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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenicity of N-(2-hydroxyethy)-2-pyrrolidone with different purity grades has been assessed in several reverse mutation assays by the manufacturer.In the first test (GLP compliant and according to OECD guideline 471), a test substance of lower purity (98.1%) was tested using Salmonella typhimurium strainsTA1535, TA100, TA1537, TA98and Escherichia coli strainE.coli WP2 uvrA in a standard plate test (BASF AG, 2001). In the first experiment, 20, 100, 500, 2500 and 5000 µg/plate were tested in all strains. In the second experiment, concentrations of 100, 500, 2500, 5000 and 7500 µg/plate were tested only in the strains TA1535 and TA100.All tests were performedboth with and without metabolic activation (Aroclor-induced rat liverS-9mix).

No precipitation of the test substance was found and no bacteriotoxic effect was observed. In strains TA1537, TA98 andE.coli WP2 uvrA, no increase in the number of his+ or trp+ revertants was reported. InTA1535, mutagenicity was observed both with and without metabolic activation from about500µg/plate (factor 2.2- 3.2)onward with an increase in the number of his+ revertantsbya factor of15.6 - 20.5at7,500µg/plate. InTA100, a slight increase in the number of revertant colonies with and withoutS-9mix over a dose range of about2,500 µg -7,500µg/plate (factor 1.4-3.4) was reported.

According to the results of this study, the test sample (purity 98.1%) was mutagenic in the Salmonella typhimurium reverse mutation assay under the experimental conditions used. The positive reactions were considered to be caused by impurities as confirmed by subsequent experiments. 

In the second test (GLP compliant and according to OECD guideline 471), the test substance with a higher purity (99.8%; in line with specification provided in chapter 1.2) was tested using the same Salmonella typhimurium strainsTA1535, TA100, TA1537, TA98and Escherichia coli strainE.coli WP2 uvrA in a standard plate test and in a preincubation test (BASF AG, 2004a). In both tests, the concentrations tested were: 20, 100, 500, 2500 and 5000 µg/plate.All tests were performedboth with and without metabolic activation (Aroclor-induced rat liverS-9mix).No precipitation of the test substance was found and no bacteriotoxic effect was observed. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolising system.According to the results of this study, the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay underthe experimental conditions used.

 

Due to the different results in the two studies as reported above a follow-up study was conducted (BASF AG, 2004b). Two different batches were tested,N-(2 -Hydroxyethyl)-2-pyrrolidonwith a purity of 99.8% (the batch which was tested in the BASF AG 2004a study) and with a purity of 98.1% (the batch which was tested in the BASF AG 2001 study) using Salmonella typhimurium strain TA1535. Concentrations of 20, 100, 500, 2500 and 5000 µg/plate were tested in the absence and presence of S-9 mix. The substance with a purity of 99.8% was negative, while the substance with a purity of 98.1% was positive both in the absence and presence of S-9 mix. The authors concluded that impurities were the cause of the positive reaction found with the test substance with the lower purity (see BASF AG, 2001), the substance itself (purity of 99.8%) did not show a positive reaction in the Ames test (BASF AG, 2004a).

In addition to these more recent studies, a reverse mutation assay had been performed with three different samples of N-(2 -hydroxyethyl)-2 -pyrrolidone, two production lots and a purified sample, in Salmonella typhimurium strains TA98, TA1537, TA1538, TA100 and TA1535 (Arthur D. Little Inc., 1982). No further information on the purity of the different lots was provided. The tested concentrations were: 0.32, 1.6, 8, 40, 200 μl/plate and the tests were performed without and with S9 -mix.

The first production lot gave a positive (mutagenic) response in TA100 and TA1535 even in the absence of metabolic activation. Toxicity (a sparse lawn) was evident at 200 µl/plate in all strains and slight toxicity was observed in TA100 at 40 µl/plate.

The second production lot gave a positive (mutagenic) response in TA100 and TA1535 even in the absence of metabolic activation, confirming the results obtained with the first lot of this material. Toxicity (a sparse lawn) was evident at 200 µl/plate in all strains and was also evident at all concentrations in TA98 (i.e., there was a reduced number of revertants in contrast to the DMSO control plates).

