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Genetic toxicity in vitro

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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
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
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
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.
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'.

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.

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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The substance N-Octylpyrrolidon was tested for its mutagenic potential based on the ability to induce point mutations in an Escherichia coli strain in a reverse mutation assay.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix (Aroclor 1254-induced)
Test concentrations with justification for top dose:
4 - 5000 µg/plate (SPT); 0.8 - 500 µg/plate (PIT)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: 4-nitroquinoline-N-oxide; with S9: 2-aminoanthracene
Details on test system and experimental conditions:
PRINCIPLE
Bacterial reverse mutation assays using amino-acid requiring strains of Escherichia coli are commonly employed as initial screening methods for detecting a point mutagenic activity of chemical substances and are used to screen for possible mammalian mutagens and carcinogens. The principle of these assays is that mutations are detected by the reversion of mutations present in the bacterial strains. This leads to a restoration of the functional capability of the bacteria to synthesize the essential amino acid and thus to the ability to grow in the absence of the amino acid required by the parent strains. Most of the substances are not mutagenic or carcinogenic themselves, but only after metabolic transformation, which in vivo is catalyzed mainly by the enzyme systems of the liver. Therefore, the tests are carried out not only directly, but also in the presence of an exogenous metabolic activation system. The most commonly used system is a cofactor supplemented postmitochondrial fraction (S-9) obtained from livers of rats treated with an enzyme-inducing agent, e.g. Aroclor 1254.

TEST SUBSTANCE ANALYSIS
The stability of the test substance throughout the study period was not determined analytically. However, the characterization was performed in October, whereas the study was conducted from September 19, 2001 (1st experiment) to October 4, 2001 (last experiment). Thus, it can be assumed that the test substance was stable throughout the study period. The stability of a comparable batch (103/90, test substance No.: 91/453) in the vehicle DMSO and in water has been verified analytically.

EXOGENOUS METABOLIC ACTIVATION
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 solution in corn oil with a concentration of 20 g/1 00 ml) per kg body weight 5 days before sacrifice. During this time, the animals are housed in Makrolon cages in air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20 – 24° C and a relative humidity of 30 – 70 %. 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 KCI solution (1 ml /1 g wet liver), then cut into small pieces and homogenized in three volumes of KCI 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 stored at -70° C to -80° C.

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:
MgCl2 8 mM
KCI 33 mM
Glucose-6-phosphatase 5 mM
NADP 4 mM
Phosphate buffer (pH 7.4) 15 mM
The phosphate buffer is prepared by mixing an Na2H-PO 4 solution with an NaH2PO4 solution in a ratio of about 4 :1. To demonstrate the efficacy of the S-9 mix in this assay, the S-9 batch was characterized with benzo(a)pyrene.

BACTERIA
For testing, deep-frozen (-70° C to -80° C) bacterial cultures are thawed at room temperature, and 0.1 ml of this bacterial suspension is inoculated in nutrient broth solution (8 g Difco nutrient broth + 5 g NaCl/liter) and incubated in the shaking water bath at 37° C for about 10 - 12 hours. As a rule, a germ density of > 10E8 bacteria/mI is reached. These cultures grown overnight are kept in iced water from the beginning of the experiment until the end in order to prevent further growth. The use of the strain mentioned is in accordance with the current scientific recommendations for the conduct of this assay.

Escherichia coli
Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E.coIi WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions. The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.

Checking out the tester strains
E.coli WP2 uvrA is checked for UV sensitivity. Tryptophan auxotrophy for both strains is automatically checked in each experiment via the spontaneous rate.

EXPERIMENTAL PROCEDURE
Choice of the vehicle
Due to the limited solubility af the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Mutagenicity tests
Standard plate test
The experimental procedure is based on the method of Ames et al.. Test tubes containing 2-mI portions of soft agar (overlay agar), which consists of 100 ml agar (0.6 % agar + 0.6 % NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 ml test solution or vehicle
0.1 ml fresh bacterial culture
0.5 ml S-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation).
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.

Composition of the minimal agar:
The composition of the minimal agar (SAl selective agar) is based an the description of Green, M.H.L. and Muriel, W.J., with the exception of solution E (tryptophan solution), which has previously been added to the soft agar:
300 ml solution B (agar)
100 ml solution A (saline solution)
8 ml solution C (glucose solution)
10 ml solution D (casein solution)
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

Preincubation Test
The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al.. 0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 -72 hours in the dark, the bacterial colonies are counted.

