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Genetic toxicity: in vitro

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Administrative data

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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
N-(n-octyl)-2-pyrrolidinone
EC Number:
403-700-8
EC Name:
N-(n-octyl)-2-pyrrolidinone
Cas Number:
2687-94-7
Molecular formula:
C12 H23 N O
IUPAC Name:
N-(n-octyl)-2-pyrrolidinone
Details on test material:
- Name of test material (as cited in study report): N-(N-Octyl)pyrrolidone; Substance No. 91/453
- Physical state: colorless liquid
- Analytical purity: 99.3 %
- Lot/batch No.: 103/90
- Storage condition of test material: refrigerator

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 fraction (Aroclor 1254-induced)
Test concentrations with justification for top dose:
25 - 150 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S9: cyclohexylamine, without S9: ethylmethanesulphonate
Details on test system and experimental conditions:
Test principle
The substance N-(n-Octyl)pyrrolidone was assessed for its potential to induce structural and/or numerical chromosomal aberrations in V79 cells in vitro both in the presence and absence of a metabolizing system.

According to pretests for the determination of the highest experimental dose and in consideration of the cytotoxicity actually found in the main experiment, 25 µg/ml, 50 µg/ml and 75 µg/ml culture medium (18 hours sampling time) and 75 µg/ml culture medium (28 hours sampling time) in the experiment without S-9 mix, or 50 µg/ml, 100 µg/ml and 150 µg/ml culture medium (18 hours sampling time) and 150 µg/ml culture medium (28 hours sampling time) in the experiment with metabolic activation, were evaluated.
Preparation of chromosomes were done 18 hours (low, intermediate and top dose) and 28 hours (top dose only) after treatment, which lasted for about 4 hours. Duplicate cultures were used for all experimental groups. About 2 - 3 hours prior to harvesting the cells, Colcemid was added to arrest cells in a metaphase-like stage of mitosis (C-metaphase). After preparation of the chromosomes and staining with Giemsa, 100 metaphases of each culture in the case of the test substance, untreated control and carrier control, or 50 cells of each culture in the case of the concurrent positive controls, were analyzed for chromosomal aberrations.

The negative controls gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control chemicals led to the expected increase in the number of cells containing structural chromosomal aberrations. According to the results of the present study, the test substance did not cause any increase in the number of structural aberrant metaphases incl. and excl. gaps at both sampling times either without S-9 mix or after adding a metabolizing system. An increase in the frequency of cells containing numerical aberrations was not demonstrated either.
Thus, under the experimental conditions chosen here N-(n-Octyl)pyrrolidone has neither a chromosome-damaging (clastogenic) effect nor an aneugenic activity in V79 cells in vitro.

TEST SUBSTANGE ANALYSIS
The stability of the test substance throughout the study period will be verified analytically by reanalysis at a later date. The results of this analysis may be requested from the sponsor.
The stability of the test substance in the carrier DMSO and in distilled water was determined analytically.


CELL SYSTEM
Cell line, storage
The V79 cell line (1, 2) being derived from the Chinese hamster has a
- high proliferation rate (doubling time of about 12-16 hours)
- high plating efficiency (.h 90%)
- stable karyotype (modal number of 22 chromosomes).

Stocks of the V79 cell line (1 ml portions) were maintained at -196°C in liquid nitrogen using 7% DMSO in culture medium as a cryoprotectant. Each batch used for cytogenetic experiments was checked for
- mycoplasma contamination
- karyotype stability
- plating efficiency (incl. vital staining).

Cell culture
Stock solutions were thawn at 37° C in a water bath and volumes of 0.5 ml were transferred into 25 cm² plastic flasks which contain about 5.0 ml MEM (Minimal Essential Medium incl. glutamine), supplemented with 10 % FCS (Fetal Calf Serum) and antibiotics. Cells were grown at 37° C with 5 % CO2 and 90 % humidity and subcultured twice weekly. Cell monolayers were suspended in culture medium after dispersion with 2.5 % trypsin solution (about 0.1 ml).

