1-ethylpyrrolidin-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

N-Ethyl-2-pyrrolidone (NEP) was found to be non-genotoxic in bacteria (Ames test) and mammalian cells (HPRT) (BASF, 1998 and 2008).

There was no increase in the number of revertants in any of the S. typhimurium (TA98, TA100, TA1535 and TA1537) or E. coli (E. coli WP2) tester strains at any dose (standard plate: 20 - 5000 µg/plate; preincubation 4 - 2500 µg/plate), neither in the standard plate test nor in the preincubation test, with or without metabolic activation. The test was conducted in compliance with OECD TG 471. Vehicle controls (DMSO) and positive controls were valid. No cytotoxicity nor confounding factors like precipitation were reported.

 

N-Ethyl-2-pyrrolidonewastested was tested for its ability to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of phenobarbital / β-naphthoflavone-induced rat liver S9 mix (exogenous metabolic activation). The study was compliant with OECD 476 and GLP. Concentrations for all tests were 0; 125; 250; 500; 1000 and 1130 μg/ml. After an attachment period of 20 – 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. Slight cytotoxicity was observed. There was no increase in mutant frequency with or with out metabolic activation. The negative result of the first experiment was confirmed by a repetition experiment. The negative controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances,and MCA, led to the expected increase in the frequencies of forward mutations.

N-Ethyl-2-pyrrolidone is considered not mutagenic under in vitro conditions in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation

Link to relevant study records

Reference

Reference 1

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

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

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; 1 000; 1 130 µg/mL
with S9 mix (4-hour exposure period)
0; 125; 250; 500; 1 000; 1 130 µg/mL

Experiment 2:
without S9 mix (24-hour exposure period)
0; 125; 250; 500; 1 000; 1 130 µg/mL
with S9 mix (4-hour exposure period)
0; 400; 600; 800; 1 000; 1 130 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 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:

The 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 teh 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'.

Exp.	Exposure period	Test groups	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6cells]	Cytotoxixity***
						CE1[%]	CE2[%]
1	4 hours	Negative control	-	-	3.97	100.0	100.0
		125 µg/ml	-	-	7.52	105.6	990.
		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	-	-	0.56	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 µg/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 MF_corr.: number of mutant colonies oper 10⁶cells corrected with the CE2 value

*** Cloning efficiency related to the respective negative control

¹300 µg/ml                                            ²MCA 10 µg/ml

Conclusions:

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.

Reference 2

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:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his +/- Trp+/-

Species / strain / cell type:

other: Salmonella typhimurium TA 100, TA 1535, TA 1537, TA 98 and E. coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

20 - 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9 mix for strain TA10, TA98, TA1537 and TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9 mix for strain E. coli WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: N-methyl-N´-nitro-N-nitrosoguanidine

Remarks:

without S-9 mix for strains TA100 and TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

without S-9 mix for strainTA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S-9 mix for strain TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S-9 mix for strain E. coli WP2 uvrA

Evaluation criteria:

In general, a substance to be characterized as positive in the bacterial tests has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Species / strain:

other: S. typhimurium TA 100, TA 1535, TA 1537, TA 98 and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

An increase in the number of his+ or trp+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system. In contrast, positive control substances caused increases in the number of revertants as expected, indicating proper test conditions.

Conclusions:

Interpretation of results (migrated information):
negative

According to the results of the present study, the test substance N-Ethyl-2-pyrrolidon is not mutagenic in the Ames test and in the Escherichia coli -
reverse mutation assay under the experimental conditions chosen here.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

NEP was non-clastogenic in two independent in-vivo experiments.
An in-vivo Micronucleus test according to OECD TG 474 with N-Ethyl-2-pyrrolidon (NEP) was conducted in NMRI mice. Male animals were administered dose levels of 500 mg/kg, 1 000 mg/kg and 2000 mg/kg bw by gavage (BASF, 2006). Cyclophosphamide for clastogenicity and vincristine for spindle poison effects were administered as positive control chemicals. Purified water was used as negative control.. Both of the positive controls chemicals led to the expected increase in the number of polychromatic erythrocytes containing small or large micronuclei.The administration of NEP led to evident signs of toxicity, but no inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.The rate of micronuclei was always close to the range of the concurrent negative control in all dose groups and at all sacrifice intervals and well within the range of the historical control data . NEP does not have any clastogenic effect, and there were no indications of any aneugenic activity in bone marrow cells in this vivo study.

 

N-Ethyl-2-pyrrolidon (NEP) was also assessed in a bone marrow chromosome analysis study in mice according to OECD 475. NEP was administered once orally to male animals at dose levels of 500 mg/kg, 1 000 mg/kg and 2 000 mg/kg bw (BASF, 2005). Cyclophosphamide was administered as positive control. Purified water was used as negative control. After preparation of the bone marrow and staining of the preparations with Giemsa, 100 metaphases were analyzed per animal . The rate of aberrant metaphases was always within the range as that of the concurrent negative control in all dose groups and at all sacrifice intervals. Also in this in-vivo study NEP does not have a clastogenic effect and there were no indications of any aneugenic activity in mice bone marrow cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

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 and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