Titer determination
In the standard plate test, 0.1 ml of the overnight cultures is diluted to 10-6 in each case. Test tubes containing 2-ml portions of soft agar containing maximal amino acid solution (5 mM tryptophan) are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 ml vehicle (without and with test substance)
0.1 ml fresh bacterial culture (dilution: 10-6)
0.5 ml S-9 mix
In the preincubation test, 0.1 ml of the overnight cultures is diluted to 10-6 in each case. 0.1 ml vehicle (with and without test substance), 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for about 20 minutes using a shaker. Subsequently, 2 ml of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan) is added. After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.

Control articles
Negative controls
Parallel with each experiment, negative controls are carried out in order to check for possible contaminants (sterility control) and to determine the spontaneous mutation rate (vehicle control).

Sterility control
Additional plates are treated with soft agar, S-9 mix, buffer , vehicle or the test substance but without the addition of tester strains.

Vehicle control
The vehicle control with and without S-9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.

Positive controls
The following positive controls are used to check the mutability of the bacteria and the activity of the S-9 mix:

With S-9 mix
2-aminoanthracene (2-AA) (SIGMA, A-1 381)
- 60 µg/plate, dissolved in DMSO
- strain: Escherichia coli WP2 uvrA

Without S-9 mix
4-nitroquinoIine-N-oxide'(4-NQO) (SIGMA, N-81 41)
- 5 µg/plate, dissolved in DMSO
- strain: E. coli WP2 uvrA
The stability of the selected positive controls is well-defined under the selected culture conditions, since they are well-established reference mutagens.

Scope of tests and test conditions
1st Experiment
Strains: E.coli WP2 uvrA
Doses: 0; 20; 100; 500; 2,500 and 5,000 µg/plate
Vehicle: DMSO
Type of test: Standard plate test with and without S-9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment
Strains: E.coli WP2 uvrA
Doses: 0; 4; 20; 100; 500 and 1,000 pg/plate
Vehicle: DMSO
Type of test: Standard plate test with and without S-9 mix
Number of plates: 3 test plates per dose or per control

3rd Experiment
Strains: E.coli WP2 uvrA
Doses: 0; 0.8; 4; 20; 100 and 500 pg/plate
Vehicle: DMSO
Type of test: Preincubation test with and without S-9 mix
Number of plates. 3 test plates per dose or per control

EVALUATION
Mutagenicity
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, five doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st experiment.

Titer
The titer is generally determined only in the experimental parts with S-9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

Toxicity
Toxicity detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced trp- background growth)
- reduction in the titer
is recorded for all test groups both with and without S-9 mix in all experiments and indicated in the tables.

Solubility
Precipitation of the test material is recorded and indicated in the tables. As long as precipitation does not interfere with the colony scoring, 5 mg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/ substantiation.

Acceptance criteria
Generally, the experiment is to be considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data.
- The titer of viable bacteria was >10E8/ml.
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
-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 either without S-9 mix or after adding a metabolizing system. A test substance is generally considered non-mutagenic in this test if:
- The number of revertants for the tester strain was within the historical negative control range under all experimental conditions in three experiments carried out independently of each other.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: A bacteriotoxic effect was observed in the PIT at doses > 100 µg/plate and in the SPT at doses > 1000 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The substance N-Octylpyrrolidon was tested for mutagenicity in the Escherichia coli reverse mutation assay both in the standard plate test and in the preincubation test with and without the addition of a metabolizing system (S-9 mix) obtained from rat liver using the Escherichia coli strain WP2 uvrA.

MUTAGENICITY
Standard plate test
Tests without S-9 mix
E. coli WP2 uvrA: No increase in the number of trp+ revertants.

Tests with S-9 mix
E. coli WP2 uvrA: No increase in the number of trp+ revertants.

Preincubation test
Tests without S-9 mix
E. coIi WP2 uvrA: No increase in the number of trp+ revertants.

Tests with S-9 mix
E. coli WP2 uvrA: No increase in the number of trp+ revertants.

TOXICITY
A bacteriotoxic effect (reduced trp- background growth, decrease in the number of trp-revertants, reduction in the titer) was observed in the standard plate test from about 1,000 µg/plate onward.
In the preincubation assay bacteriotoxicity (slight decrease in the number of trp+ revertants, slight reduction in the titer) was observed with S-9 mix from about 100 µg/plate onward.

