2-pyrrolidone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1. Gene mutation in bacteria: Jagannath, 1987. Mutagenicity Test on 2-Pyrrolidone in the Ames-Salmonella/Microsome Reverse Mutation Assay. GLP, comparable to the OECD guideline 471, S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100, with and without metabolic activation (Ames test), negative
2. Cytogenicity in mammalian cells in vitro: BASF AG, 1987. In Vitro Cytogenetic Investigations of 2-Pyrrolidon in Human Lymphocytes. Report No. 30M0286/8617. According to the OECD guideline 473, Chromosome Aberration Assay, with and without metabolic activation, negative
3. Gene mutation in mammalian cells: Wells, 2019. 2-pyrrolidone in vitro gene mutation test in CHO cells (HPRT locus assay), OECD 476, GLP, up to 851 µg/mL, negative

Link to relevant study records

Reference

Reference 1

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study (GLP) with acceptable restrictions (strain combination not suitable for the detection of cross links)

Principles of method if other than guideline:

According to Ames et al. Mut. Res., 31, 347-364, 1975

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

0, 110, 1100, 5500, 11000, 27500, 55000, 110000 and 165000 µg/plate (= 0, 0.1, 1.0, 5.0, 10, 25, 50, 100 and 150 µL/plate)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: without metabolic activation: Quinacrine mustard and with metabolic activation: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

Evaluations considered if a dose-response was observed and strain-specific evaluation criteria. For strains TA-1535, TA-1537 and TA-1538, the data set is evaluated as positive if a dose-response is observed over a minimum of three test concentrations and the increase in revertants is equal to or greater than three times the solvent control value at the peak of the dose-response. The solvent control value should be within the normal range for evaluating the results. For strains TA-98 and TA-100, the data set is evaluated as positive if a dose-response is observed over a minimum of three test concentrations and the increase in revertants achieves a doubling of the solvent control value at the peak of the dose-response. The solvent control value should be within the normal range for evaluating the results.

Statistics:

Formal statistical methods were not used to evaluate the data.

Species / strain:

other: Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

165000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Standard plate test (110-16500 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 98	no	25.7	1.2	no	negative
	yes	46.3	1.0	no	negative
TA 100	no	147.7	1.0	no	negative
	yes	140.3	1.0	no	negative
TA 1535	no	25.3	1.0	no	negative
	yes	17.3	1.0	no	negative
TA 1537	no	5	1.7	no	negative
	yes	10.3	1.1	no	negative
TA 1538	no	13	1.1	no	negative
	yes	16	1.8	no	negative
Standard plate test II(110-16500 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 98	no	28.3	1.1	no	negative
	yes	49	1.0	no	negative
TA 100	no	127	1.2	no	negative
	yes	142.7	1.1	no	negative
TA 1535	no	20.7	1.2	no	negative
	yes	15	1.2	no	negative
TA 1537	no	7.7	1.6	no	negative
	yes	12	1.0	no	negative
TA 1538	no	12	1.2	no	negative
	yes	22	1.2	no	negative

Conclusions:

Interpretation of results: negative
2-Pyrrolidone is negative in this Ames Test

Executive summary:

The test item was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver).

The experiments were carried out as plate incorporation tests. The test item was completely dissolved in distilled water. The solvent served as the negative control.

Preliminary test

Concentrations of test substance were selected based on a preliminary toxicity assay at 14 concentration levels using two-fold dilutions from a high concentration of 150 microliter per plate (for liquids). As no significant toxicity was observed, 150 microliters per plate was used as the top concentration in the studies.

Main study

Eight concentrations ranging from 0.1 to 150 μL test substance/plate were employed in the plate incorporation test (in detail the following concentrations were employed: 0, 0.1, 1.0, 5.0, 10, 25, 50, 100 and 150 microliters per plate for all strains in both of the two independent repeats: with and without metabolic activation).

Cytotoxicity was noted in the plate incorporation test, carried out without and with metabolic activation at 165000 µg/plate..

No increase in revertant colony numbers as compared with control counts was observed for the test substance, tested up to a concentration of 150 μL/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively. The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

In conclusion, under the present test conditions the test substance 2 -pyrrolidone tested up to a concentration of 150 µL/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in the plate incorporation test carried out without and with metabolic activation.

