1-butylpyrrolidin-2-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test (GLP, OECD 471): negative;
- MNT (GLP, OECD487): negative;
- Mouse Lymphoma Test (OECD 476): negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-01 to 2012-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): "Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (EMA/CHMP/ICH/126642/2008)"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Freie und Hansestadt Hamburg. Behörde für Soziales, Familie, Gesundheit und Verbraucherschutz

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: DNA repair mutation uvrB (TA 1535, TA 1537, TA 98, TA 100) and rfa mutation (TA 102)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix derived from Aroclor 1254-induced rat liver

Test concentrations with justification for top dose:

- Cytotoxicity test: ten concentrations ranging from 0.316 to 5000 μg plate
- Main Test: 31.6, 100, 316, 1000, 3160 and 5000 μg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the test item is completely soluble in acetone

Untreated negative controls:

yes

Remarks:

vehicle control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-amino-anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation (two independent experiments were conducted).

DURATION
- Preincubation period: 20 min (preincubation method)
- Exposure duration: 48 to 72 hours


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.

Evaluation criteria:

described in "Statistics".

Statistics:

A test item is considered to show a positive response if:
- at one or more concentrations the number of revertants is reproducibly increased in at least one strain with or without metabolic activation. A 2- fold increase in comparison to the solvent control is regarded as being relevant for a positive response in the strains TA 98, TA 100 and TA 102. For the strains TA 1535 and TA 1537 a 3-fold increase represents a biological relevant effect. The Mann and Whitney test (p ≤ 0.05) may be used to determine statistical significance;
- a concentration-related increase of the revertants is observed. The Spearman's rank correlation coefficient may be applied.
Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates. A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test. Ten concentrations ranging from 0.316 to 5000 μg test substance/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 μg/plate. Hence, 5000 μg /plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

COMPARISON WITH HISTORICAL CONTROL DATA: the number of revertant colonies are in the range of historical control data.

Remarks on result:

other: all strains/cell types tested

Table 1. Preliminary cytotoxicity test. Plate incorporation test (without metabolic activation)

Test item (µg/plate)	Background lawn	Revertants per plate (TA 100) (cytotoxicity)
		plate 1 / plate 2
0.316	normal	135 / 131
1.0	normal	130/ 135
3.16	normal	140 / 134
10.0	normal	136 / 139
31.6	normal	112 / 113
100	normal	148 / 155
316	normal	143 / 142
1000	normal	119 / 141
3160	normal	113 / 121
5000	normal	141 / 116
Vehicle control 100 (µL/plate)	normal	155 / 165

 

Table 2. Main test. Plate incorporation test (without metabolic activation)

Test item (µg/plate)	Number of reverted colonies (mean values ± SD)
	TA 98	TA 100	TA 102	TA 1535	TA 1537
31.6	31.3 ± 1.5	123.0 ± 7.2	258.3 ± 2.9	12.0 ± 1.7	4.7 ± 0.6
100	32.3 ±1.5	115.7 ± 3.2	252.7 ± 2.1	15.3 ± 4.2	4.3 ± 1.5
316	23.3 ± 1.5	144.0 ± 5.6	271.0 ± 8.7	13.3 ± 3.5	4.0 ± 1.7
1000	31.3 ± 5.5	122.0 ± 18.2	278.3 ± 4.0	13.0 ± 3.6	4.3 ± 1.2
3160	25.3 ± 4.0	124.7 ± 4.2	277.0 ± 1.0	18.0 ±1.0	4.0 ± 1.7
5000	27.0 ± 1.7	160.7 ± 10.0	271.0 ± 2.6	16.3 ± 0.6	4.0 ± 1.0
Vehicle control (100 µL/plate)	28.0 ± 5.2	148.0 ± 3.6	275.0 ± 2.6	16.7 ± 3.2	7.7 ± 0.6
Positive reference item	2-Nitro-fluorene	Sodium azide	Mito- mycin C	Sodium azide	9-Amino-acridine
Concentration µg/plate	10	10	10	10	100
	123.7 ± 1.5	1013.7 ±19.5	1076.3 ±35.6	196.0 ±1.0	108.0 ± 4.6
SD - standard deviation mean (n = 3)

 