SOLUBILITY
No test substance precipitation was found.

DISCUSSION
According to the results of the present study, the test substance did not lead to an increase in the number of revertant colonies either without S-9 mix or after adding a metabolizing system in several experiments carried out independently of each other (standard plate test and preincubation assay).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Escherichia coli reverse mutation assay (standard plate test and preincubation test)

CONCLUSION

Thus, under the experimental conditions chosen here, it is concluded that N-Octylpyrrolidon is not a mutagenic agent in a bacterial reverse mutation test.

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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 strains tested; no tester strain included to detect crosslinking mutagens
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix (Aroclor 1254-induced)
Test concentrations with justification for top dose:
4 - 5000 µg/plate (SPT); 0.8 - 500 µg/plate (PIT)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

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 1 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 3:2.

Bacteria
The rate of induced back mutations of several bacteria mutants from histidine auxotrophy to histidine prototrophy is determined. The indicator organisms TA 1535, TA 1537, TA 98 and TA 100 selected by Ames especially for this purpose are derivatives of Salmonella typhimurium. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances. The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frame-shift mutagens. These strains carry different frame-shift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type his D 3052 in the case of TA 98. The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 (4) and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity. For testing, deep-frozen (-70°C to -80°C) bacterial cultures are thawed at room temperature, 0.1 ml of this bacterial suspension is inoculated in nutrient broth solution (8 g Difco nutrient broth + 5 g NaCl/liter) and incubated in the shaking water bath at 37° C for about 10 - 12 hours. As a rule, a germ density of >10E8 bacteria/ ml is reached.

Standard plate test
The experimental procedure is based on the method of Ames et al. Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0.6 % agar + 0.6 % NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 ml test solution or solvent
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

Preincubation test
The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al.. 0.1 ml test solution or solvent, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds. Composition of the minimal glucose agar:
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37°C for 48 hours in the dark, the bacterial colonies (his revertants) are counted.

Titer determination
In general, the titer is determined only in the experiments with S-9 mix both without test substance (solvent only) and after adding the two highest amounts of substance. For this purpose, in the case of the Ames standard plate test 0.1 ml of the overnight cultures (see 3.3.) is diluted to 10 in each case. Test tubes containing 2 ml portions of soft agar containing maximal amino acid solution (5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 ml solvent (without and with test substance)
0.1 ml bacterial suspension (dilution: 10)
0.5 ml S-9 mix
In the preincubation assay 0.1 ml of the overnight cultures is diluted to 10E6 in each case. 0.1 ml solvent (with and without test substance), 0.1 ml bacterial Suspension and 0.5 ml S-9 mix are incubated at 37°C for 20 minutes. Subsequently, 2 ml of soft agar which consists of 100 ml agar (0.6 % agar + 0.6 % NaCl) and 10 ml amino acid solution (maximal amino acid solution for titer determination: 5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds. After incubation at 37° for 48 hours in the dark, the bacterial colonies are counted.

Checking out the tester strains
The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (A uvrB); ampicillin resistance (R factor plasmid). Histidine auxotrophy is automatically checked in each experiment via the spontaneous rate.

Negative control
Parallel with each experiment with and without S-9 mix, a negative control (solvent control, sterility control) is carried out for each tester strain in order to determine the spontaneous mutation rate.

Positive controls
The following positive control substances are used to check the mutability of the bacteria and the activity of the S-9 mix:

with S-9 mix 2.5 µg 2-aminoanthracene (2-AA) (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535

without S-9 mix:
5 µg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (dissolved in DM50) for the strains TA 100 and TA 1535
10 µg nitro-o-phenylendiamine (NPD) (dissolved in DMSO) for the strain TA 98
100 µg 9-aminoacridine chloride monohydrate (AAC) (dissolved in DM50) for the strain TA 1537.