Reference 2

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:

other: Guideline study (GLP)

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

Target gene:

his-

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Test concentrations with justification for top dose:

1250, 2500 und 3500 µg/mlL(without S9-Mix), 2500, 5000 und 6000 µg/mL (with S9-Mix)

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 (positive controls: 50) per replicate

Statistics:

The Fisher exact test was applied to determine significant differences between the relative frequencies of a characteristic of two groups.

Species / strain:

lymphocytes: human

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

Without metabolic activation

Treatm.	% aber. + gaps	% aber. - gaps	% exchanges
negative	5	1	0
solvent	6	2.5	0
1250 ug	8.5	1	0.5
2500 ug	7	3	0
3500 ug	4	1	0
positive	44**	37**	5*

Multiple abberation metaphases were detected in the 1250 µg treatment  (0.5 %) and the positive control (2 %).
Aneuploid metaphases were detected in the solvent control (1 %) and the  2500 µg treatment (1 %). 
Polyploid metaphases were detected in the negative control (1 %), the 1250  µg and the 2500 ug treatment (0.5 % each).

With metabolic activation

Treatm.	% aber. + gaps	% aber. - gaps	% exchanges
negative	2	0	0
solvent	7.5	2	0
2500 ug	6.5	0.5	0
5000 ug	8#	0.5	0
6000 ug	8.5#	1	0

Multiple aberration metaphases were not detected in any treatment or control.
Aneuploid metaphases were detected in the 5000 µg treatment (0.5 %). 
Polyploid metaphases were detected in the solvent control (1 %), the 5000 µg and the 6000 µg treatment (0.5 % each).

**/* = statistically different from both the negative and solvent control (significance level 99/95%)
# = statistically different from the negative control (significance level  95 %)

Conclusions:

Interpretation of results: negative
2-pyrrolidone is not genotoxic in human lymphocyte chromosome aberation assay.

Executive summary:

2 -pyrrolidone was tested in the Human Lymphocytes Chromosome Aberation Assay in the presence and absence of metabolic activation. There were statistically significant differences between both negative and solvent control in the two high dose groups without and with metabolic activation. However, there were also aberration observable in the negative control. Therefore, these significant differences are considered to be of no importance for genotoxicity of the test material. 2 -pyrrolidone does not induce clastogenic effects and chromosomal aberrations in this assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 May 2018 - 17 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MAFF Japan, 59 Nousan Number 4200 (1985)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: supplied by Taminco
- Expiration date of the lot/batch: unknown
- Purity test date: 23/02/2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: received at ambient conditions and stored at room temperature (19 °C to 25 °C), protected from light
- Stability under storage conditions: not specified
- Stability under test conditions: concentration analysis of dosing formulations was performed
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: 2-Pyrrolidone was prepared as a stock solution in sterile water at a concentration of 85.1 mg/mL on the day of each assay. Lower concentrations for each assay were prepared by sequential serial dilution in sterile water.
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): not specified

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (Hprt)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO-K1-BH4 cell line (a proline auxotroph with a modal chromosome number of 20)
- Suitability of cells: given
- Normal cell cycle time (negative control): population doubling time of 12 to 14 hours ;cloning efficiency generally > 80%.

For cell lines:
- Absence of Mycoplasma contamination: yes, tested
- Number of passages if applicable: up to 20 passages before they are discarded
- Methods for maintenance in cell culture: not specified
- Cell cycle length, doubling time or proliferation index: population doubling time of 12 to 14 hours
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: [yes]
- Periodically ‘cleansed’ of spontaneous mutants: [not specified]

This cell line was originally isolated by A.W. Hsie (Oak Ridge National Laboratories; Oak Ridge, TN) and the cells in use at Charles River were provided by M.J. LeBaron (The Dow Chemical
Company; Midland, MI).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: The exogenous metabolic activation system (MUTAZYME™; S9)
- source of S9 : Molecular Toxicology, Inc. (Lot Nos. 3932 and 3984); consisted of Aroclor™ 1254-induced male rat liver S9 lyophilized with NADPH-regenerating system cofactors and phosphate buffer
- method of preparation of S9 mix: stored and reconstituted as per the manufacturer’s instructions (to yield the same recipe as described by Ames et al.5), except that it also contained 10 mM CaCl2, which has been demonstrated to enhance the mutagenicity of some promutagens. Reconstituted S9 mix was prepared fresh on the day of testing and was maintained refrigerated (2 °C to 8 °C) or on ice.
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