TISSUE PREPARATION
S-9 fraction
The S-9 fraction is prepared according to Ames et al. At least 5 male Sprague-Dawley rats (200 - 300 g) receive a single intraperitoneal injection of 500 mg Aroclor 1254 (as a 20 % solution in peanut oil - w/v) per kg body weight 5 days before sacrifice. During this time the animals are housed in Makrolon cages in air-conditioned rooms. The day/night rhythm is 12 hours (light period from 6.00 - 18.00 hours and dark period from 18.00 - 6.00 hours). Standardized pelleted feed and tap water from bottles are available ad libitum. 5 days after administration the rats are sacrificed, and the livers are prepared (all preparation steps for obtaining the liver microsome enzymes are carried out using sterile solvents and glassware at a temperature of +4°C). The livers are weighed and washed in an equivalent volume of a 150 mM KC1 solution (1 ml/g wet liver), then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 ml portions of the supernatant (so-called S-9 fraction) are quickly deep-frozen in dry ice and stored at -70°C to -80°C. Preparation of S-9 fraction: March 24, 1992.

S-9 mix
The S-9 mix is prepared freshly prior to each experiment. For this purpose, a sufficient amount of S-9 fraction is thawed at room temperature and 1 volume of S-9 fraction is mixed with 9 volumes of S-9 supplement (cofactors). This preparation, the so-called S-9 mix, is kept on ice until used. The concentrations of the cofactors in the S-9 mix are:
MgCl 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM.
The phosphate buffer is prepared by mixing an Na2HPO4 solution with an NaH2PO4 solution in a ratio of about 4:1.

EXPERIMENTAL PERFORMANCE
Pretest for dose selection
The doses were determined from appropriate pretests with cultures exposed to a wide dose range of the test article, i.e. 0.1 µ/ml - 2000 µg/ml culture medium both without S-9 mix and with S-9 mix. In the course of this, various parameters were checked for all or at least for some selected doses; the results were given in the following tables. As a rule the highest test substance
concentration should not exceed a limit of 10 mM as recommended by an ICPEMC Task group.
According to the findings of the pretests, 50 µg/ml without S-9 mix and 100 µg/ml with metabolic activation were selected as top doses both after harvest times of 18 and 28 hours. This selection was based on the number of analyzable cells.

Cell attachment to the slides:
At 100 µg/ml there was a reduced attachment of the cells to the slides, indicated by cell morphology, i.e. cells were partly rounded. At 500 µg/ml most cells were detached and rounded.
At doses of 1000 µg/ml or 2000 µg/ml there was no detachment of the cells to the slides; i.e. all cells were rounded.

Thus, for the main experiment the following doses were selected:
Doses without S-9 mix Harvest times
75 µg/ml* (0.38 mM) 18 hours, 28 hours
50 µg/ml (0.25 mM) 18 hours, 28 hours
25 µg/ml (0.13 mM) 18 hours
5 µg/Ml (0.025 mM) 18 hours
1 µg/ml* (0.005 MM) 18 hours
Doses with S-9 mix Harvest times
150 µg/ml* (0.16 mM) 18 hours, 28 hours
100 µg/ml (0.51 MM) 18 hours, 28 hours
50 µg/ml (0.25 mM) 18 hours
10 µg/ml (0.05 MM) 18 hours
2 µg/ml* (0.01 mM) 18 hours
The additionally selected higher and lower doses (marked with *<), i.e. 75 µg/ml and 1 µg/ml (without S-9 mix) or 150 µg/ml and 2 µg/ml (with S-9 mix), were planned to be evaluated only if the results obtained regarding cytotoxicity deviated from those obtained in the pretest.
In addition, in cases of questionable or conflicting results an evaluation of more than 3 dose levels might also become necessary.

Sampling times
Chromosomal aberrations were generally analyzed in the first metaphase after they were formed to avoid loss during mitoses or conversion of the initial aberrations into more complex derivatives during subsequent cell cycles. Since aberrations were induced by the majority of chemicals during DNA replication, harvest time must allow cells to progress through S-phase after treatment. Because V79 cells are asynchronous and different chemicals might affect different stages of the cell cycle more than one sampling time is necessary. Furthermore, mitotic delay may result from clastogen exposure and thus considerably delay the first post-treatment mitosis. Therefore, samples taken at 18 hours (low, intermediate and top dose) and 28 hours (top dose only) after the beginning of a 4-hour treatment will cover the intervals in which maximum aberration frequencies are expected.