Tester strains, doses, number of plates
1st Experiment:
Strains: TA 1535, TA 100, TA 1537, TA 98
Doses: 0, 20, 100, 500, 2500 and 5000 µg/plate
Solvent: DMSO
Type of test, standard plate test with and without test condition: S-9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment:
Strains: TA 1535, TA 100, TA 1537, TA 98
Doses: 0, 4, 20, 100, 500 and 1000 µg/plate
Solvent: DMSO
Type of test, standard plate test with and without test condition: S-9 mix
Number of plates: 3 test plates per dose or per control

3rd Experiment:
Strains: TA 1535, TA 100, TA 1537, TA 98
Doses: 0, 0.8, 4, 20, 100 and 500 µg/plate
Solvent: DMSO
Type of test, preincubation test with and without test condition: S-9 mix
Number of Plates: 3 test Plates per dose or per control

Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his +revertants) was observed at doses > 1000 µg (standard plate test) or at 500 µg (preincubation test).

No test substance precipitation was found.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: standard plate test and preincubation test
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 September to 03 December 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Guidline was revised in July 2015, now 490.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced Rat Liver S-9
Test concentrations with justification for top dose:
Range finding test: 100, 50, 10, 5.0, 1.0, 0.5, 0.1, 0.05, 0.01 and 0.005 μL/mL
Mutant Assay: 0.2 to 0.02 μL/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol; DMSO; acetone.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulfonate
Remarks:
In the absence of metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
In the presence of metabolic activation
Details on test system and experimental conditions:
Range Finding Test
In order to determine the test article concentration that would produce 0-100% cytotoxicity, a Range Finding Test was performed. The test article was tested with and without S – 9 activation at concentrations of 100, 50, 10, 5.0, 1.0, 0.5, 0.1, 0.05, 0.01, and 0.005 uL/mL.
Treatment was performed by adding 4 mL of F0 P or 4 mL of S – 9 mix to the culture and then adding the appropriate volume of test article or test article/vehicle mixture. Two control cultures were run simultaneously with the treated cultures. The cultures were gassed with approximately 5% CO2 and 95% air and incubated at 37±1℃ on a roller drum apparatus rotating at 25±2 rpm.
After a 4-hour exposure period, the cells were pelleted by centrifuging them at approximately 1000 rpm for 10 minutes, and the test article was removed by pouring off the supernatant. Two rinses in 10 mL of F10P were performed, after which the cells were resuspended in 20 mL of F10P, gassed with approximately 5% CO2 and 95% air, and incubated at 37±1℃ on a roller drum rotating at 25±2 rpm. At approximately 20 hours and 44 hours post treatment, 1 mL samples were removed from each culture to determine the cell population density of each. The 1 mL sample was placed in a vial containing 19 mL of 0.1% trypsin. The vials were incubated for 10 minutes at 37±1℃, after which they were placed on an automatic cell counter. Three coincidence-corrected counts were made, and the average count was used to determine the concentration of cells per mL for each culture . After the determination of cell numbers at 20 hours post treatment, each culture having greater than 0.3E6 cells/mL. This was performed by retaining the volume of culture that would result in a final concentration of 0.3E6 cells/mL when fresh medium was added to it to yield a combined final volum of 20 mL.
Based on the results of the Range Finding Test, the test article was tested in the first Assay at doses ranging from 0.5 to 0.025 uL/mL in both the absence and presence of S – 9 activation mix. Based on the toxicity observed in the first Assay, the doses were adjusted for subsequent Assays to include 0.20, 0.18, 0.16, 0.14, 0.12, 0.10, 0.08, 0.06, 0.04 and 0.02uL/mL.

