberation Assay in the presence and absence of metabolic activation. There were statistically significant differences between both negative and solvent control in the two high dose groups without and with metabolic activation. However, there were also aberration observable in the negative control. Therefore, these significant differences are considered to be of no importance for genotoxicity of the test material. 2 -pyrrolidone does not induce clastogenic effects and chromosomal aberrations in this assay.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17 May 2018 - 17 May 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: MAFF Japan, 59 Nousan Number 4200 (1985)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: supplied by Taminco
- Expiration date of the lot/batch: unknown
- Purity test date: 23/02/2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: received at ambient conditions and stored at room temperature (19 °C to 25 °C), protected from light
- Stability under storage conditions: not specified
- Stability under test conditions: concentration analysis of dosing formulations was performed
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: 2-Pyrrolidone was prepared as a stock solution in sterile water at a concentration of 85.1 mg/mL on the day of each assay. Lower concentrations for each assay were prepared by sequential serial dilution in sterile water.
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): not specified - Target gene:
- hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (Hprt)
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: CHO-K1-BH4 cell line (a proline auxotroph with a modal chromosome number of 20)
- Suitability of cells: given
- Normal cell cycle time (negative control): population doubling time of 12 to 14 hours ;cloning efficiency generally > 80%.
For cell lines:
- Absence of Mycoplasma contamination: yes, tested
- Number of passages if applicable: up to 20 passages before they are discarded
- Methods for maintenance in cell culture: not specified
- Cell cycle length, doubling time or proliferation index: population doubling time of 12 to 14 hours
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: [yes]
- Periodically ‘cleansed’ of spontaneous mutants: [not specified]
This cell line was originally isolated by A.W. Hsie (Oak Ridge National Laboratories; Oak Ridge, TN) and the cells in use at Charles River were provided by M.J. LeBaron (The Dow Chemical
Company; Midland, MI). - Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
The exogenous metabolic activation system (MUTAZYME™; S9)
- source of S9 : Molecular Toxicology, Inc. (Lot Nos. 3932 and 3984); consisted of Aroclor™ 1254-induced male rat liver S9 lyophilized with NADPH-regenerating system cofactors and phosphate buffer
- method of preparation of S9 mix: stored and reconstituted as per the manufacturer’s instructions (to yield the same recipe as described by Ames et al.5), except that it also contained 10 mM CaCl2, which has been demonstrated to enhance the mutagenicity of some promutagens. Reconstituted S9 mix was prepared fresh on the day of testing and was maintained refrigerated (2 °C to 8 °C) or on ice.
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) - Test concentrations with justification for top dose:
- 106, 213, 426, 638, and 851 μg/mL with and without S9 (based on dose range finder)
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: aqueous solvents (sterile water, CAS No. 7732-18-5; MediaTech Lot Nos. 26017006, 30917005, and 06718006)
- Justification for choice of solvent/vehicle: not specified
- Justification for percentage of solvent in the final culture medium: not specified - Negative solvent / vehicle controls:
- yes
- Remarks:
- sterile water
- Positive controls:
- yes
- Positive control substance:
- N-dimethylnitrosamine
- ethylmethanesulphonate
- Remarks:
- Neat positive control substances were stored according to the manufacturers’ suggested specifications. Positive control substances were freshly prepared in DMSO. All unused material was discarded.
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 1
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Cells for treatment were seeded (on Day -1) in 75-cm² flasks at a density of approximately 2.5 × 10E6 cells in 10 mL F12FBM5.
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: 3.5 to 4.5 hours
- Harvest time after the end of treatment (sampling/recovery times): After the 4-hour treatment, the treatment media was removed, the cultures were washed twice with 10 mL CMF-DPBS, washed once with 5 mL trypsin,and incubated at 36 °C to 38 °C for 3 to 5 minutes (or until the cells detached). Four mL F12FBM5 was added and the cells were resuspended and counted using a Coulter counter.
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Approximately 2.4 × 10E6 cells (or all available) from each culture also were added to a 225-cm² flask in 30 mL F12FBM5 for phenotypic expression and incubated under standard conditions. The cultures were subcultured on Day 3 and either Day 5 or Day 6 to maintain logarithmic growth and permit expression of the mutant phenotype. At each subculture, the cultures were washed twice with 15 mL CMF-DPBS, washed once with 5 mL trypsin and incubated at 36 °C to 38 °C for 3 to 5 minutes (or until the cells detached). End of the phenotypic expression period on Day 7 or Day 8.
