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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 487
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
N-(2-Hydroxyethyl) pyrrolidon: 99.795 %

Method

Species / strain
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.
Controls
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.

Results and discussion

Test results
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'.

Any other information on results incl. tables

 

 

 

 

Applicant's summary and conclusion

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.