Mutation Assay
The test article was solubilized and a serial dilution was performed as previously described. Cultures containing 6 mL of cells at a concentration of 1E6 cells/mL were prepared. These cultures contained 50% conditioned F10P and 50% fresh F0P. Immediately prior to adding the appropriate aliquots of the test article dosing solution, 4 mL of either F0P medium of 4 mL S – 9 mix was added to each culture, depending on whether or not it was to be treated in conjunction with the exogenous metabolic activation system. Immediately after addition of the test article, the culture was gassed with approximately 5% co2 and 95% air. After all of the cultures had been treated, they were placed on a roller drum apparatus and rotated at a speed of 25±2 rpm in an environment of 37± 1℃. Two solvent control cultures were included in each treatment group, i.e., with activation and without activation. In addition, two sets of cultures were treated with positive control chemicals. As indicated earlier, EMS was the positive control chemical for the none-activated portion of the Assay, and DMBA was the positive control chemical for the S – 9 activated portion of the Assay.
After a 4-hour exposure period, the cells were pelleted by centrifugation at approximately 1000 rpm for 10 minutes, and the test article was removed by pouring off the supernatant. Two rinses in 10 mL of F10P were performed. The final rinse was followed by resuspension in 20 mL F10P, gassing with approximately 5% CO2 and 95% air, and incubation at 37±1℃ on a roller drum apparatus set at 25±2 rpm. Approximately 20 hours and 44hours post treatment, 1 mL samples were removed from each culture to determine the cell population density of each. The 1 mL sample was placed in a vial containing 19 mL of 0.1% trypsin. The vials were incubated for 10 minutes at 37±1℃, after which they were placed on an automatic cell counter. Three coincidence-corrected counts were made, and the average count was used to determine the concentration of cells per mL for each culture. After the determination of cell numbers, each culture having a population greater than 0.3E6 cells/mL was adjusted to 0.3E6 cells/mL. At the 20-hour point the final volume after adjustment was 20 mL. For the 44-hour point the final volum was 10 mL.
Evaluation criteria:
The following criteria were used in evaluating the acceptability of the Assay.
Solvent Control Cultures:
1. The average Cloning Efficiency of the solvent control cultures should be 50% or higher.
2. The average MF of the solvent control cultures should be less than 100 per 1000,000 viable cells.
Positive Controls:
The results for the positive control cultures should be considered acceptable if:
1. The treated cultures have MF's that are three times or greater than the average of their solvent control cultures.
2. Their solvent controls have an average Cloning Efficiency of 50% or greater.
The following criteria were used as guidelines in evaluating the results of the Assay for a negative, positive or equivocal response. Since it is impossible to write criteria that would apply to every configuration of data generated by the Assay, the Study Director is responsible for the ultimate decision in the evaluation of the results.
Criteria for a Negative Response:
1. All of the cultures exhibiting TG of 10% and greater have MF's that are less than twice that of the mean MF of the corresponding solvent control cultures and,
2. There is no evidence of a dose-dependent response.
Criteria for a Positive Response:
A response is considered positive if at least one culture has an MF that is two times or more greater than the average MF of the corresponding solvent control cultures and the response is dose dependent.
Criteria for an Equivocal Response:
A response is considered equivocal if it does not fulfill the criteria of either a negative or a positive response, and/or the Study Director does not consider the response to be either positive or negative.
Statistics:
None stated
Species / strain:
mouse lymphoma L5178Y cells
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:
not valid
Positive controls validity:
valid
Additional information on results:
Miscibility test:
The results of the miscibility test indicated that the test article was miscible in DMSO, acetone and ethanol. Of these three solvents, ethanol is the preferred solvent for this test system, therefore ethanol was used to prepare the dosing solutions.

Range finding test:
Both in the absence and presence of exogenous activation, the concentrations of 0.5 μL/mL and above were completely toxic. These cultures had 0% RSG. The cultures treated with 0.1 μL/mL without and with activation had 39% and 96% RSG, respectively. The doses of 0.05 μL/mL and below were relatively nontoxic.
It should be noted that the four highest concentrations, 100, 50, 10 and 5.0 μL/mL, formed thick precipitate in the medium. The 100 and 50 μL/mL concentrations also acidified the medium, therefore requiring the addition of NaOH to adjust the cultures to their normal pH. These physical characteristics, however, did not affect assay conditions since the test article was toxic at much lower concentrations.
Based on the results of the Range Finding Test and suspension growth data obtained from an assay that was aborted, the Assays were conducted at doses spanning a range of 0.2 to 0.02 μL/mL without and with activation. The test article produced an extremely steep toxic response curve, so the serial concentrations that were prepared differed by 0.02 μL/mL.