Regarding the purified sample, a weak mutagenic activity was evident in TA1535 both in the absence and presence of metabolic activation. A doubling of the number of revertants over the control background was observed only at the highest concentrationtested (200 µl/plate). Toxicity was evident in TA100 at 200 µl/plate (lawn effect) and at all the lower concentrations tested (number of revertants lower than the number obtained in the DMSO control backgrounds). The purified sample was significantly less potent as the production lots of N-(2 -hydroxyethyl)-2 -pyrrolidone at a technical grade which supports the assumption that impurities had caused the positive reaction.

N-(2 -hydroxyethyl)-2 -pyrrolidone) with a purity >= 99.5% was studied in a mammalian gene mutation assay (V79/HPRT) according to OECD Guideline 476 and under GLP in two independent experiments, using two parallel cultures each(Harlan, 2012).The first experiment was performed with and without S9-mix and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. Cytotoxicity was not observed. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Therefore, N-(2-Hydroxyethyl)-2-pyrrolidon is considered to be non-mutagenic in this HPRT assay.

In a recent Micronucleus test, N-2 -hydroxyethyl-2 -pyrrolidon was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Two independet experiments were carried out, both with and without the addition of liver S9 mix from induced rats. According to an initial range finding cytotoxicity test concentrations up to 1300 µg/ml (10 mM) were chosen. A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group.The negative controls gave frequencies within the negative control data range for V79 cells. Both positive substances, EMS and cyclophosphamide led to the expected increase in the number of cells containing micronuclei. No cytotoxicity indicated by reduced relative increase in cell count , proliferation index or replicative index was observed up to the higest required test substance concentration. The test substrance did not cause any biologically relevant increase in the number of cells containing micronuclei either without or after adding a metabolizing system (BASF SE, 2014).

N-2-hydroxyethyl-2-pyrrolidone specified as a technical grade (no further data on purity)was also evaluated in fourin vitromammalian short-term genotoxicity assays measuring different endpoints (Arthur D. Little Inc., 1982). Three assays were conducted with Chinese hamster ovary (CHO) cell lines and consisted of a mammalian gene mutation assay (HGPRT) and two cytogenetic assays (chromosome aberrations and sister chromatid exchanges). The fourth experiment consisted of aneoplastic transformation assay using a mouse fibroblast cell line. All tests were performed in absence of an exogenous metabolic activation system.

 

In the chromosome aberration test, the substance was tested at concentrations of 125 – 1000 µg/ml. The substance was not cytotoxic to mass cultures of CHO cells from 0.01 - 250 µg/mL. Since the substance was not toxic, the highest concentration used in the cytogenetic assays was 1000 µg/ml. Of 100 metaphases examined, 3 chromosome aberrations were found in CHO cells treated with 125 μg/ml of the substance. This number was calculated to be statistically significant (P<0.05) over the zero aberration of the control. However, no aberrations were found in cells treated with higher concentrations of the chemical. According to the authors, one or two aberrations were often observed in control cultures historically, therefore the aberrations scored at this particular concentration were considered to be not biologically significant. The positive control induced the expected number of chromosome aberrations.

In the mammalian gene mutation (HGPRT) assay, the substance (technical grade) was tested at concentrations of 125 – 1000 µg/ml. All tested concentrations of the substance uniformly induced a higher mutation frequency than the control in CHO cells. However, no dose-dependent effect was observed. Since the substance was not cytotoxic, the mean number of mutant colonies per plate between the treated cultures and the control can be compared. When tabulated as such, cultures treated with the substance showed a 2-3 fold increase in mutants per plate over the untreated control. This increase, although weak, was found to be statistically significant (p<0.05), with the exception of the 500 μg/ml concentration which had a high standard deviation.