- Selection time (if incubation with a selective agent): Thioguanine (6-thioguanine)
- Fixation time (start of exposure up to fixation or harvest of cells): from day 7 to day 10 (4 days)
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
At the end of the phenotypic expression period (on Day 7 or Day 8), cultures were trypsinized as above (except using 5 mL HX-F12FBM5 to inactivate the trypsin) and 2.4 × 10E6 cells from each culture were seeded at a density of 6 × 10E5 cells/150-mm plate (4 plates total) in 30 mL HX-F12FBM5 containing 10 μM Thioguanine
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2.4 × 106 cells from each culturen were seeded at a density of 6 × 105 cells/150-mm plate (4 plates total). Mutant frequencies are expressed as the number of TGr mutants/106 clonable cells.
- Criteria for small (slow growing) and large (fast growing) colonies: not specified
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, determination of clonable cells in triplicate 60-mm plates
- Any supplementary information relevant to cytotoxicity: Cytotoxicity of the test substance to the test system was determined in order to allow the selection of appropriate dose levels to be tested in the definitive mutagenicity assay. Cells were treated with 2-Pyrrolidone at concentrations of 1.66, 3.32, 6.65, 13.3, 26.6, 53.2, 106, 213, 426, and 851 μg/mL with and without S9. The highest concentration evaluated approximated the limit dose for this assay (10 mM)
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Evaluation criteria:
- Vehicle Control Cultures: The average absolute cloning efficiency of vehicle control substance must be ≥ 50 % (at initial survival and selection). In addition, the average spontaneous mutant frequency of the vehicle control substance must be comparable to the 95 % control interval of the historical negative controls. Spontaneous mutant frequencies are calculated separately for cultures with and without S9.
Positive Control Cultures: The positive control substances must induce a statistically significant increase compared with the concurrent vehicle controls (p ≤ 0.01). A significant increase in the absence of S9 indicates the test system can identify a mutagen, while a significant increase in the presence of S9 will be considered to have demonstrated the efficacy of the S9 mix as well as the ability of the test system to detect a mutagen.
Maximum Concentration Evaluated: The highest test substance concentrations evaluated must be the limit dose for this assay (10 mM), or be the highest that is able to be prepared and administered as a uniform fine suspension, or be minimally insoluble in treatment medium, or induce 10 % to 20 % relative survival (the cytotoxicity limit for this assay), whichever is lowest. Lower concentrations may be justified for test substances which clearly show mutagenic activity.
Number of Concentrations: A minimum of 4 concentrations with acceptable relative survival is required for a valid assay. Fewer concentrations may be justified for test substances which clearly show mutagenic activity. - Statistics:
- A one-tailed Fisher’s Exact test was performed to compare the mutant frequencies observed in the treated cultures with those of the concurrent vehicle control substance cultures. The Cochran-Armitage test was used to assess dose dependency.
- 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
- Positive controls validity:
- valid
- Conclusions:
- 2-Pyrrolidone was negative in the In Vitro Mammalian Cell Gene Mutation Test (CHO/HPRT Assay).
- Executive summary:
2-Pyrrolidone was evaluated for the potential to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (Hprt) in Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).
2-Pyrrolidone was prepared in sterile water and evaluated in a preliminary dose range-finding assay at concentrations of 1.66, 3.32, 6.65, 13.3, 26.6, 53.2, 106, 213, 426, and 851 μg/mL with and without S9. The highest concentration evaluated approximated the limit dose for this assay (10 mM). Sterile water was evaluated concurrently as the vehicle control. Relative survivals at the concentration of 851 μg/mL were 106.85 % without S9 and 89.49 % with S9. The test substance was found to be freely soluble at all concentrations evaluated.
Based upon these results, 2-Pyrrolidone was evaluated in the definitive mutagenicity assay at concentrations of 106, 213, 426, 638, and 851 μg/mL with and without S9. All test substance concentrations, as well as the concurrent positive and vehicle controls, were evaluated in duplicate cultures. In the treatment with S9, mutant frequency data between replicate cultures of the positive control and high dose test article was variable, and appeared that cultures from these 2 groups may have been interchanged during plating. Therefore, this treatment was not considered in the evaluation of mutagenicity, and the treatment was repeated at the same concentrations.