Mutation Assay:
In the absence of activation, all of the cultures except two had Mutant Frequencies that were within the normal range of the mean Mutant Frequency of the corresponding solvent control cultures. The Relative Total Growth (RTG) of these cultures ranged from 55% to 116%. The two cultures that had mutant frequencies that were more than two-fold greater than the mean of the solvent controls had been treated with midrange doses of 0.06 and 0.04 μL/mL and exhibited 70% and 44% RTG, respectively. These data points were not considered to be indicative of a positive response because: 1) the mutant frequencies observed were well within the range of mutant frequencies observed for SITEK's historical solvent control values, 2) the data presented no indication of a dose response trend, and 3) there was no significant increase in the absolute number of mutant colonies observed in the nonactivated portion are considered to be negative.
The response for cultures treated in the presence of S-9 also was negative. All of the cultures had Mutant Frequencies that were similar to the mean Mutant Frequency of the corresponding solvent controls. The RTG for these cultures ranged from 74% to 186%. Although these results were negative, the average cloning efficiency of the solvent controls for the S-9 activated portion of the assay did not meet the minimum acceptable criteria of 50% cloning efficiency. Therefore, the S-9 activated portion of the assay was repeated.
The repeated assay with S-9 activation also produced a negative response. The RTG ranged from 10% to 98%. One culture, 0.14 μL/mL A, having 57% RTG had a Mutant Frequency that was greater than twice the mean Mutant Frequency of the corresponding solvent controls, but this was not considered significant since it was a culture treated with a midrange dose and it exhibited moderate toxicity.
The cultures treated with the positive controls EMS and DMBA exhibited acceptable positive responses.
For the colony sizing for the TK-/- colonies, the test article produced a negative response, the colony sizing profiles for the test article-treated cultures are similar to those of solvent control cultures.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test substance produced a negative response in cultures treated in the absence and presence of S-9 under the test conditions.
Executive summary:

In a GLP compliant Mouse Lymphoma Assay according to OECD guideline 476 (revised in July 2015, now 490), L5178Y cells were treated with 0.02 - 0.2 µl/ml test substance in ethanol +/- metabolic activation (1991). The dose range was determined by the results of a Range-Finder-Study, showing severe toxicity at higher concentrations. The test substance produced a negative response +/- metabolic activation under the test conditions chosen.

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 26 October to 12 December 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River U.K. Limited, Margate, Kent, England
- Age at study initiation: approximately 35 days old
- Weight at study initiation: between 22 and 24 grams
- Assigned to test groups randomly: yes
- Fasting period before study: overnight
- Housing: kept in a plastic disposable cage
- Diet (e.g. ad libitum): pelleted Labsure LAD 1 rodent breeding diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: approximately 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%):
- Air changes (per hr): 30 changes of air per hour.
- Photoperiod (hrs dark / hrs light): The room was illuminated by artificial light for 12 hours per day.

IN-LIFE DATES: From: 26 October 1988 To: 12 December 1988
Route of administration:
oral: gavage
Vehicle:
Corn oil
Duration of treatment / exposure:
Immediately

Frequency of treatment:
Single dose
Post exposure period:
24, 48 or 72 hours


Remarks:
Doses / Concentrations:
1720 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Preliminary toxicity test:
2 males and 2 females per dose for Phase I.
5 males and 5 females per dose for Phase II.

Micronucleus test:
15 males and 15 females for vehicle control.
20 males and 20 females for the test substance.
5 males and 5 females for positive control.

Control animals:
yes
Positive control(s):
Mitomycin C
- Justification for choice of positive control(s):
- Route of administration: Oral by intragastric gavage.
- Doses / concentrations: 12 mg/kg

Details of tissue and slide preparation:
The femurs were cleared of tissue and one epiphysis removed from each bone. A direct bone marrow smear was made onto a slide containing a drop of calf serum. One smear was made from each femur. The prepared smears were fixed in methanol. The smears were air-dried and stained for 10 minutes in 10% Giemsa. Following rinsing in distilled water and differentiation in buffered distilled water, the smears were air-dried and mounted with coverslips using DPX.
Evaluation criteria:
None stated

Statistics:
For a comparison of an individual treated group with a concurrent control group, Wilcoxon's sum of ranks test is used.

Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Signs and mortalities:
One male and three female animals died after treatment with the test substance in the main study. On post mortem examination, none of the animals showed signs of having been misdosed. None of the animals in the positive control or vehicle control groups showed any clinical signs for the duration of the test.

Micronucleated polychromatic erythrocyte counts (mnp):
The test substance did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes at any of the three skill times (P>0.05 using Wilcoxon's sum of ranks test). Mitomycin C caused large, highly significant increases (P<0.001) in the frequence of micronucleated polychromatic erythrocytes.

Micronucleated normochromatic erythrocytes (mnn):
The test substance did not cause any substantial increases in the incidence of micronucleated normochromatic erythrocytes at any of the three kill times.