In the sister chromatid exchange (SCE) assay, the substance of technical purity was tested at concentrations of 125 – 1000 µg/ml. The substance was not cytotoxic to mass cultures of CHO cells from 0.01 - 250 µg/ml. Since the substance was not toxic, the highest concentration used in the cytogenetic assays was 1000 µg/ml.At the two highest concentrations of 500 and 1000 μg/ml, the substance induced a slight but statistically significant increase in the number of SCE above the control. No apparent cytotoxicity of the target cells was associated with these high concentrations as judged by the mitotic indexes of the control and treated cells.

 

In the cell transformation assay with BALB/c-3T3 cells, the substance of technical purity was tested at concentrations of 2 – 250 µg/ml.The test compound produced a negative response. The positive control MCA induced a mean of 3.00 Type III foci per plate which is within the normal range of transformation induced by this carcinogenic arylaromatic hydrocarbon. The results of the cytotoxicity assay showed higher surviving fractions of cells treated with the substance than the control. The lower cell counts in controls may be due to incomplete trypsinisation of cells from the plates in this particular experiment.


Based on all the available genotoxicity studies in bacteria and mammalian cell cultures, N-(2-hydroxyethyl)-2-pyrrolidon is not considered genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct 2013-April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guidline study under GLP conditions
Qualifier:
according to
Guideline:
other: OECD 487
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
1st Experiment:
4 hours exposure; 24 hours harvest time; without S9 mix: 0; 162.50, 325.00, 650.00, 1 300.00 µg/mL
4 hours exposure, 24 hours harvest time, with S9 mix: 0; 162.50, 325.00, 650.00, 1 300.00 µg/mL

2nd Experiment:
24 hours exposure, 24 hours harvest time, without S9 mix 0; 162.50, 325.00, 650.00, 1 300.00 µg/mL
4 hours exposure, 44 hours harvest time, with S9 mix 0; 162.50, 325.00, 650.00, 1 300.00 µg/mL
Vehicle / solvent:
Due to the good solubility of the test substance in water, culture medium (Minimal Essential Medium: MEM) was selected as vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
Without metabolic act: 300, 400, 500 µg/mL ethyl methanesulfonate dissolved in MEM without FCS (1.5, 2.0 or 2.5 mg/mL, respectively) With metabolic act.: 0.5 and 1.0 µg/mL cyclophosphamide CPP was dissolved in MEM without FCS (2.5 and 5.0 µg/mL).
Details on test system and experimental conditions:
V79 cell line is a permanent cell line derived from the Chinese hamster.

Culture media:
MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with
- 10% (v/v) fetal calf serum (FCS)
- 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 µg/mL)
- 1% (v/v) amphotericine B (250 µg/mL)
During exposure to the test substance in the presence of S9 mix MEM medium was used without FCS supplementation.

Cell culture:
Deep-frozen cell stocks were thawed at 37°C in a water bath, and volumes of 0.5 mL were transferred into 25 cm2 plastic flasks containing about 5 mL MEM supplemented with 10% (v/v) fetal calf serum (FCS). Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity and subcultured twice weekly. Cell monolayers were suspended in culture medium after detachment with 0.25% (w/v) trypsin solution.

Cell cycle and harvest time:
The cell cycle of the untreated V79 cells lasts for about 12 - 14 hours under the selected culture conditions (last measurement based on the BrdU method of Speit et al. [15]: 12 hours; May 2012). Thus, a harvest time of 24 hours is about 2 times the normal cell cycle length.
V79 cells are an asynchronous cell population, i.e. at the time of test substance treatment there are different cell stages (G1-, S-, G2-phase and mitosis). Since the effect on these cell stages may vary for different test substances, more than one harvest time after treatment may be appropriate.
Furthermore, substance-induced mitotic delay may considerably delay the first post- treatment mitosis. Therefore, delayed harvest times (e.g. 44 hours) and prolonged exposure periods (e.g. 24 hours treatment) may be required for the detection of several substances.