Test concentrations with justification for top dose:

106, 213, 426, 638, and 851 μg/mL with and without S9 (based on dose range finder)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqueous solvents (sterile water, CAS No. 7732-18-5; MediaTech Lot Nos. 26017006, 30917005, and 06718006)
- Justification for choice of solvent/vehicle: not specified
- Justification for percentage of solvent in the final culture medium: not specified

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Remarks:

Neat positive control substances were stored according to the manufacturers’ suggested specifications. Positive control substances were freshly prepared in DMSO. All unused material was discarded.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Cells for treatment were seeded (on Day -1) in 75-cm² flasks at a density of approximately 2.5 × 10E6 cells in 10 mL F12FBM5.
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment: 3.5 to 4.5 hours
- Harvest time after the end of treatment (sampling/recovery times): After the 4-hour treatment, the treatment media was removed, the cultures were washed twice with 10 mL CMF-DPBS, washed once with 5 mL trypsin,and incubated at 36 °C to 38 °C for 3 to 5 minutes (or until the cells detached). Four mL F12FBM5 was added and the cells were resuspended and counted using a Coulter counter.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Approximately 2.4 × 10E6 cells (or all available) from each culture also were added to a 225-cm² flask in 30 mL F12FBM5 for phenotypic expression and incubated under standard conditions. The cultures were subcultured on Day 3 and either Day 5 or Day 6 to maintain logarithmic growth and permit expression of the mutant phenotype. At each subculture, the cultures were washed twice with 15 mL CMF-DPBS, washed once with 5 mL trypsin and incubated at 36 °C to 38 °C for 3 to 5 minutes (or until the cells detached). End of the phenotypic expression period on Day 7 or Day 8.
- Selection time (if incubation with a selective agent): Thioguanine (6-thioguanine)
- Fixation time (start of exposure up to fixation or harvest of cells): from day 7 to day 10 (4 days)
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
At the end of the phenotypic expression period (on Day 7 or Day 8), cultures were trypsinized as above (except using 5 mL HX-F12FBM5 to inactivate the trypsin) and 2.4 × 10E6 cells from each culture were seeded at a density of 6 × 10E5 cells/150-mm plate (4 plates total) in 30 mL HX-F12FBM5 containing 10 μM Thioguanine
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2.4 × 106 cells from each culturen were seeded at a density of 6 × 105 cells/150-mm plate (4 plates total). Mutant frequencies are expressed as the number of TGr mutants/106 clonable cells.
- Criteria for small (slow growing) and large (fast growing) colonies: not specified

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, determination of clonable cells in triplicate 60-mm plates
- Any supplementary information relevant to cytotoxicity: Cytotoxicity of the test substance to the test system was determined in order to allow the selection of appropriate dose levels to be tested in the definitive mutagenicity assay. Cells were treated with 2-Pyrrolidone at concentrations of 1.66, 3.32, 6.65, 13.3, 26.6, 53.2, 106, 213, 426, and 851 μg/mL with and without S9. The highest concentration evaluated approximated the limit dose for this assay (10 mM)

METHODS FOR MEASUREMENTS OF GENOTOXICIY

Evaluation criteria:

Vehicle Control Cultures: The average absolute cloning efficiency of vehicle control substance must be ≥ 50 % (at initial survival and selection). In addition, the average spontaneous mutant frequency of the vehicle control substance must be comparable to the 95 % control interval of the historical negative controls. Spontaneous mutant frequencies are calculated separately for cultures with and without S9.
Positive Control Cultures: The positive control substances must induce a statistically significant increase compared with the concurrent vehicle controls (p ≤ 0.01). A significant increase in the absence of S9 indicates the test system can identify a mutagen, while a significant increase in the presence of S9 will be considered to have demonstrated the efficacy of the S9 mix as well as the ability of the test system to detect a mutagen.
Maximum Concentration Evaluated: The highest test substance concentrations evaluated must be the limit dose for this assay (10 mM), or be the highest that is able to be prepared and administered as a uniform fine suspension, or be minimally insoluble in treatment medium, or induce 10 % to 20 % relative survival (the cytotoxicity limit for this assay), whichever is lowest. Lower concentrations may be justified for test substances which clearly show mutagenic activity.
Number of Concentrations: A minimum of 4 concentrations with acceptable relative survival is required for a valid assay. Fewer concentrations may be justified for test substances which clearly show mutagenic activity.