Ratio of polychromatic to normochromatic erythrocytes (p/n):
Both the test substance and mitomycin C failed to cause any significant decreases in the ratio of polychromatic to normochromatic erythrocytes (P>0.05 using Wilcoxon's sum of ranks test).
Conclusions:
Interpretation of results (migrated information): negative
From the results obtained it is concluded that the test substance shows no evidence of mutagenic potential or bone marrow cell toxicity when administered orally in this in vivo test procedure.
Executive summary:

In a GLP compliant Mammalian Erythrocyte Micronucleus test according to OECD guideline 474, mice were once orally dosed with 1720 mg/kg bw test substance in corn oil and bone marrow cells were examined 24, 48 and 72 h later (1989). In contrast to the positive control, no increase of micronucleated PCE was observed, but one male and 3 female animals died after test substance administration. No substantial increase of NCEs and no significant decrease of the PCE/NCE ration was observed after administration of the test substance or the positive control.

Therefore, the test substance showed no evidence of mutagenic potential or bone marrow cell toxicity under the conditions of this test.

Mode of Action Analysis / Human Relevance Framework

​No information available

Additional information

Valid experimental data were available to assess the genetic toxicity in vitro and in vivo.

 

Gene mutation in bacteria

N-(n-Octyl)pyrrolidone was not mutagenic in a standard plate and pre-incubation Ames test (1992_RL1 and 2001_RL1) with and without metabolic activation according to OECD guideline 471 (tested up to 5000 μg/plate in the standard plate test (SPT) and up to 500 µg/ml in the preincubation test (PIT) with Salmonella typhimurium TA1535, TA 1537, TA 98, TA 100 and E.coli WP2; metabolic activation: liver S-9 mix from Aroclor-induced male Sprague-Dawley rats). Cytotoxicity (reduction of the background lawn) was observed at the concentration > 1000 µg/ml in the standard plate test and at 500 µg/ml in the preincubation test. With E.coli WP2 a bacteriotoxic effect was observed in the PIT at doses >100 µg/plate and in the SPT at doses >1000 µg/plate.

Two other Ames tests (1986_RL2 and 1989_RL1) support the negative outcome of the key studies.

Cytogenicity in mammalian cells

In an in vitro Chromosome Abberation Test. according to OECD guideline 473, N-(n-Octyl)pyrrolidone was also not cytogenic in Chinese hamster lung fibroblasts (V79) when incubated at concentrations up to 150 µg/ml with and without metabolic activation (liver S-9 mix from Aroclor-induced rats) (1992_RL1).

Gene mutation in mammalian cells

In a Mouse Lymphoma Assay according to OECD guideline 476 (revised in July 2015, now 490), L5178Y cells were treated with 0.02 - 0.2 µl/ml test substance in ethanol +/- metabolic activation (1991_RL1). The dose range was determined by the results of a Range-Finder-Study, showing severe toxicity at higher concentrations. The test substance produced a negative response +/- metabolic activation under the test conditions chosen.

 

Genetic toxicity in vivo

In a Mammalian Erythrocyte Micronucleus test according to OECD guideline 474, mice were once orally dosed with 1720 mg/kg bw test substance in corn oil and bone marrow cells were examined 24, 48 and 72 h later (1989_RL1). In contrast to the positive control, no increase of micronucleated PCE was observed, but one male and 3 female animals died after test substance administration. No substantial increase of NCEs and no significant decrease of the PCE/NCE ration was observed after administration of the test substance or the positive control.

Therefore, the test substance showed no evidence of mutagenic potential or bone marrow cell toxicity under the conditions of this test.


Short description of key information:
- Gene mutation in bacteria: Ames tests with and without metabolic activation (1992_RL1 and 2001_RL1), supported by two other Ames tests (1989_RL1 and 1986_RL2): negative
- Cytogenicity in mammalian cells: V79 cells with and without metabolic activation (Chromosome aberration test, 1992_RL1): negative
- Gene mutation in mammalian cells: MLA in L5178Y cells with and without metabolic activation (1991_RL1): negative.
- Cytogenicity in vivo: oral in vivo MNT (1989_RL1): negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance was not mutagenic or cytogenic in in vitro experiments using bacteria and rodent cells (Ames test, in vitro CA, MLA) and non-mutagenic in vivo (MNT). Therefore, based on the information currently available, there is no need for classification of N-(n-Octyl)pyrrolidone for mutagenic effects.