2-Pyrrolidone again was found to be freely soluble at all concentrations evaluated. The average relative survivals at the highest concentration evaluated for mutagenicity were approximately 98.48 % and 89.71 % with and without S9, respectively. In the treatment with S9, statistically significant and dose-dependent increases in mutant frequency were observed at 638 and 851 μg/mL. However, the highest mutant frequency observed was 6.67 × 10E-6, within the 95 % confidence limit of the historical control for the vehicle (-2.13 × 10E-6 to 27.47 × 10E-6). Therefore, these increases are not considered to be biologically relevant. No statistically significant or dose-dependent increases in mutant frequency, as compared to the concurrent vehicle control, were observed at any concentration evaluated without S9 (p > 0.05), and all observed values were within historical control limits. In contrast, the DMN and EMS positive controls induced significant increases in mutant frequencies (p < 0.01).
All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.
These results indicate 2-Pyrrolidone was negative in the In Vitro Mammalian Cell Gene Mutation Test (CHO/HPRT Assay) under the conditions, and according to the criteria, of the test protocol.
Referenceopen allclose all
Standard plate test (110-16500 µg/plate) | |||||
Strain | Metabolic activation system | mean revertants in Controls | maximum revertant factor | dose dependency | Assessment |
TA 98 | no | 25.7 | 1.2 | no | negative |
yes | 46.3 | 1.0 | no | negative | |
TA 100 | no | 147.7 | 1.0 | no | negative |
yes | 140.3 | 1.0 | no | negative | |
TA 1535 | no | 25.3 | 1.0 | no | negative |
yes | 17.3 | 1.0 | no | negative | |
TA 1537 | no | 5 | 1.7 | no | negative |
yes | 10.3 | 1.1 | no | negative | |
TA 1538 | no | 13 | 1.1 | no | negative |
yes | 16 | 1.8 | no | negative | |
Standard plate test II(110-16500 µg/plate) | |||||
Strain | Metabolic activation system | mean revertants in Controls | maximum revertant factor | dose dependency | Assessment |
TA 98 | no | 28.3 | 1.1 | no | negative |
yes | 49 | 1.0 | no | negative | |
TA 100 | no | 127 | 1.2 | no | negative |
yes | 142.7 | 1.1 | no | negative | |
TA 1535 | no | 20.7 | 1.2 | no | negative |
yes | 15 | 1.2 | no | negative | |
TA 1537 | no | 7.7 | 1.6 | no | negative |
yes | 12 | 1.0 | no | negative | |
TA 1538 | no | 12 | 1.2 | no | negative |
yes | 22 | 1.2 | no | negative |
Without metabolic activation
Treatm. |
% aber. + gaps |
% aber. - gaps |
% exchanges |
negative |
5 |
1 |
0 |
solvent |
6 |
2.5 |
0 |
1250 ug |
8.5 |
1 |
0.5 |
2500 ug |
7 |
3 |
0 |
3500 ug |
4 |
1 |
0 |
positive |
44** |
37** |
5* |
Multiple abberation metaphases were detected in the 1250 µg treatment (0.5 %) and the positive control (2 %).
Aneuploid metaphases were detected in the solvent control (1 %) and the 2500 µg treatment (1 %).
Polyploid metaphases were detected in the negative control (1 %), the 1250 µg and the 2500 ug treatment (0.5 % each).
With metabolic activation
Treatm. |
% aber. + gaps |
% aber. - gaps |
% exchanges |
negative |
2 |
0 |
0 |
solvent |
7.5 |
2 |
0 |
2500 ug |
6.5 |
0.5 |
0 |
5000 ug |
8# |
0.5 |
0 |
6000 ug |
8.5# |
1 |
0 |
Multiple aberration metaphases were not detected in any treatment or control.
Aneuploid metaphases were detected in the 5000 µg treatment (0.5 %).
Polyploid metaphases were detected in the solvent control (1 %), the 5000 µg and the 6000 µg treatment (0.5 % each).
**/* = statistically different from both the negative and solvent control (significance level 99/95%)
# = statistically different from the negative control (significance level 95 %)
Dose Range-Finding Assay
2-Pyrrolidone was prepared in sterile water and evaluated in a preliminary dose range-finding assay at concentrations of 1.66, 3.32, 6.65, 13.3, 26.6, 53.2, 106, 213, 426, and 851 μg/mL with and without S9. The highest concentration evaluated approximated the limit dose for this assay (10 mM). Sterile water was evaluated concurrently as the vehicle control. Relative survivals at the concentration of 851 μg/mL were 106.85 % without S9 and 89.49 % with S9. The test substance was found to be freely soluble at all concentrations evaluated.