S9 fraction
The S9 fraction was prepared according to Ames et al. (1975, Mut. Res, 31, 347 pp)) at BASF SE in an AAALAC- approved laboratory.3.4.2. Pretests for dose selection

Pretest:
In the pretest for toxicity based on the purity and the molecular weight of the test substance 1 300 µg/mL (approx. 10 mM) N-(2-Hydroxyethyl)-2-pyrrolidon was used as top concentration. The cells were prepared at a harvest time of 24 hours (about 2 cell cycles) after 4 and 24 hours exposure time without S9 mix and after 4 hours exposure time with S9 mix. The pretest was performed following the method described for the main experiment. As indication of test substance toxicity relative increase in cell count and cell attachment (morphology) were determined for dose selection.
In the pretest various additional parameters were checked or determined for all or at least some selected doses. The following parameters are available: pH, solubility

Dose selection
Following the requirements of the current guidelines a test substance should be tested up to a maximum concentration of 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. In case of toxicity, the top concentration should produce 55% ± 5% cytotoxicity (relative increase in cell count [RICC] and/or proliferation index [CBPI] and/or replicative index [RI]) compared to the respective vehicle control. In the pretest the pH value was not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations tested. In addition, no test substance precipitation was observed up to the highest required test substance concentration. No cytotoxicity indicated by reduced RICC of about or below 40 - 50% was observed either after 4 hours and 24 hours treatment in the absence of S9 mix or after 4 hours treatment in the presence of S9 mix. The concentrations are given as rounded values by using a dilution factor of 2. At least three concentrations were evaluated to detect a possible dose-response relationship. At least 2 cultures were prepared per test group, and at least 1 000 cells per culture were evaluated for the occurrence of micronucleated cells.

Test substance preparation
The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution.
The substance was dissolved in culture medium (MEM). To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted from the stock solution according to the planned doses. All test substance formulations were prepared immediately before administration.

3.6.3. Preparation of test cultures

The stocks of cells (1.0-mL portions) were thawed at 37°C in a water bath. 0.5 mL were pipetted into 25 cm2 cell culture flasks containing 5 mL MEM (incl. 10% [v/v] FCS). The flasks were subsequently incubated at 37°C, 5% (v/v) CO2 and relative humidity of ≥ 90% until they have reached confluency of at least 50% (duration about 2 – 4 days). The medium was replaced after about 24 - 30 hours to remove any dead cells. Prior to the preparation of the final test cultures, the cells may run through max. 15 routine passages.
After the "last" routine passage, there was another passage to prepare test cultures:

Seeding of the cells
A single cell suspension with the required cell count (3 - 5x105 cells per culture, depending on the schedule) was prepared in MEM incl. 10% (v/v) FCS. 5 mL cell suspension was transferred into 25 cm² cell culture flasks using a dispenser. Subsequently, the test cultures were incubated at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity. The cultures were visually checked for attachment and viability before treatment of the test cultures.

Definition of test cultures:
A test culture consists of two cytospin preparations on glass slides. A test group consists of two separately treated test culture flasks, means four preparations and thus of four slides.

Treatment of test cultures:
After the attachment period, about 20 - 24 hours after seeding, the medium was removed from the flasks and the treatment medium was added. The cultures were incubated for the respective exposure period at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity.

Treatment of the cultures

Test groups MEM medium (with or without FCS)*
[mL] Vehicle or
test substance preparation in vehicle [mL] S9 mix

[mL]
Without S9 mix
Vehicle control 4.0 1.0 -
Test groups 4.0 1.0 -
Positive control (EMS) 4.0 1.0 -
With S9 mix
Vehicle control 3.0 1.0 1.0
Test groups 3.0 1.0 1.0
Positive control (CPP) 3.0 1.0 1.0
* For exposure in the absence of S9 mix MEM medium with 10% (v/v) FCS was used.

At the end of the exposure period, the medium was removed and the cultures were rinsed twice with 5 mL HBSS (Hanks Balanced Salt Solution). Subsequently, 5 mL MEM (incl. 10% [v/v] FCS) supplemented with CytB (final concentration: 3 µg/mL; stock: 0.6 mg/mL in DMSO was added and incubated at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity for the respective recovery time. In the case of 24-hour continuous exposure, CytB was added to the treatment medium at start of treatment, and cell preparation was started directly at the end of exposure. At 44 hours preparation interval in the presence of S9 mix the supplementation of CytB was 24 hours before preparation of the cultures.