Statistics:

A one-tailed Fisher’s Exact test was performed to compare the mutant frequencies observed in the treated cultures with those of the concurrent vehicle control substance cultures. The Cochran-Armitage test was used to assess dose dependency.

Species / strain:

Chinese hamster Ovary (CHO)

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

Positive controls validity:

valid

Dose Range-Finding Assay

2-Pyrrolidone was prepared in sterile water and evaluated in a preliminary dose range-finding assay at concentrations of 1.66, 3.32, 6.65, 13.3, 26.6, 53.2, 106, 213, 426, and 851 μg/mL with and without S9. The highest concentration evaluated approximated the limit dose for this assay (10 mM). Sterile water was evaluated concurrently as the vehicle control. Relative survivals at the concentration of 851 μg/mL were 106.85 % without S9 and 89.49 % with S9. The test substance was found to be freely soluble at all concentrations evaluated.

Table 1
Dose Range-Finding Assay
Dose Level			Initial Survival
			Cells/mL	Cloning Efficiency				Relative Survival
Treatment	(µg/mL)	S9	(x 10E6 )	(Colonies/Plate)			%	(adj.,%)
di-H2O	10.0^a	-	1.292	106	82	106	49.00	100.00
2-Pyrrolidinone	1.66	-	1.086	105	86	85	46.00	78.90
2-Pyrrolidinone	3.32	-	1.887	48	53	46	24.50	73.02
2-Pyrrolidinone	6.65	-	0.939	141	130	132	67.17	99.65
2-Pyrrolidinone	13.3	-	1.105	97	95	85	46.17	80.62
2-Pyrrolidinone	26.6	-	1.076	81	86	93	43.33	73.69
2-Pyrrolidinone	53.2	-	0.944	119	111	98	54.67	81.52
2-Pyrrolidinone	106	-	1.090	111	109	97	52.83	90.95
2-Pyrrolidinone	213	-	1.077	90	94	101	47.50	80.81
2-Pyrrolidinone	426	-	0.981	92	102	111	50.83	78.75
2-Pyrrolidinone	851	-	1.012	135	143	123	66.83	106.85
di-H2O	10.0^a	+	1.071	134	132	127	65.50	100.00
2-Pyrrolidinone	1.66	+	1.172	101	120	115	56.00	93.56
2-Pyrrolidinone	3.32	+	1.132	134	135	111	63.33	102.24
2-Pyrrolidinone	6.65	+	1.111	139	144	147	71.67	113.55
2-Pyrrolidinone	13.3	+	1.171	104	107	122	55.50	92.63
2-Pyrrolidinone	26.6	+	1.033	122	121	130	62.17	91.56
2-Pyrrolidinone	53.2	+	1.076	116	114	120	58.33	89.46
2-Pyrrolidinone	106	+	1.014	125	108	107	56.67	81.93
2-Pyrrolidinone	213	+	1.019	135	123	129	64.50	93.70
2-Pyrrolidinone	426	+	0.995	139	144	112	65.83	93.40
2-Pyrrolidinone	851	+	1.013	137	118	117	62.00	89.49
^aµL/mL