Table 1 |
||||||||
Dose Range-Finding Assay |
||||||||
Dose Level |
Initial Survival |
|||||||
Cells/mL |
Cloning Efficiency |
Relative Survival |
||||||
Treatment |
(µg/mL) |
S9 |
(x 10E6 ) |
(Colonies/Plate) |
% |
(adj.,%) |
||
di-H2O |
10.0a |
- |
1.292 |
106 |
82 |
106 |
49.00 |
100.00 |
2-Pyrrolidinone |
1.66 |
- |
1.086 |
105 |
86 |
85 |
46.00 |
78.90 |
2-Pyrrolidinone |
3.32 |
- |
1.887 |
48 |
53 |
46 |
24.50 |
73.02 |
2-Pyrrolidinone |
6.65 |
- |
0.939 |
141 |
130 |
132 |
67.17 |
99.65 |
2-Pyrrolidinone |
13.3 |
- |
1.105 |
97 |
95 |
85 |
46.17 |
80.62 |
2-Pyrrolidinone |
26.6 |
- |
1.076 |
81 |
86 |
93 |
43.33 |
73.69 |
2-Pyrrolidinone |
53.2 |
- |
0.944 |
119 |
111 |
98 |
54.67 |
81.52 |
2-Pyrrolidinone |
106 |
- |
1.090 |
111 |
109 |
97 |
52.83 |
90.95 |
2-Pyrrolidinone |
213 |
- |
1.077 |
90 |
94 |
101 |
47.50 |
80.81 |
2-Pyrrolidinone |
426 |
- |
0.981 |
92 |
102 |
111 |
50.83 |
78.75 |
2-Pyrrolidinone |
851 |
- |
1.012 |
135 |
143 |
123 |
66.83 |
106.85 |
di-H2O |
10.0a |
+ |
1.071 |
134 |
132 |
127 |
65.50 |
100.00 |
2-Pyrrolidinone |
1.66 |
+ |
1.172 |
101 |
120 |
115 |
56.00 |
93.56 |
2-Pyrrolidinone |
3.32 |
+ |
1.132 |
134 |
135 |
111 |
63.33 |
102.24 |
2-Pyrrolidinone |
6.65 |
+ |
1.111 |
139 |
144 |
147 |
71.67 |
113.55 |
2-Pyrrolidinone |
13.3 |
+ |
1.171 |
104 |
107 |
122 |
55.50 |
92.63 |
2-Pyrrolidinone |
26.6 |
+ |
1.033 |
122 |
121 |
130 |
62.17 |
91.56 |
2-Pyrrolidinone |
53.2 |
+ |
1.076 |
116 |
114 |
120 |
58.33 |
89.46 |
2-Pyrrolidinone |
106 |
+ |
1.014 |
125 |
108 |
107 |
56.67 |
81.93 |
2-Pyrrolidinone |
213 |
+ |
1.019 |
135 |
123 |
129 |
64.50 |
93.70 |
2-Pyrrolidinone |
426 |
+ |
0.995 |
139 |
144 |
112 |
65.83 |
93.40 |
2-Pyrrolidinone |
851 |
+ |
1.013 |
137 |
118 |
117 |
62.00 |
89.49 |
aµL/mL |
Definitive Mutagenicity Assay
Based upon the results of the dose range-finding assay, 2-Pyrrolidone was evaluated in the definitive mutagenicity assay at concentrations of 106, 213, 426, 638, and 851 μg/mL with and without S9. All test substance concentrations, as well as the concurrent positive and vehicle controls, were evaluated in duplicate cultures. In the treatment with S9, mutant frequency data between replicate cultures of the positive control and high dose test article was variable, and appeared that cultures from these 2 groups may have been interchanged during plating. Therefore, this treatment was not considered in the evaluation of mutagenicity, and the treatment was repeated at the same concentrations. 2-Pyrrolidone again was found to be freely soluble at all concentrations evaluated. The average relative survivals at the highest concentration evaluated for mutagenicity were approximately 98.48 % and 89.71 % with and without S9, respectively. In the treatment with S9, statistically significant and dose-dependent increases in mutant frequency were observed at 638 and 851 μg/mL. However, the highest mutant frequency observed was 6.67 × 10E-6, within the 95 % confidence limit of the historical control for the vehicle (-2.13 × 10E-6 to 27.47 × 10E-6). Therefore, these increases are not considered to be biologically relevant. No statistically significant or dose-dependent increases in mutant frequency, as compared to the concurrent vehicle control, were observed at any concentration evaluated without S9 (p > 0.05), and all observed values were within historical control limits. In contrast, the DMN and EMS positive controls induced significant increases in mutant frequencies (p < 0.01).