The investigations were carried out in healthy male CIr:NMRI mice. Animals with a mean weight of about 30 g (with an age range of about 5 - 8 weeks according to the information of the breeder) were used for the study. For the duration of at least 5 days the animals were housed in Makrolon cages. During this time the animals were accommodated in fully air-conditioned rooms in which central air conditioning guaranteed a range of 20 - 24°C for temperature and a range of 30 - 70% for relative humidity . Before the start of the treatment the animals were transferred to Makrolon cages, type MI , and housed individually under the same conditions until the end of the test. The day/night rhythm was 12 hours (12 hours light from 6 .00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours). Standardized pelleted feed and drinking water from bottles were available ad libitum. Due to individual housing in appropriately labeled cages using cage cards, there was no identification of the animals.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2000 mg/kg body weight and in the vehicle controls. In the test groups of 1000 mg/kg and 500 mg/kg body weight and in the positive control group, the 24-hour sacrifice interval was investigated only. About 2 - 3 hours before the animals were sacrificed, they were intraperitoneally injected with 3.3 mg Colcemid/kg body weight in order to arrest mitosis at the stage of metaphase. After preparation of the bone marrow and staining of the preparations with Giemsa, 100 metaphases were analyzed per animal.

Duration of treatment / exposure:

single oral administration

Frequency of treatment:

The animals were treated once and samples of bone marrow were taken 24 hours and 48 hours after the treatment.

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

As a positive control chemical, cyclophosphamide for clastogenicity was administered to male animals once orally in a volume of 10 ml/kg body weight.

Evaluation criteria:

The test is considered valid if the following criteria art met:
- The quality of slides allowed the identification and evaluation of a sufficient number of analyzable cells.
- The proportion of chromosomally damaged cells in negative control animals was within the expected range (<= 2 % incl. gaps).

The test chemical is consideres positive in the assay if the following criteria are met:
- A dose-related and significant increase in the number of metaphases containing chromosomal aberrations at any of the intervals.
- The proportion of aberrant metaphases must significantly exeed the value of the concurrent negative control range.

A test substance is generally considered negative in this test system if:
- There was no significant increase in the number of chromosomally damaged cells at any dose above concurrent control frequencies an at any time.
- The frequencies of cells containing structurally damaged chromosomes were within th negative control range.
- The positive control article induced a significant increase in the number of aberrant metaphases.

Statistics:

The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to aberrant metaphases. The relative frequencies of aberrant metaphases of each animal was used as a criterion for the rank determination for the U test.

Sex:

male

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results (migrated information): negative

Reference 2

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 and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

The investigations were carried out in healthy male CrI:NMRI mice. Animals with a mean weight of about 30 g (with an age range of about 5 - 8 weeks according to the information from the breeder) were used for the study. For the duration of at least 5 days the animals were housed in Makrolon cages. During this time the animals were accommodated in fully air-conditioned rooms in which central air conditioning guaranteed a range of 20 - 24°C for temperature and a range of 30 - 70% for relative humidity. Before the start of the treatment the animals were transferred to Makrolon cages, type MI, and housed individually under the same conditions until the end of the test. The day/night rhythm was 12 hours (12 hours light from 6 .00 - 18 .00 hours and 12 hours darkness from 18 .00 - 6 .00 hours). Standardized pelleted feed and drinking water from bottles were available ad libitum. Due to individual housing in appropriately labeled cages using cage cards, the animals were not individually marked for identification. The animals were assigned to the test groups according to a randomization plan prepared with an appropriate computer program.

Route of administration:

oral: gavage

Vehicle:

water

Duration of treatment / exposure:

single administration

Frequency of treatment:

The animals were treated once and samples of bone marrow were taken 24 hours and 48 hours after the treatment.

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPP) and Vincristine Sulphate (VCR)

Evaluation criteria:

The mouse micronucleus test is considered valid if the following criteria are met:
- The qualitiy of the slides must allow the identification and evaluation of a sufficient number of analyzable cells, i.e. >= 2000 PCEs an a clear
differentiation between PCEs and NCEs.
- The ratio of PCEs/NCE/s in the untreated animals (negative control) has to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for PCEs and
for NCEs
- The two posititive control substances have to induce a significant increase in the number of PCEs containing small and large micronuclei within tht
range of the historical control data and above.

A finding is considered positive if the following criteria are met:
- Significant and dose-related increase in the number of PCEs containing micrinuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control
range

A test substance is considered negative if the following criteria are met:
- The number fof cells containing micronuclei in the dose groups is not siginificantly above the negative control and is within the historical conrol
data.

Statistics:

The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determination for the U test.

Sex:

male

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Animals which were administered the vehicle or the positive control substances cyclophosphamide or vincristine did not show any clinical signs of toxicity.

The administration of the test substance led to evident signs of toxicity.

The negative controls gave frequencies of micronucleated polychromatic erythrocytes within the historical control range.

Both of the positive controls chemicals led to the expected increase in the number of polychromatic erythrocytes containing small or large micronuclei.

According to the results of the present study, the single oral administration of N-Ethyl-2-pyrrolidon did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always close to the

range of the concurrent negative control in all dose groups and at all sacrifice intervals and well within the range of the historical control data.

No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.

Conclusions:

Interpretation of results (migrated information): negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Classification is not warranted according to the criteria of EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.