Definitive Mutagenicity Assay

Based upon the results of the dose range-finding assay, 2-Pyrrolidone was evaluated in the definitive mutagenicity assay at concentrations of 106, 213, 426, 638, and 851 μg/mL with and without S9. All test substance concentrations, as well as the concurrent positive and vehicle controls, were evaluated in duplicate cultures. In the treatment with S9, mutant frequency data between replicate cultures of the positive control and high dose test article was variable, and appeared that cultures from these 2 groups may have been interchanged during plating. Therefore, this treatment was not considered in the evaluation of mutagenicity, and the treatment was repeated at the same concentrations. 2-Pyrrolidone again was found to be freely soluble at all concentrations evaluated. The average relative survivals at the highest concentration evaluated for mutagenicity were approximately 98.48 % and 89.71 % with and without S9, respectively. In the treatment with S9, statistically significant and dose-dependent increases in mutant frequency were observed at 638 and 851 μg/mL. However, the highest mutant frequency observed was 6.67 × 10E-6, within the 95 % confidence limit of the historical control for the vehicle (-2.13 × 10E-6 to 27.47 × 10E-6). Therefore, these increases are not considered to be biologically relevant. No statistically significant or dose-dependent increases in mutant frequency, as compared to the concurrent vehicle control, were observed at any concentration evaluated without S9 (p > 0.05), and all observed values were within historical control limits. In contrast, the DMN and EMS positive controls induced significant increases in mutant frequencies (p < 0.01).

All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

Table 2
Definitive Mutagenicity Assay (Treatment Without S9)
Treatment	Dose Level (µg/mL)	S9	Initial Survival									Selection
			Cells/mL (x 10^6)	Cloning Efficiency							Relative Survival (%, adj.)	TG^r					Total Mutant Colonies	Cloning Efficiency			Mutant Frequency (x 10^-6)
				(Colonies/Plate)					%									(Colonies/Plate)		(%)	Individual		Average
di-H2O	10.0^a	-	1.435	137	146		112		65.83		101.25	3	7	4		2	16	95	130	126	58.50	11.40
di-H2O	10.0^a	-	1.410	119	148		125		65.33		98.75	5	7	5		4	21	140	103	149	65.33	13.39	12.39
2-pyrrolidinone	106	-	0.968	135	125		145		67.50		70.06	1	2	6		4	13	104	122	119	57.50	9.42
2-pyrrolidinone	106	-	0.940	138	146		122		67.67		68.15	1	0	0		0	1	90	110	108	51.33	0.81	5.12
2-pyrrolidinone	213	-	1.457	121	130		109		60.00		93.70	3	4	2		9	18	166	153	157	79.33	9.45
2-pyrrolidinone	213	-	1.213	116	111		133		60.00		78.04	5	3	6		3	17	139	154	117	68.33	10.37	9.91
2-pyrrolidinone	426	-	1.467	132	117		137		64.33		101.13	6	4	2		10	22	164	183	190	89.50	10.24
2-pyrrolidinone	426	-	1.300	126	132		143		66.83		93.15	4	5		3	4	16	131	112	137	63.33	10.53	10.38
2-pyrrolidinone	638		1.312	100		85	98	47.17		66.33		7	1		2	4	14	126	112	113	58.50	9.97
2-pyrrolidinone	638		1.318	132		128	120	63.33		89.49		4	8		10	7	29	125	119	141	64.17	18.83	14.40
2-pyrrolidinone	851		1.376	115		136	125	62.67		92.40		10	7		12	4	33	178	171	156	84.17	16.34
2-pyrrolidinone	851		1.338	117		122	125	60.67		87.02		2	2		1	2	7	144	144	141	71.50	4.08	10.21
EMS	200		1.307	84		94	83	43.50		60.95		79	123		77	83	362	136	132	151	69.83	215.99
EMS	200		1.235	116		100	111	54.50		72.17		53	65		52	52	222	63	59	68	31.67	292.11	254.05**
^aµg/mL
*= p < 0.05, statistical increase over concurrent vehicle control using one-tailed Fisher's Exact Test.
**= p < 0.01, statistical increase over concurrent vehicle control using one-tailed Fisher's Exact Test.