All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.
Table 2 |
|||||||||||||||||||||||
Definitive Mutagenicity Assay (Treatment Without S9) |
|||||||||||||||||||||||
Treatment |
Dose Level (µg/mL) |
S9 |
Initial Survival |
Selection |
|||||||||||||||||||
Cells/mL (x 106) |
Cloning Efficiency |
Relative Survival (%, adj.) |
TGr |
Total Mutant Colonies |
Cloning Efficiency |
Mutant Frequency (x 10-6) |
|||||||||||||||||
(Colonies/Plate) |
% |
(Colonies/Plate) |
(%) |
Individual |
Average |
||||||||||||||||||
di-H2O |
10.0a |
- |
1.435 |
137 |
146 |
112 |
65.83 |
101.25 |
3 |
7 |
4 |
2 |
16 |
95 |
130 |
126 |
58.50 |
11.40 |
|
||||
di-H2O |
10.0a |
- |
1.410 |
119 |
148 |
125 |
65.33 |
98.75 |
5 |
7 |
5 |
4 |
21 |
140 |
103 |
149 |
65.33 |
13.39 |
12.39 |
||||
2-pyrrolidinone |
106 |
- |
0.968 |
135 |
125 |
145 |
67.50 |
70.06 |
1 |
2 |
6 |
4 |
13 |
104 |
122 |
119 |
57.50 |
9.42 |
|
||||
2-pyrrolidinone |
106 |
- |
0.940 |
138 |
146 |
122 |
67.67 |
68.15 |
1 |
0 |
0 |
0 |
1 |
90 |
110 |
108 |
51.33 |
0.81 |
5.12 |
||||
2-pyrrolidinone |
213 |
- |
1.457 |
121 |
130 |
109 |
60.00 |
93.70 |
3 |
4 |
2 |
9 |
18 |
166 |
153 |
157 |
79.33 |
9.45 |
|
||||
2-pyrrolidinone |
213 |
- |
1.213 |
116 |
111 |
133 |
60.00 |
78.04 |
5 |
3 |
6 |
3 |
17 |
139 |
154 |
117 |
68.33 |
10.37 |
9.91 |
||||
2-pyrrolidinone |
426 |
- |
1.467 |
132 |
117 |
137 |
64.33 |
101.13 |
6 |
4 |
2 |
10 |
22 |
164 |
183 |
190 |
89.50 |
10.24 |
|
||||
2-pyrrolidinone |
426 |
- |
1.300 |
126 |
132 |
143 |
66.83 |
93.15 |
4 |
5 |
3 |
4 |
16 |
131 |
112 |
137 |
63.33 |
10.53 |
10.38 |
||||
2-pyrrolidinone |
638 |
|
1.312 |
100 |
85 |
98 |
47.17 |
66.33 |
7 |
1 |
2 |
4 |
14 |
126 |
112 |
113 |
58.50 |
9.97 |
|
||||
2-pyrrolidinone |
638 |
|
1.318 |
132 |
128 |
120 |
63.33 |
89.49 |
4 |
8 |
10 |
7 |
29 |
125 |
119 |
141 |
64.17 |
18.83 |
14.40 |
||||
2-pyrrolidinone |
851 |
|
1.376 |
115 |
136 |
125 |
62.67 |
92.40 |
10 |
7 |
12 |
4 |
33 |
178 |
171 |
156 |
84.17 |
16.34 |
|
||||
2-pyrrolidinone |
851 |
|
1.338 |
117 |
122 |
125 |
60.67 |
87.02 |
2 |
2 |
1 |
2 |
7 |
144 |
144 |
141 |
71.50 |
4.08 |
10.21 |
||||
EMS |
200 |
|
1.307 |
84 |
94 |
83 |
43.50 |
60.95 |
79 |
123 |
77 |
83 |
362 |
136 |
132 |
151 |
69.83 |
215.99 |
|
||||
EMS |
200 |
|
1.235 |
116 |
100 |
111 |
54.50 |
72.17 |
53 |
65 |
52 |
52 |
222 |
63 |
59 |
68 |
31.67 |
292.11 |
254.05** |
||||
aµg/mL |
|||||||||||||||||||||||
*= p < 0.05, statistical increase over concurrent vehicle control using one-tailed Fisher's Exact Test. |
|||||||||||||||||||||||
**= p < 0.01, statistical increase over concurrent vehicle control using one-tailed Fisher's Exact Test. |
Table 3 |
|||||||||||||||||||||||||||||
Repeat Definitive Mutagenicity Assay (Treatment With S9) |
|||||||||||||||||||||||||||||
Treatment |
Dose Level (µg/mL) |
S9 |
Initial Survival |
Selection |
Mutant Frequency (x 10-6) |
||||||||||||||||||||||||
Cells /ml (x 10E6) |
Cloning Efficiency |
Relative Survival (%, adj.) |
TGr |
Total Mutant Colonies |
Cloning Efficiency |