Preparation of the test cultures:

Cell harvest and preparation of slides:
The cells were prepared based on the method described by Fenech (1995, Mut Res., 285, 35 pp). Just before preparation the culture medium was completely removed. Single cell suspensions were prepared from each test group by trypsination. Then, the cell numbers per flask of each single cell suspension were determined using a cell counter. Subsequently, 5x104 cells per slide were centrifuged at 1 400 rpm for 7 minutes onto labelled slides using a Cytospin centrifuge. After drying, the slides were fixed in 90% (v/v) methanol for 10 minutes.

Staining:
Before scoring, the slides were stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI, stock 5 mg/mL) and propidium iodide (PI; stock: 5 mg/mL) in Fluoroshield™ (Sigma-Aldrich) at a concentration of 0.25 µg/mL each. By the use of the combination of both fluorescence dyes it can be differentiated between DNA (DAPI; excitation: 350 nm, emission: 460 nm) and cytoplasm (PI; excitation: 488 nm, emission: 590 nm).












Evaluation criteria:
Assessment criteria
A test substance is considered "positive" if the following criteria are met:
- A significant, dose-related and reproducible increase in the number of cells containing micronuclei was observed.
- The number of micronucleated cells exceeded both the value of the concurrent negative control and the range of our laboratory’s recent negative control data.

A test substance generally is considered "negative" if the following criteria are met:
• The number of micronucleated cells in the test groups is not distinctly increased above the concurrent negative control and is within our laboratory’s recent negative control data range
Statistics:
The statistical evaluation of the data was carried out using the MUVIKE program system. The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test were statistically significant compared with the respective negative control, labels (* p ≤ 0.05, ** p ≤ 0.01) were printed in the tables of the report.
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Additional information on results:
MICRONUCLEUS ANALYSIS
No biologically relevant increase in the number of micronucleated cells was observed either without S9 mix or after the addition of a metabolizing system. In both experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.2 – 0.6% micronucleated cells) were close to the concurrent negative control values (0.5 – 0.8% micronucleated cells) and clearly within our historical negative control data range (0.1 - 1.8% micronucleated cells).
The positive control substances EMS (without S9 mix; 300 and 400 µg/mL) and CPP (with S9 mix; 0.5 µg/mL) induced statistically significant increased micronucleus frequencies in both independently performed experiments. In the absence and presence of metabolic activation the frequency of micronucleated cells (2.5 – 4.0% micronucleated cells) was clearly above the range of the lab's historical negative control data range (0.1 - 1.8% micro- nucleated cells) and within the historical positive control data range (2.3 – 26.6% micronucleated cells).

RELATIVE INCREASE IN CELL COUNT (RICC)
In addition, in both main experiments in the absence and the presence of S9 mix cell growth indicated by the parameter relative increase in cell count was not adversely influenced after test substance exposure under any experimental condition.

PROLIFERATION INDEX (CBPI)
No reduced proliferative activity was observed either after 4 hours exposure interval in the absence and presence of S9 mix or after 24 hours continuous test substance treatment in the test groups scored for cytogenetic damage.

REPLICATIVE INDEX (RI)
In addition, in both main experiments in the absence and the presence of S9 mix the replicative index was not relevant decreased after test substance exposure under any experimental condition.

CELL MORPHOLOGY
In this study, cell attachment or cell morphology was not adversely influenced (grade > 2) at any concentration tested for the occurrence of micronuclei.

According to the results of the present in vitro micronucleus assay, the test substance N-(2- Hydroxyethyl)-2-pyrrolidon did not lead to a biologically relevant increase in the number of micronucleated cells either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. The frequencies of micronuclei after test substance treatment were close to the range of the concurrent negative control values at both exposure times and clearly within the range of our historical negative control data

The number of micronucleated cells in the vehicle control groups were within the historical negative control data range and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of micronuclei induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system 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.