Table 3
Repeat Definitive Mutagenicity Assay (Treatment With S9)
Treatment	Dose Level (µg/mL)	S9	Initial Survival						Selection																			Mutant Frequency (x 10^-6)
			Cells /ml (x 10E6)	Cloning Efficiency				Relative Survival (%, adj.)	TG^r														Total Mutant Colonies	Cloning Efficiency
				(Colonies/Plate)			(%)																	(Colonies/P		late)	(%)	Individual Average
di-H2O	10.0^a	+	1.103	111	89	98	49.67	100.43	0	0											1	0	1	123	131	117	61.83	0.67
di-H2O	10.0^a	+	1.021	118	108	93	53.17	99.57	1	1											1	0	3	121	123	117	60.17	2.08	1.38
2-pyrrolidinone	106	+	0.931	98	99	106	50.50	86.21	1	1										0		0	2	133	128	118	63.17	1.32
2-Pyrrolidinone	106	+	0.940	96	81	110	47.83	82.44	1	0									3			0	4	140	91	130	60.17	2.77	2.04
2-Pyrrolidinone	213	+	0.977	109	108	97	52.33	93.77	1	0								0				0	1	116	111	128	59.17	0.70
2-Pyrrolidinone	213	+	0.939	113	106	89	51.33	88.39	0	0							0					0	0	108	117	108	55.50	0.00	0.35
2-Pyrrolidinone	426	+	1.004	117	75	117	51.50	94.82	0	0							0					1	1	119	106	112	56.17	0.74
2-Pyrrolidinone	426	+	0.932	103	104	111	53.00	90.59	0	0						0						0	0	114	120	116	58.33	0.00	0.37
2-Pyrrolidinone	638	+	1.023	109	112	116	56.17	105.38	3	1					5							2	11	120	125	131	62.67	7.31
2-Pyrrolidinone	638	+	1.026	104	86	96	47.67	89.65	1	1				0								0	2	114	115	119	58.00	1.44	4.38 *
2-Pyrrolidinone	851	+	0.990	101	105	113	53.17	96.55	0	0			0									0	0	113	117	107	56.17	0.00
2-Pyrrolidinone	851	+	0.987	94	121	118	55.50	100.41	5	5		3										3	16	104	97	99	50.00	13.33	6.67 **
DMN	100	+	0.959	43	33	46	20.33	35.74	34	28	34											38	134	101	78	97	46.00	121.38
DMN	100	+	0.927	29	40	42	18.50	31.46	32	34	21											25	112	82	89	83	42.33	110.24	115.81 **
^aµL/mL
*= p < 0.05, statistical increase over concurrent vehicle control using one-tailed Fisher's Exact Test.
**= p < 0.01, statistical increase over concurrent vehicle control using one-tailed Fisher's Exact Test.

Dose Formulation Analysis Results

Concentration analysis demonstrated that all dose formulations from all trials analyzed were 94.4 % to 113 % of target, and met the acceptance criteria with the following exception. The 10.6 mg/mL (low-dose) formulation samples from the definitive mutagenicity assay (conducted on 08 Jun 2018) quantified at 71.7 % of the nominal concentration. However, as both the high- and mid-dose formulations from this assay date were acceptable, ascertaining the limit dose for the assay was achieved, there is no impact on the validity or integrity of the study. Test substance was not detected in any of the vehicle control samples. The Analyses of Dosing Formulations is depicted below.

Conclusions

These results indicate 2-Pyrrolidone was negative in the In Vitro Mammalian Cell Gene Mutation Test (CHO/HPRT Assay) under the conditions, and according to the criteria, of the test protocol.

Conclusions:

2-Pyrrolidone was negative in the In Vitro Mammalian Cell Gene Mutation Test (CHO/HPRT Assay).

Executive summary:

2-Pyrrolidone was evaluated for the potential to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (Hprt) in Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).

2-Pyrrolidone was prepared in sterile water and evaluated in a preliminary dose range-finding assay at concentrations of 1.66, 3.32, 6.65, 13.3, 26.6, 53.2, 106, 213, 426, and 851 μg/mL with and without S9. The highest concentration evaluated approximated the limit dose for this assay (10 mM). Sterile water was evaluated concurrently as the vehicle control. Relative survivals at the concentration of 851 μg/mL were 106.85 % without S9 and 89.49 % with S9. The test substance was found to be freely soluble at all concentrations evaluated.

Based upon these results, 2-Pyrrolidone was evaluated in the definitive mutagenicity assay at concentrations of 106, 213, 426, 638, and 851 μg/mL with and without S9. All test substance concentrations, as well as the concurrent positive and vehicle controls, were evaluated in duplicate cultures. In the treatment with S9, mutant frequency data between replicate cultures of the positive control and high dose test article was variable, and appeared that cultures from these 2 groups may have been interchanged during plating. Therefore, this treatment was not considered in the evaluation of mutagenicity, and the treatment was repeated at the same concentrations.