||||||||||||||||||||||||
(Colonies/Plate) |
(%) |
(Colonies/P |
late) |
(%) |
Individual Average |
||||||||||||||||||||||||
di-H2O |
10.0a |
+ |
1.103 |
111 |
89 |
98 |
49.67 |
100.43 |
0 |
0 |
1 |
0 |
1 |
123 |
131 |
117 |
61.83 |
0.67 |
|
||||||||||
di-H2O |
10.0a |
+ |
1.021 |
118 |
108 |
93 |
53.17 |
99.57 |
1 |
1 |
1 |
0 |
3 |
121 |
123 |
117 |
60.17 |
2.08 |
1.38 |
||||||||||
2-pyrrolidinone |
106 |
+ |
0.931 |
98 |
99 |
106 |
50.50 |
86.21 |
1 |
1 |
0 |
0 |
2 |
133 |
128 |
118 |
63.17 |
1.32 |
|
||||||||||
2-Pyrrolidinone |
106 |
+ |
0.940 |
96 |
81 |
110 |
47.83 |
82.44 |
1 |
0 |
3 |
0 |
4 |
140 |
91 |
130 |
60.17 |
2.77 |
2.04 |
||||||||||
2-Pyrrolidinone |
213 |
+ |
0.977 |
109 |
108 |
97 |
52.33 |
93.77 |
1 |
0 |
0 |
0 |
1 |
116 |
111 |
128 |
59.17 |
0.70 |
|
||||||||||
2-Pyrrolidinone |
213 |
+ |
0.939 |
113 |
106 |
89 |
51.33 |
88.39 |
0 |
0 |
0 |
0 |
0 |
108 |
117 |
108 |
55.50 |
0.00 |
0.35 |
||||||||||
2-Pyrrolidinone |
426 |
+ |
1.004 |
117 |
75 |
117 |
51.50 |
94.82 |
0 |
0 |
0 |
1 |
1 |
119 |
106 |
112 |
56.17 |
0.74 |
|
||||||||||
2-Pyrrolidinone |
426 |
+ |
0.932 |
103 |
104 |
111 |
53.00 |
90.59 |
0 |
0 |
0 |
0 |
0 |
114 |
120 |
116 |
58.33 |
0.00 |
0.37 |
||||||||||
2-Pyrrolidinone |
638 |
+ |
1.023 |
109 |
112 |
116 |
56.17 |
105.38 |
3 |
1 |
5 |
2 |
11 |
120 |
125 |
131 |
62.67 |
7.31 |
|
||||||||||
2-Pyrrolidinone |
638 |
+ |
1.026 |
104 |
86 |
96 |
47.67 |
89.65 |
1 |
1 |
0 |
0 |
2 |
114 |
115 |
119 |
58.00 |
1.44 |
4.38 * |
||||||||||
2-Pyrrolidinone |
851 |
+ |
0.990 |
101 |
105 |
113 |
53.17 |
96.55 |
0 |
0 |
0 |
0 |
0 |
113 |
117 |
107 |
56.17 |
0.00 |
|
||||||||||
2-Pyrrolidinone |
851 |
+ |
0.987 |
94 |
121 |
118 |
55.50 |
100.41 |
5 |
5 |
3 |
3 |
16 |
104 |
97 |
99 |
50.00 |
13.33 |
6.67 ** |
||||||||||
DMN |
100 |
+ |
0.959 |
43 |
33 |
46 |
20.33 |
35.74 |
34 |
28 |
34 |
38 |
134 |
101 |
78 |
97 |
46.00 |
121.38 |
|
||||||||||
DMN |
100 |
+ |
0.927 |
29 |
40 |
42 |
18.50 |
31.46 |
32 |
34 |
21 |
25 |
112 |
82 |
89 |
83 |
42.33 |
110.24 |
115.81 ** |
||||||||||
aµL/mL |
|||||||||||||||||||||||||||||
*= p < 0.05, statistical increase over concurrent vehicle control using one-tailed Fisher's Exact Test. |
|||||||||||||||||||||||||||||
**= p < 0.01, statistical increase over concurrent vehicle control using one-tailed Fisher's Exact Test. |
Dose Formulation Analysis Results
Concentration analysis demonstrated that all dose formulations from all trials analyzed were 94.4 % to 113 % of target, and met the acceptance criteria with the following exception. The 10.6 mg/mL (low-dose) formulation samples from the definitive mutagenicity assay (conducted on 08 Jun 2018) quantified at 71.7 % of the nominal concentration. However, as both the high- and mid-dose formulations from this assay date were acceptable, ascertaining the limit dose for the assay was achieved, there is no impact on the validity or integrity of the study. Test substance was not detected in any of the vehicle control samples. The Analyses of Dosing Formulations is depicted below.