Table 3. Main test. Plate incorporation test (with metabolic activation)

Test item (µg/plate)	Number of reverted colonies (mean values ± SD)
	TA 98	TA 100	TA 102	TA 1535	TA 1537
31.6	26.0 ± 0.0	126.3 ± 2.5	295.0 ± 7.5	17.3 ± 1.5	4.7 ± 2.5
100	23.7 ± 0.6	127.7 ± 3.8	294.7 ± 8.0	18.0 ± 3.5	4.7 ± 2.9
316	27.0 ± 2.6	126.3 ± 7.4	280.0 ± 7.9	14.0 ± 3.6	4.3 ± 1.2
1000	24.7 ± 4.2	114.7 ± 11.2	284.3 ± 3.2	15.3 ± 1.5	6.0 ± 2.6
3160	30.3 ± 1.2	116.7 ± 6.4	278.0 ± 7.8	14.0 ± 6.1	3.7 ± 2.1
5000	30.3 ± 7.1	118.3 ± 4.2	265.3 ± 9.0	14.3 ± 4.9	4.0 ± 1.7
Vehicle control 100 µL/plate	25.3 ± 2.1	131.3 ± 8.1	255.7 ± 5.5	15.7 ± 2.3	7.0 ± 2.0
Positive reference item	Benzo(a)pyrene	2-Amino-anthracene	Benzo(a)pyrene	2-Amino-anthracene	Benzo(a)pyrene
Concentration µg/plate	10	2	10	2	10
	208.7 ± 8.1	1042.7 ± 10.7	951.7 ± 18.5	191.0 ± 6.9	107.3 ± 10.0
SD - standard deviation mean (n = 3)

 

Table 4. Main test. Preincubation test (without metabolic activation)

Test item (µg/plate)	Number of reverted colonies (mean values ± SD)
	TA 98	TA 100	TA 102	TA 1535	TA 1537
31.6	22.7 ± 1.5	128.0 ± 6.2	269.3 ± 4.5	14.0 ± 1.7	3.7 ± 1.2
100	22.7 ± 3.8	117.0 ± 16.8	265.7 ± 4.0	20.3 ± 1.2	3.7 ± 0.6
316	37.0 ± 1.7	122.3 ± 12.0	265.3 ± 4.0	16.0 ± 2.6	3.0 ± 1.0
1000	29.3 ± 6.7	107.0 ± 3.6	263.3 ± 4.2	16.0 ± 0.0	4.3 ± 3.2
3160	34.0 ± 6.1	115.3 ± 5.9	280.3 ± 24.8	17.0 ± 3.5	4.7 ± 1.5
5000	23.7 ± 1.2	123.0 ± 9.5	251.3 ± 21.9	15.3 ± 4.9	6.0 ± 4.0
Vehicle control (50 µL/plate)	28.0 ± 5.6	133.3 ± 2.5	252.7 ± 1.5	15.3 ± 3.8	5.3 ± 4.2
Positive reference item	2-Nitrofluorene	Sodium azide	Mitomycin C	Sodium azide	9-Aminoacridine
Concentration (µg/plate)	10	10	10	10	100
	136.0 ± 2.0	1002.0 ± 25.2	947.0 ± 7.8	137.7 ± 3.2	107.7 ± 2.1
SD - standard deviation mean (n = 3)

 

Table 5. Main test. Preincubation test (with metabolic activation)

Test item (µg/plate)	Number of reverted colonies mean values ± SD
	TA 98	TA 100	TA 102	TA 1535	TA 1537
31.6	21.7 ± 0.6	107.7 ± 4.2	270.7 ± 1.5	23.3 ± 0.6	3.7 ± 1.2
100	22.3 ± 1.5	127.3 ± 11.6	270.3 ± 1.5	24.0 ± 2.0	6.7 ± 0.6
316	26.0 ± 1.0	133.0 ± 7.8	269.7 ± 1.5	21.0 ± 5.2	6.7 ± 1.2
1000	32.7 ± 4.6	116.0 ± 9.8	270.3 ± 3.5	22.0 ± 3.6	5.3 ± 2.5
3160	27.7 ± 1.2	118.0 ± 2.6	287.0 ± 1.7	20.3 ± 4.9	4.3 ± 1.5
5000	25.3 ± 3.8	114.3 ± 4.5	300.3 ± 1.5	18.7 ± 2.9	5.3 ± 3.2
Vehicle control (50 µL/plate)	26.7 ± 3.8	115.7 ± 5.5	272.0 ± 7.9	23.0 ± 2.0	7.0 ± 1.0
Positive reference item	Benzo(a)pyrene	2-Aminoanthracene	Benzo(a)pyrene	2-Aminoanthracene	Benzo(a) pyrene
Concentration (µg/plate)	10	2	10	2	10
	130.0 ± 6.1	1004.7 ± 20.0	986.7 ± 5.5	137.3 ± 2.1	111.0 ± 2.0
SD - standard deviation mean (n = 3)