2-Pyrrolidone again was found to be freely soluble at all concentrations evaluated. The average relative survivals at the highest concentration evaluated for mutagenicity were approximately 98.48 % and 89.71 % with and without S9, respectively. In the treatment with S9, statistically significant and dose-dependent increases in mutant frequency were observed at 638 and 851 μg/mL. However, the highest mutant frequency observed was 6.67 × 10E-6, within the 95 % confidence limit of the historical control for the vehicle (-2.13 × 10E-6 to 27.47 × 10E-6). Therefore, these increases are not considered to be biologically relevant. No statistically significant or dose-dependent increases in mutant frequency, as compared to the concurrent vehicle control, were observed at any concentration evaluated without S9 (p > 0.05), and all observed values were within historical control limits. In contrast, the DMN and EMS positive controls induced significant increases in mutant frequencies (p < 0.01).

All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

These results indicate 2-Pyrrolidone was negative in the In Vitro Mammalian Cell Gene Mutation Test (CHO/HPRT Assay) under the conditions, and according to the criteria, of the test protocol.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

1. Cytogenicity in mammalian cells in vivo: BASF AG, 1993. Cytogenetic Study In Vivo of Pyrrolidon-2 in Mice; Micronucleus Test, Single Intraperitoneal Administration. Report No.26M0014/924193, GLP, according to the OECD guideline 474, NMRI mouse, up to 2000 mg/kg i.p.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Route of administration:

intraperitoneal

Vehicle:

water

Details on exposure:

Single intraperitoneal administration with a volume of 10 mL/kg body weight.

Duration of treatment / exposure:

16, 24 and 48 hours

Frequency of treatment:

single

Post exposure period:

- Sacrifice intervals per dose-group were:
2000 mg/kg: 16, 24 and 48 hours
1000 mg/kg: 24 hours
500 mg/kg: 24 hours
Controls: 24 hours

Dose / conc.:

500 mg/kg bw/day

Remarks:

intraperitoneal

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

intraperitoneal

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

intraperitoneal

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

20 mg cyclophosphamide/kg bw or 0.15 mg vincristine/kg bw, both dissolved in distilled water

Tissues and cell types examined:

bone marrow, erythrocytes (see below in "Details of tissue and slide preparation"

Details of tissue and slide preparation:

- Preparation of bone marrow: After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum which was at 37 °C (about 2 mL/femur). The suspension was mixed thoroughly with a pipette, centrifuged at 1500 rpm for 5 minutes, the supernatant removed and the cells were resuspended. One drop of this suspension was dropped onto clean microscopic slides.
Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained in eosin and methylene blue solution for 5 minutes, rinsed, placed in fresh distilled water for 2 or 3 minutes and finally stained in Giemsa solution for 12 minutes. After being rinsed twice and clarified with xylene, the preparations were embedded in Corbit-Balsam. Slides were coded before microscopic analysis.

-Evaluations: In general, 1000 polychromatic erythrocytes from each male and female animal of every test group was evaluated and investigated for micronuclei. The normochromatic erythrocytes which occur were also scored. The following parameters were recorded:
Number of polychromatic erythrocytes
Number of polychromatic erythrocytes containing micronuclei,
Number of normochromatic erythrocytes,
Number of normochromatic erythrocytes containing micronuclei,
Ratio of polychromatic to normochromatic erythrocytes,
Number of small micronuclei (d < D/4) and of large micronuclei (d >= D/4)

CRITERIA FOR DOSE SELECTION:
Doses were selected on the basis of a preliminary toxicity study: Here, a dose of 2000 mg/kg bw led to signs of toxicity including irregular respiration, piloerection abdominal position, apathy and squatting posture; the general state was poor.

Statistics:

No statistical methods were employed in data analysis.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

observed in the highest dose tested

CLINICAL EXAMINATIONS
The single intraperitoneal administration of the solvent in a volume of 10 mL/kg body weight was tolerated by all animals without any signs or symptoms. A dose of 2000 mg/kg bw. of test substance led to irregular respiration, piloerection, abdominal position and apathy about 30 minutes after administration; the general state of some animals was poor. After treatment of the animals with 1000 or 500 mg/kg, only irregular respiration and piloerection were observed after about 30 minutes. After about 1 -2 hours clinical signs were no longer observed. None of the positive control substances caused any evident signs of toxicity.