Conclusions
These results indicate 2-Pyrrolidone was negative in the In Vitro Mammalian Cell Gene Mutation Test (CHO/HPRT Assay) under the conditions, and according to the criteria, of the test protocol.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
1. Cytogenicity in mammalian cells in vivo: BASF AG, 1993. Cytogenetic Study In Vivo of Pyrrolidon-2 in Mice; Micronucleus Test, Single Intraperitoneal Administration. Report No.26M0014/924193, GLP, according to the OECD guideline 474, NMRI mouse, up to 2000 mg/kg i.p.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Route of administration:
- intraperitoneal
- Vehicle:
- water
- Details on exposure:
- Single intraperitoneal administration with a volume of 10 mL/kg body weight.
- Duration of treatment / exposure:
- 16, 24 and 48 hours
- Frequency of treatment:
- single
- Post exposure period:
- - Sacrifice intervals per dose-group were:
2000 mg/kg: 16, 24 and 48 hours
1000 mg/kg: 24 hours
500 mg/kg: 24 hours
Controls: 24 hours - Dose / conc.:
- 500 mg/kg bw/day
- Remarks:
- intraperitoneal
- Dose / conc.:
- 1 000 mg/kg bw/day
- Remarks:
- intraperitoneal
- Dose / conc.:
- 2 000 mg/kg bw/day
- Remarks:
- intraperitoneal
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- 20 mg cyclophosphamide/kg bw or 0.15 mg vincristine/kg bw, both dissolved in distilled water
- Tissues and cell types examined:
- bone marrow, erythrocytes (see below in "Details of tissue and slide preparation"
- Details of tissue and slide preparation:
- - Preparation of bone marrow: After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37 °C (about 2 mL/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant removed and the cells were resuspended. One drop of this suspension was dropped onto clean microscopic slides.
Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained in eosin and methylene blue solution for 5 minutes, rinsed, placed in fresh distilled water for 2 or 3 minutes and finally stained in Giemsa solution for 12 minutes. After being rinsed twice and clarified with xylene, the preparations were embedded in Corbit-Balsam. Slides were coded before microscopic analysis.
-Evaluations: In general, 1000 polychromatic erythrocytes from each male and female animal of every test group was evaluated and investigated for micronuclei. The normochromatic erythrocytes which occur were also scored. The following parameters were recorded:
Number of polychromatic erythrocytes
Number of polychromatic erythrocytes containing micronuclei,
Number of normochromatic erythrocytes,
Number of normochromatic erythrocytes containing micronuclei,
Ratio of polychromatic to normochromatic erythrocytes,
Number of small micronuclei (d < D/4) and of large micronuclei (d >= D/4)
CRITERIA FOR DOSE SELECTION:
Doses were selected on the basis of a preliminary toxicity study: Here, a dose of 2000 mg/kg bw led to signs of toxicity including irregular respiration, piloerection abdominal position, apathy and squatting posture; the general state was poor. - Statistics:
- No statistical methods were employed in data analysis.