CONCLUSION
Thus, under the experimental conditions chosen, the conclusion was drawn that N-(2- Hydroxyethyl)-2-pyrrolidon has not the potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

 

 

 

 

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Thus, under the experimental conditions chosen here, the conclusion is drawn that N-(2- Hydroxyethyl)-2-pyrrolidon has not the potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.
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 compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
20, 100, 500, 2500 and 5000 µg/plate (standard plate test and preincubation test)
Vehicle / solvent:
- Vehicle used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, water was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
with S-9 mix for strains TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
Remarks:
without S-9 mix for strains TA 1535, TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenyldiamine (NOPD)
Remarks:
without S-9 mix for strain TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S-9 mix for strain TA 1537

Migrated to IUCLID6: (AAC)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S-9 mix for strain E. coli WP2 uvrA

Migrated to IUCLID6: (4-NQO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
Plate incorcoration method:
- Exposure duration: ca. 48-72 hours at 37°C in the dark
Preincubation method:
- Preincubation period: 20 minutes at 37°C
- Exposure duration: ca. 48-72 hours at 37°C in the dark

NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Decrease in the number of revertants
- Reduced his- or trp- background growth
- Reduction in the titer
Evaluation criteria:
The test chemical is considered positive in this assay if a dose-related and reproducible increase in the number of revertant colonies (i.e. about doubling of the spontaneous mutation rate) in at least one tester strain is observed either without S-9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if the number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation: No precipitation of test substance was found.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No bacteriotoxic effect was observed.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
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 compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/ß-naphthoflavone induced rat liver S9-mix
Test concentrations with justification for top dose:
40.3, 80.6, 161.3, 322.5, 645.0, 1290.0 µg/mL
Vehicle / solvent:
Deionised local tap water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation

Migrated to IUCLID6: Dissolved in nutrient medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
With metabolic activation

Migrated to IUCLID6: Dissolved in Dimethylsulfoxide
Details on test system and experimental conditions:
The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
Evaluation criteria:
The gene mutation assay is considered acceptable if it meets the following criteria:
- The numbers of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data.
- The positive control substances should produce a significant increase in mutant colony frequencies.
- The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. An increase of the induction factor exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the second experiment without metabolic activation at 161.3, 322.5, and 645.0 µg/mL in culture I, and at 322.5, 645.0, and 1290 µg/mL in culture
II, and in culture II with metabolic activation at 80.6 and 161.3 µg/mL. However, these increases were based on rather low mutation frequencies of the corresponding solvent controls of just 2.5, 2.4, and 4.1 colonies per 10^6 cells. Furthermore, the absolute values of the mutation frequency induced by the test item remained well within the historical range of solvent controls and there was no dose dependent increase as indicated by the lacking statistical significance. Therefore, the increase of the induction factor was judged as biologically irrelevant fluctuation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The mutagenicity of N-(2-hydroxyethy)-2-pyrrolidone with different purity grades has been assessed in several reverse mutation assays by the manufacturer. In the first test (GLP compliant and according to OECD guideline 471), a test substance of lower purity (98.1%) was tested using Salmonella typhimurium strains TA1535, TA100, TA1537, TA98 and Escherichia coli strain E.coli WP2 uvrA in a standard plate test (BASF AG, 2001). In the first experiment, 20, 100, 500, 2500 and 5000 µg/plate were tested in all strains. In the second experiment, concentrations of 100, 500, 2500, 5000 and 7500 µg/plate were tested only in the strains TA1535 and TA100. All tests were performed both with and without metabolic activation (Aroclor-induced rat liver S-9mix).

No precipitation of the test substance was found and no bacteriotoxic effect was observed. In strains TA1537, TA98 and E.coli WP2 uvrA, no increase in the number of his+ or trp+ revertants was reported. In TA1535, mutagenicity was observed both with and without metabolic activation from about 500 µg/plate (factor 2.2- 3.2) onward with an increase in the number of his+ revertants by a factor of 15.6 - 20.5 at 7,500 µg/plate. In TA100, a slight increase in the number of revertant colonies with and without S-9 mix over a dose range of about 2,500 µg -7,500 µg/plate (factor 1.4-3.4) was reported.