MICRONUCLEI
Mean polychromatic (normochromatic) erythrocytes containing micronuclei were:
Negative control (24 hrs) 1.5 % (1.5 %)
2000 mg/kg (16 hrs) 1.2 % (1.1 %)
2000 mg/kg (24 hrs) 1.7 % (0.25 %)
2000 mg/kg (48 hrs) 1.6 % (1.29 %)
1000 mg/kg (24 hrs) 2.4 % (1.0 %)
 500 mg/kg (16 hrs) 1.2 % (0.26 %)
Cyclophosphamide (24 hrs) 13.6 % (2.33 %)
Vincristine (24 hrs) 83.2 % (1.72 %)

Conclusions:

Interpretation of results: negative
Cytogenetic analysis of bone marrow cells of mice in vivo show negative results.

Executive summary:

To assess the cytotoxicity, 2 -pyrrolidone was tested in the Micronucleus Test in Mice in vivo. The study was conducted according to the OECD Guideline 474 and is compliant to GLP requirements.

Administration of test substance did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d D/4) or large micronuclei (d > D/4) did not deviate from the solvent control value at any sacrifice interval. No inhibition of erythropoiesis induced by the treatment of mice with Pyrrolidon-2 was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups. The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Gene mutation in bacteria

2-Pyrrolidone was not mutagenic in a plate incorporation Ames test with and without metabolic activation (tested up to 16500 μg/plate in Salmonella typhimurium TA1535, TA 1537, TA 1538, TA 98 and TA 100; metabolic activation: liver S-9 mix from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters; Jagannath 1987). Cytotoxicity (reduction of the background lawn) was observed at the highest concentration tested.

Cytogenicity in mammalian cells

In a GLP conform assay according to OECD guideline 473, human lymphocytes were tested for chromosome aberrations in doses up to 3500 µg/mL of 2 -pyrrolidone without metabolic activation and doses up to 6000 mg/mL with metabolic activation (BASF AG 1987). There were no increases of chromosome aberrations detected in any treatment if compared with the solvent controls in the assays with and without metabolic activation. Due to the very low mean number of aberrations in the negative control with metabolic activation, statistically significant effects were found in the two highest dose groups, but they were considered as not relevant.

Gene mutation in mammalian cells

2 -pyrrolidone was tested in HPRT locus assay to detect gene mutation in CHO cells (Welly, 2019). The test substance did not lead to an increase in the number of mutant colonies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. The mutant frequencies at any concentration were close to or within the range of that of the concurrent negative control values and within the range of the historical negative control data. The mutation frequencies of the negative control groups were within the historical negative control data range including all vehicles used in their laboratory and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMN clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. In both experiments in the absence and the presence of S9 mix, no cytotoxicity indicated by reduced relative cloning efficiency of below 20 % relative survival was observed.

In vivo

Cytogenicity in mammalian cells

Cytogenicity in vivo was tested in a mouse micronucleus test conducted according to GLP requirements and OECD 474 (BASF AG 1993). The test substance was administered once i.p. to 5 male and 5 female NMRI mice at doses up to 500, 1000, 2000 mg/kg body weight, respectively. Bone marrow cells were harvested after 16, 24 or 48 hours. Administration of the test substance did not lead to any increase in the rate of micronuclei. The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d<D/4) or large micronuclei (d>D/4) did not deviate from the solvent control value at any sacrifice interval. No inhibition of erythropoiesis induced by the treatment of mice with Pyrrolidon-2 was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups. The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.Positive and negative controls all produced appropriate responses. The test substance did not produce any chromosome-damaging (clastogenic) effect.

Justification for classification or non-classification

The test substance did not cause gene mutations in bacterial cells, did not cause cytogenicity in human cells and did not cause gene mutations at the hprt locus of CHO cells in vitro or in vivo experiments in mice.

Therefore, no indication is given for a gene mutation potential of 2-Pyrrolidone and classification is not warranted according to the criteria of EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.