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- observed in the highest dose tested
- Conclusions:
- Interpretation of results: negative
Cytogenetic analysis of bone marrow cells of mice in vivo show negative results. - Executive summary:
To assess the cytotoxicity, 2 -pyrrolidone was tested in the Micronucleus Test in Mice in vivo. The study was conducted according to the OECD Guideline 474 and is compliant to GLP requirements.
Administration of test substance did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d D/4) or large micronuclei (d > D/4) did not deviate from the solvent control value at any sacrifice interval. No inhibition of erythropoiesis induced by the treatment of mice with Pyrrolidon-2 was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups. The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.
Reference
CLINICAL EXAMINATIONS
The single intraperitoneal administration of the solvent in a volume of
10 mL/kg body weight was tolerated by all animals without any signs or
symptoms. A dose of 2000 mg/kg bw. of test substance led to irregular
respiration, piloerection, abdominal position and apathy about 30
minutes after administration; the general state of some animals was
poor. After treatment of the animals with 1000 or 500 mg/kg, only
irregular respiration and piloerection were observed after about 30
minutes. After about 1 -2 hours clinical signs were no longer observed.
None of the positive control substances caused any evident signs of
toxicity.
MICRONUCLEI
Mean polychromatic (normochromatic) erythrocytes containing micronuclei
were:
Negative control (24 hrs) 1.5 % (1.5 %)
2000 mg/kg (16 hrs) 1.2 % (1.1 %)
2000 mg/kg (24 hrs) 1.7 % (0.25 %)
2000 mg/kg (48 hrs) 1.6 % (1.29 %)
1000 mg/kg (24 hrs) 2.4 % (1.0 %)
500 mg/kg (16 hrs) 1.2 % (0.26 %)
Cyclophosphamide (24 hrs) 13.6 % (2.33 %)
Vincristine (24 hrs) 83.2 % (1.72 %)
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro
Gene mutation in bacteria
2-Pyrrolidone was not mutagenic in a plate incorporation Ames test with and without metabolic activation (tested up to 16500 μg/plate in Salmonella typhimurium TA1535, TA 1537, TA 1538, TA 98 and TA 100; metabolic activation: liver S-9 mix from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters; Jagannath 1987). Cytotoxicity (reduction of the background lawn) was observed at the highest concentration tested.
Cytogenicity in mammalian cells
In a GLP conform assay according to OECD guideline 473, human lymphocytes were tested for chromosome aberrations in doses up to 3500 µg/mL of 2 -pyrrolidone without metabolic activation and doses up to 6000 mg/mL with metabolic activation (BASF AG 1987). There were no increases of chromosome aberrations detected in any treatment if compared with the solvent controls in the assays with and without metabolic activation. Due to the very low mean number of aberrations in the negative control with metabolic activation, statistically significant effects were found in the two highest dose groups, but they were considered as not relevant.
Gene mutation in mammalian cells
2 -pyrrolidone was tested in HPRT locus assay to detect gene mutation in CHO cells (Welly, 2019). The test substance did not lead to an increase in the number of mutant colonies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. The mutant frequencies at any concentration were close to or within the range of that of the concurrent negative control values and within the range of the historical negative control data. The mutation frequencies of the negative control groups were within the historical negative control data range including all vehicles used in their laboratory and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMN clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. In both experiments in the absence and the presence of S9 mix, no cytotoxicity indicated by reduced relative cloning efficiency of below 20 % relative survival was observed.
In vivo
Cytogenicity in mammalian cells
Cytogenicity in vivo was tested in a mouse micronucleus test conducted according to GLP requirements and OECD 474 (BASF AG 1993). The test substance was administered once i.p. to 5 male and 5 female NMRI mice at doses up to 500, 1000, 2000 mg/kg body weight, respectively. Bone marrow cells were harvested after 16, 24 or 48 hours. Administration of the test substance did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d<D/4) or large micronuclei (d>D/4) did not deviate from the solvent control value at any sacrifice interval. No inhibition of erythropoiesis induced by the treatment of mice with Pyrrolidon-2 was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups. The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.Positive and negative controls all produced appropriate responses. The test substance did not produce any chromosome-damaging (clastogenic) effect.
Justification for classification or non-classification
The test substance did not cause gene mutations in bacterial cells, did not cause cytogenicity in human cells and did not cause gene mutations at the hprt locus of CHO cells in vitro or in vivo experiments in mice.
Therefore, no indication is given for a gene mutation potential of 2-Pyrrolidone and classification is not warranted according to the criteria of EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
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