Control articles
Untreated controls
Nothing was added to the negative controls with and without S-9 mix which contained cells and culture medium only.

Carrier controls
The carrier controls with and without S-9 mix only contained the vehicle for the test substance at the same concentration and volume used in the test culture.

Positive controls
The following positive control substances were used to demonstrate the sensitivity of the test method and the activity of the S-9 mix:

- Without metabolic activation
750 µg ethyl-methane-sulfonate (EMS)/ml culture medium added in a volume of 1.0 ml

- With metabolic activation (S-9 mix)
1 µg cyclophosphamide/ml culture medium added in a volume of 1.0 ml

Preparation of test cultures
*Logarithmically growing cultures more than 50 % confluent were trypsinized (2.5 % trypsin solution and Ca-Mg-free Hanks Balanced Salt Solution HBSS). Prior to trypsin treatment the cells were rinsed once with 5 ml Ca-Mg-free HBSS.
* This process was stopped by adding MEM supplemented with 10% FCS.
* A single suspension was prepared and about 5 ml MEM supplemented with 10% FCS and containing about 50 000 cells were seeded in each chamber of Quadriperm dishes. Two chambers of a Quadriperm dish were used for one test culture.
* The Quadriperm dishes were incubated at 37°C with 5 % CO2 and 90 % humidity.

Treatment of the test cultures
24 hours after seeding and incubating the cells the medium was replaced by serum-free medium. The test article, dissolved in 50 µl DMS0, was added to the culture medium with or without 1 ml S-9 mix. Concurrent negative and positive controls were tested in parallel. After incubation (37°C, 5 %, CO2, 90 % humidity) for 4 hours the serum-free medium was replaced by MEM supplemented with 10 % FCS after rinsing twice with Hanks balanced salt solution (HBSS). Subsequently, the Quadriperm dishes were incubated again until the cells were harvested.

Cell harvest and preparation of metaphase spreads
The cells were prepared based on the method described by SCHMID, W and SPEIT, G and HAUPTER, S.
- 2 - 3 hours prior to harvesting the cells, 0.2 µg Colcemid/ml culture medium (= 1 µg Colcemid dissolved in 0.1 ml PBS/culture) was added in each chamber in order to arrest mitosis in the metaphase.
- After incubation at 37°C the culture medium was completely removed.
- For hypotonic treatment 5 ml of a 0.4% KCl solution which was at 37°C was added for about 20 minutes.
- Subsequently 5 ml of fixative (methanol : glacial acetic acid / 3 :1) which was at 4°C was added and kept for at least 15 minutes and then replaced. After about another 10 minutes fixative was replaced again and kept for at least 5 minutes at room temperature for complete fixation.
- The slides were taken out of the Quadriperm chambers, briefly dripped off and than rapidly passed through a Bunsen burner flame.
- The preparations were dried in the air and subsequently stained in a solution of Giemsa and Titrisol (15 ml Giemsa, 185 ml Titrisol pH 7.2) for 10 minutes.
- After being rinsed twice in aqua dest. and clarified in xylene, the preparations were mounted in Corbit-Balsam.

EVALUATION
Chromosome analysis
As a rule, the first 100 consecutive well-spread metaphases of each culture for the test substance, negative and carrier controls or 50 cells of each culture for the positive controls were counted, and if the cell had 20 -22 chromosomes, they were analyzed for chromosome aberrations according to the following definitions:
Structural chromosome aberrations
-G' and GV chromatid gap and isochromatid gap unstained regions (so-called achromatic lesions) without dislocation of the segment which appears to be separated.
-B' and B" chromatid break and chromosome break visible discontinuity in chromatid or chromosome structure with lateral or longitudinal dislocation of the fragment.
-F' and V' chromatid fragment and chromosome fragment acentric chromosome segments which
occur singly or in pairs.
-D' and DV chromatid deletion and chromosome deletion loss of a segment on the level of
chromatids or chromosomes.
-m. A. multiple aberrations metaphases with 5 or more aberrations excl. gaps.
-disintegration The chromosomes being present as of chromosomal irregular particles, a chromosomal structure cannot be detected any (pulverization) longer.

