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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).
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.
3. N-Ethyl-2-pyrrolidone in vitro gene mutation test in CHO cells (HPRT locus assay), OECD 476, GLP, up to 1130 µg/mL

Link to relevant study records

Referenceopen allclose all

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:
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/ml (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:
other: without S-9 mix (0.2 µg mitomycin C/mL culture medium); with metabolic activation (6 µg cyclophosphamide/mL culture medium)
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
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'.

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 ug treatment  (0.5%) and the positive control (2%).
Aneuploid metaphases were detected in the solvent control (1%) and the  2500 ug treatment (1%).
Polyploid metaphases were detected in the negative control (1%), the 1250  ug 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 ug treatment (0.5%).
Polyploid metaphases were detected in the solvent control (1%), the 5000 ug and the 6000 ug 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%)
Conclusions:
Interpretation of results (migrated information):
negative

2-pyrrolidone is not genotoxic in human lymphocyte chromosome abberation assay.
Executive summary:
2 -pyrrolidone was tested in the Human Lymphocytes Cromosome Aberation Assay in the present and absent 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 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:
other: Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100
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:
other: see freetext for details
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 ug/plate
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'.

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

Conclusions:
Interpretation of results (migrated information):
negative

2-Pyrrolidone is negative in Ames Test
Executive summary:

2 -Pyrrolidone did not induce revertants in the Ames test.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
hprt
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9-mix derived from the livers of rats
Test concentrations with justification for top dose:
Experiment 1:
without S9 mix (4-hour exposure period): 0; 125; 250; 500; 1000; 1130 µg/mL
with S9 mix (4-hour exposure period): 0; 125; 250; 500; 1000; 1130 µg/mL

Experiment 2:
without S9 mix (24-hour exposure period): 0; 125; 250; 500; 1000; 1130 µg/mL
with S9 mix (4-hour exposure period): 0; 400; 600; 800; 1000; 1130 µg/mL
Vehicle / solvent:
Culture medium (Ham's F12)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ethyl methanesulfonate; methylcholanthrene
Details on test system and experimental conditions:
This assay was performed in two independent experiments, using identical procedures, both with and without rat liver microsomal activation.
Evaluation criteria:
A finding is assessed as positive if the following criteria are met:
- Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range (see Appendix 5).
- Evidence of reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.

The test substance is considered non-mutagenic according to the following criteria:
The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.
Statistics:
Due to the clearly negative findings, a statistical evaluation was not carried out.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Additional information on results:
According to the results of the present in vitro study, the test substance N-Ethyl-2-pyrrolidone 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 our historical negative control data. The mutation frequencies of the negative control groups were within our historical negative control data range including all vehicles used in our 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 MCA 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. Cytotoxicity: 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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

 Table1: Genotoxicity of 2 -Pyrrolidone
 Experiment No.  Exposure period  Test groups  S9 mix Prec.*   Genotoxicity **MFcorr [per 106 cells]  Cytotoxicity   
           CE1 [%]  CE2 [%]
1  4 hours  Negative control  -  3.97  100.0 100.0 
     125 µg/ml  -  -  7.52 105.6   99.0
     500 µg/ml  -  2.17  79.4  92.5
     500 µg/ml  -  4.61 72.1  92.5
     1000 µg/ml  -  0.71  90.9  95.2
     1130 µg/ml  -  2.20  83.1  90.9
     Positive control-1  -  62.43  80.7  92.2
               
 2  24 hours  Negative control  -  -  3.97  100.0  100.0
     125 µg/mL  -  -  0.00  101.0  83.5
     500 µg/mL  -  -  2.23  84.7  94.0
     500 µg/mL  -  -  2.45  94.8  98.6
     1000 µg/mL  -  -  3.55  87.2  88.7
     1130 µg/mL  -  -  1.44  81.3  99.1
     Positive control-1  -  -  297.48  73.6  65.6
               
 1  4 hours  Negative control  +  -  1.27  100.0  100.0
     125 µg/mL  +  -  3.60  101.2  95.3
     500 µg/mL  +  -  0.61  91.2  104.7
     500 µg/mL  +  -  1.64  81.5  97.8
     1000 µg/mL  +  -  2.46  90.8  102.5
     1130 µg/mL  +  -  0.88  92.3  103.6
     Positive control-2  +  -  44.37  88.6  100.3
               
 2  4 hours  Negative control  +  -  1.02  100.0  100.0
     125 mg/mL  +  -  0.27  110.2  92.3
     500 µg/mL  +  -  0.00  113.2  93.0
     500 µg/mL  +  -  2.61  113.5  97.3
     1000 µg/mL  +  -  3.23  109.1  89.2
     1130 µg/mL  +  -  3.12  104.1  90.9
     Positive control-2  +  -  40.16  104.3  78.3
             

*Precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: number of mutant colonies per 10^6cells corrected with the CE2 value

*** Cloning efficiency related to the respective negative control:1- 300 µg/ml; 2 - MCA -10 µg/ml

Conclusions:
Interpretation of results (migrated information):
negative

Thus, under the experimental conditions chosen here, the conclusion is drawn that N-Ethyl-2-pyrrolidone is not mutagenic under in vitro conditions in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation.
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
Reference
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:
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
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg
Basis:
nominal conc.
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
   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%)



Conclusions:
Interpretation of results (migrated information): 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.
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

N-ethyl-2 -pyrrolidone, a related substance to 2 -pyrrolidone was tested in HPRT locus assay to detect gene mutation in CHO cells (BASF SE, 2008). 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 MCA 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 caused gene mutations in bacterial cells and did not cause cytogenicity in human cells in vitro or in vivo experiments in mice. Therefore, no indication is given for a gene mutation potential of 2-Pyrrolidone. Subsequently, there is no need for classification for mutagenic effects.