According to the results of this study, the test sample (purity 98.1%) was mutagenic in the Salmonella typhimurium reverse mutation assay under the experimental conditions used. The positive reactions were considered to be caused by impurities as confirmed by subsequent experiments.  

In the second test (GLP compliant and according to OECD guideline 471), the test substance with a higher purity (99.8%; in line with specification provided in chapter 1.2) was tested using the same Salmonella typhimurium strains TA1535, TA100, TA1537, TA98 and Escherichia coli strain E.coli WP2 uvrA in a standard plate test and in a preincubation test (BASF AG, 2004a). In both tests, the concentrations tested were: 20, 100, 500, 2500 and 5000 µg/plate. All tests were performed both with and without metabolic activation (Aroclor-induced rat liver S-9 mix). No precipitation of the test substance was found and no bacteriotoxic effect was observed. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolising system. According to the results of this study, the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay under the experimental conditions used.

 

Due to the different results in the two studies as reported above a follow-up study was conducted (BASF AG, 2004b). Two different batches were tested, N-(2 -Hydroxyethyl)-2-pyrrolidon with a purity of 99.8% (the batch which was tested in the BASF AG 2004a study) and with a purity of 98.1% (the batch which was tested in the BASF AG 2001 study) using Salmonella typhimurium strain TA1535. Concentrations of 20, 100, 500, 2500 and 5000 µg/plate were tested in the absence and presence of S-9 mix. The substance with a purity of 99.8% was negative, while the substance with a purity of 98.1% was positive both in the absence and presence of S-9 mix. The authors concluded that impurities were the cause of the positive reaction found with the test substance with the lower purity (see BASF AG, 2001), the substance itself (purity of 99.8%) did not show a positive reaction in the Ames test (BASF AG, 2004a).

In addition to these more recent studies, a reverse mutation assay had been performed with three different samples of N-(2 -hydroxyethyl)-2 -pyrrolidone, two production lots and a purified sample, in Salmonella typhimurium strains TA98, TA1537, TA1538, TA100 and TA1535 (Arthur D. Little Inc., 1982). No further information on the purity of the different lots was provided. The tested concentrations were: 0.32, 1.6, 8, 40, 200 μl/plate and the tests were performed without and with S9 -mix.

The first production lot gave a positive (mutagenic) response in TA100 and TA1535 even in the absence of metabolic activation. Toxicity (a sparse lawn) was evident at 200 µl/plate in all strains and slight toxicity was observed in TA100 at 40 µl/plate.

The second production lot gave a positive (mutagenic) response in TA100 and TA1535 even in the absence of metabolic activation, confirming the results obtained with the first lot of this material. Toxicity (a sparse lawn) was evident at 200 µl/plate in all strains and was also evident at all concentrations in TA98 (i.e., there was a reduced number of revertants in contrast to the DMSO control plates).

Regarding the purified sample, a weak mutagenic activity was evident in TA1535 both in the absence and presence of metabolic activation. A doubling of the number of revertants over the control background was observed only at the highest concentration tested (200 µl/plate). Toxicity was evident in TA100 at 200 µl/plate (lawn effect) and at all the lower concentrations tested (number of revertants lower than the number obtained in the DMSO control backgrounds). The purified sample was significantly less potent as the production lots of N-(2 -hydroxyethyl)-2 -pyrrolidone at a technical grade which supports the assumption that impurities had caused the positive reaction.

N-(2 -hydroxyethyl)-2 -pyrrolidone) with a purity >= 99.5% was studied in a mammalian gene mutation assay (V79/HPRT) according to OECD Guideline 476 and under GLP in two independent experiments, using two parallel cultures each (Harlan, 2012). The first experiment was performed with and without S9-mix and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. Cytotoxicity was not observed. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Therefore, N-(2-Hydroxyethyl)-2-pyrrolidon is considered to be non-mutagenic in this HPRT assay.