-Exchanges (translocations)
These exchange aberrations (Ex) are divided into intrachanges and interchanges:
- Int' and Int" intrachanges on the level of chromatids and chromosomes the joining of broken ends capable of reuniting two or several chromatid regions within a chromosome, e.g., centric ring chromosomes, pericentric inversions.
- I' and V' interchanges on the level of chromatids and chromosomes the joining of broken ends capable of reuniting two or several chromosomes.
They are classified as:
- symmetric interchanges, e.g., reciprocal translocations between non-homologous chromosomes,
centric fusions, quadriradial structures
- asymmetric interchanges, e.g., dicentric and polycentric chromosomes, triradial and quadriradial structures.

Numerical chromosome aberrations (so-called heteroploidies)
- Aneuploidy metaphases with absent (hypoploid) or additional (hyperploid) chromosomes; Only hyperploid metaphases are registered.
- Euploidy changes in the number of chromosomes by whole chromosome sets.

Slides were coded before microscopic analysis. If only a few cells were found or if the metaphases were of low quality, a chromosome analysis was not carried out.

Mitotic index
A mitotic index based on 1500 cells/culture was determined for all dose groups that yielded metaphase cells, for the carrier and untreated controls (= negative controls) and for the positive controls.

Cell counts
For determination of cytotoxicity additional cell cultures (using 25 cm² plastic flasks) were treated in the same way as in the main experiment. Growth inhibition was estimated by counting the number of cells in the dose groups in comparison to the concurrent carrier control at the end of the culture period using a counting chamber.

Cell morphology
About 3 hours after test substance treatment cultures of all test groups were checked regarding cell morphology, which is an indication of attachment of the cells to the slides .

Treatment conditions
pH values and osmolality were measured. The solubility of the test substance in the carrier used and in the aqueous culture medium was checked to ensure proper culturing and to avoid extreme treatment conditions.
Evaluation criteria:
see above
Statistics:
The statistical evaluation of the data was carried out using the MUCHAN program system (BASF AG). For each group the proportion of metaphases with aberrations was calculated. A comparison of each dose group with the solvent control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferoni-Holm corrected over the dose groups separately for each time point and was performed one-sided. If the results of this test are significant, labels (* p < 0.05, ** p < 0.01) were printed in the tables.

Results and discussion

Test results
Species / strain:
mammalian cell line, other: V79 cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Results:
Assay without S-9 mix; 18 hours harvest time

Untreated control:
5 (2.5 %) metaphases incl. gaps
2 (1.0 %) metaphases excl. gaps, i.e. 1 x FV; 1 x Ex
1 (0.5 %) hyperploid cell
No (0.0 %) polyploid cells

Carrier control:
9 (4.5 %) metaphases incl. gaps
6 (3.0 %) metaphases excl. gaps, i.e. 1 x B'; 1 x B"; 4 x Ex
1 (0.5 %) hyperploid cell
3 (1.5 %) polyploid cells

25 µg/ml:
6 (3.0%) metaphases incl. gaps
4 (2.0%) metaphases excl. gaps, i.e. 1 x B'; 1 x B"; 2 x Ex
No (0.0) hyperploid cells
1 (0.5%) polyploid cell

50 µg/ml:
7 (3.5%) metaphases inl. gaps
3 (1.5%) metaphases excl. gaps, i.e. 1 x B'; 1 x B"; 1 x V"
2 (1.0%) hyperploid cells
2 (1.0%) polyploid cells

75 µg/ml:
4 (2.0%) metaphases incl. gaps
2 (1.0%) metaphases excl. gaps, i.e. 2 x Ex
No (0.0%) hyperploid cells
1 (0.5%) polyploid cell

750 µg EMS/ml:
With 21 (21%) aberrant cells incl. gaps and 17 (17%) aberrant mitosis excl. gaps including 16 cells with exchanges, the positive control substance led to the expected increase in the number of chromosomally damaged cells.

Assay without S-9 mix; 28 hours harvest time

Untreated control:
8 (4.0%) metaphases incl. gaps
2 (I.0) metaphases excl. gaps, i.e. 1 x B'; 1 x 2Ex
No (0.0%) hyperploid cells
1 (0.5%) polyploid cell

Carrier control:
10 (5.0%) metaphases incl. gaps
2 (1.0) metaphases excl. gaps, i.e. 1 x 0V; 1 x Ex
2 (1.0%) hyperploid cells
4 (1.9%) polyploid cells

75 µg/ml:
5 (2.5%) metaphases inl. gaps
2 (1.0%) metaphases excl. gaps, i.e. 1 x 0"; 1 x B"
No (0.0%) hyperploid cells
2 (1.0) polyploid cells

Assay with S-9 mix; 18 hours harvest time
Untreated control:
7 (3.5%) metaphases incl. gaps
4 (2.0%) metaphases excl. gaps, i.e. 1 x B'; 1 x B"; 1 x FV;
1 x Ex
No (0.0) hyperploid cells
No (0.0%) polyploid cells

Carrier control:
9 (4.5%) metaphases incl. gaps
5 (2.5%) metaphases excl. gaps, i.e. 1 x BV; 2 x DV; 1 x FV;
1 x Ex
No (0.0%) hyperploid cells
2 (2.0%) polyploid cells

50 µg/ml:
8 (4.0%) metaphases incl. gaps
3 (1.5%) metaphases excl. gaps, i.e. 1 x B"; 2 x V"
No (0.0%) hyperploid cells
3 (1.5%) polyploid cells

100 µg/ml:
5 (2.5%) metaphases incl. gaps
3 ( 1.5%) metaphases excl . gaps, i.e. 2 x B" ; 1 x m.A. incl. Ex
No (0.0) hyperploid cells
1 (0.5%) polyploid cell

150 mg/ml:
10 (5.0%) metaphases incl. gaps
3 (1.5%) metaphases excl. gaps, i.e. 1 x B'; 1 x FV; 1 x Ex
No (0.0%) hyperploid cells
6 (2.9%) polyploid cells

1 µg cyclophosphamide/ml:
With 22 (22%) aberrant cells incl. gaps and 19 (19) aberrant metaphases excl. gaps including 11 cells with exchanges, the positive control substance led to the expected increase in the number of chromosomally damaged cells.

Assay with S-9 mix; 28 hours harvest time
Untreated control:
8 (4.0%) metaphases incl. gaps
7 (3.5%) metaphases excl. gaps, i.e. 2 x B'; 1 x BV; 1 x DV;
2 x FV; 1 x 6V"
No (0.0) hyperploid cells
No (0.0%) polyploid cells

Carrier control:
5 (2.5%) metaphases incl. gaps
1 (0.5%) metaphase excl. gaps, i.e. 1 x B"
No (0.0%) hyperploid cells
No (0.0%) polyploid cells

150 µg/ml:
1 (0.5%) metaphase incl. gaps
1 (0.5%> metaphase excl. gaps, i.e. 1 x V"
No (0.0%) hyperploid cells
No (0.0%) polyploid cells

MITOTIC INDEX
The mitotic index based on 1500 cells per culture for the different test groups without and with metabolic activation can be seen an the following tables. The numbers of mitotic cells in the samples scored are given as "absolute" values. The “relative" figures are related to the corresponding solvent controls which are set 100 %. According to this, a suppression of the mitotic activity was not observed under any of the experimental conditions.

Remarks on result:
other: other: V79 cells
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

CONCLUSIONS

According to the results of the present in vitro cytogenetic study, the test substance N-(n-Octyl)pyrrolidone did not lead to an increase of structural chromosomal aberrations incl. and excl. gaps either without S-9 mix or after the addition of a metabolizing system; types and frequency of aberrations were within the range of that of the concurrent negative control values at both sampling times and within the range of the historical control data. An increase in the number of cells containing numerical chromosomal aberrations were not demonstrated either. Thus, under the experimental conditions chosen here N-(n-Octyl)pyrrolidone is evaluated neither to be a chromosome-damaging (clastogenic) agent nor to have any aneugenic activity under in vitro conditions using V79 cells.

Applicant's summary and conclusion