In a recent Micronucleus test, N-2 -hydroxyethyl-2 -pyrrolidon was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity). Two independet experiments were carried out, both with and without the addition of liver S9 mix from induced rats. According to an initial range finding cytotoxicity test concentrations up to 1300 µg/ml (10 mM) were chosen. A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. 2000 cells for each test group.The negative controls gave frequencies within the negative control data range for V79 cells. Both positive substances, EMS and cyclophosphamide led to the expected increase in the number of cells containing micronuclei. No cytotoxicity indicated by reduced relative increase in cell count , proliferation index or replicative index was observed up to the higest required test substance concentration. The test substrance did not cause any biologically relevant increase in the number of cells containing micronuclei either without or after adding a metabolizing system (BASF SE, 2014).

N-2-hydroxyethyl-2-pyrrolidone specified as a technical grade (no further data on purity) was also evaluated in four in vitro mammalian short-term genotoxicity assays measuring different endpoints (Arthur D. Little Inc., 1982). Three assays were conducted with Chinese hamster ovary (CHO) cell lines and consisted of a mammalian gene mutation assay (HGPRT) and two cytogenetic assays (chromosome aberrations and sister chromatid exchanges). The fourth experiment consisted of a neoplastic transformation assay using a mouse fibroblast cell line. All tests were performed in absence of an exogenous metabolic activation system.

 

In the chromosome aberration test, the substance was tested at concentrations of 125 – 1000 µg/ml. The substance was not cytotoxic to mass cultures of CHO cells from 0.01 - 250 µg/mL. Since the substance was not toxic, the highest concentration used in the cytogenetic assays was 1000 µg/ml. Of 100 metaphases examined, 3 chromosome aberrations were found in CHO cells treated with 125 μg/ml of the substance. This number was calculated to be statistically significant (P<0.05) over the zero aberration of the control. However, no aberrations were found in cells treated with higher concentrations of the chemical. According to the authors, one or two aberrations were often observed in control cultures historically, therefore the aberrations scored at this particular concentration were considered to be not biologically significant. The positive control induced the expected number of chromosome aberrations.

In the mammalian gene mutation (HGPRT) assay, the substance (technical grade) was tested at concentrations of 125 – 1000 µg/ml. All tested concentrations of the substance uniformly induced a higher mutation frequency than the control in CHO cells. However, no dose-dependent effect was observed. Since the substance was not cytotoxic, the mean number of mutant colonies per plate between the treated cultures and the control can be compared. When tabulated as such, cultures treated with the substance showed a 2-3 fold increase in mutants per plate over the untreated control. This increase, although weak, was found to be statistically significant (p<0.05), with the exception of the 500 μg/ml concentration which had a high standard deviation.

In the sister chromatid exchange (SCE) assay, the substance of technical purity was tested at concentrations of 125 – 1000 µg/ml. The substance was not cytotoxic to mass cultures of CHO cells from 0.01 - 250 µg/ml. Since the substance was not toxic, the highest concentration used in the cytogenetic assays was 1000 µg/ml. At the two highest concentrations of 500 and 1000 μg/ml, the substance induced a slight but statistically significant increase in the number of SCE above the control. No apparent cytotoxicity of the target cells was associated with these high concentrations as judged by the mitotic indexes of the control and treated cells.

 

In the cell transformation assay with BALB/c-3T3 cells, the substance of technical purity was tested at concentrations of 2 – 250 µg/ml.The test compound produced a negative response. The positive control MCA induced a mean of 3.00 Type III foci per plate which is within the normal range of transformation induced by this carcinogenic arylaromatic hydrocarbon. The results of the cytotoxicity assay showed higher surviving fractions of cells treated with the substance than the control. The lower cell counts in controls may be due to incomplete trypsinisation of cells from the plates in this particular experiment.


Short description of key information:
Based on all the available genotoxicity studies in bacteria and mammalian cell cultures, N-(2-hydroxyethyl)-2-pyrrolidon is not considered genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The substance when tested at high purity meeting the specification provided in chapter 1.2, was found to be non-mutagenic in the Ames test and in the mammalian gene mutation assay (HPRT). In an in vitro micronucleus test with and without liver S9 mix and in a chromosomal aberration assay without S9 mix, no clastogenicity or aneugenic activity was observed. Based on the results of the available data, N-(2-hydroxyethyl)-2-pyrrolidon does not need to be classified according to Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.