 

Conclusions:

The test item was negative in the bacterial reverse mutation test (Ames Test) with and without metabolic activation.

Executive summary:

The test item was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test item was completely dissolved in acetone. The vehicle served as the negative control.

Preliminary test

The test substance was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test. Ten concentrations ranging from 0.316 to 5000 μg test substance/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 μg/plate. Hence, 5000 μg test substance/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 31.6 to 5000 μg test substance/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 μg test substance/plate in all test strains.

No increase in revertant colony numbers as compared with control counts was observed for the test substance, tested up to a concentration of 5000 μg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test). 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 tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-12-11 to 2014-01-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals No. 487 "In Vitro Mammalian Cell Micronucleus Test".

Deviations:

no

Principles of method if other than guideline:

An in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable.

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin В and 10% foetal bovine serum (FBS)
- Properly maintained: yes

Additional strain / cell type characteristics:

other: The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Metabolic activation:

with and without

Metabolic activation system:

The S9 Microsomal fraction prepared from male rats induced with Phenobarbitone/p-Naphthoflavone at 80/100 mg/kg/day (2% of final concentration)

Test concentrations with justification for top dose:

- The dose range was 5.51, 11.03, 22.05, 44.1, 88.2, 176.4, 352.8, 705.6 and 1411.2 µg/mL (Preliminary Toxicity Test);
- 44.1, 88.2, 176.4, 352.8, 705.6 and 1411.2 µg/mL (Experiment 1 and 2).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Minimal Essential Medium (MEM).
- Justification for choice of solvent/vehicle: the test item was soluble in culture media at 14.11 mg/mL in solubility checks performed in-house.

Untreated negative controls:

yes

Remarks:

vehicle control

Negative solvent / vehicle controls:

yes

Remarks:

medium control

True negative controls:

no

Positive controls:

yes

Remarks:

in the absence of S9 mix in Experiment 1.

Positive control substance:

mitomycin C

Remarks:

was used at 0.2 µg/mL. It was dissolved in Minimal Essential Medium.

Positive controls:

yes

Remarks:

in the presence of S9 mix

Positive control substance:

cyclophosphamide

Remarks:

was used at 5 µg/mL. It was dissolved in dimethyl sulphoxide (DMSO).

Positive controls:

yes

Remarks:

In the absence of S9, in Experiment 2,

Positive control substance:

other: demecolcine (DC)

Remarks:

was dissolved in water and used at a concentration of 0.075 µg/mL.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours (all experiments).
- Exposure duration: 4 hours (with and without S9 mix) in the Preliminary Toxicity test and in the Experiment 1; 24 hours continuous exposure (without S9 mix) in the Preliminary Toxicity test and in the Experiment 2.
- Expression time (cells in growth medium): 28 hours (all experiments).
- Fixation time (start of exposure up to fixation or harvest of cells): 32 hours (with and without S9 mix) in the Preliminary Toxicity test and in Experiment 1; 52 hours (without S9 mix) in the Preliminary Toxicity test and in the Experiment 2.

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin В.
STAIN (for cytogenetic assays): in 5% Giemsa for 5 minutes.

NUMBER OF CELLS EVALUATED: 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls (see "Any other information on materials and methods").
The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration) (see "Any other information on materials and methods").

DETERMINATION OF CYTOTOXICITY
- Method: calculation of cytokinesis block proliferation index (CBPI).
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the experiments of the main test.

Evaluation criteria:

The following criteria was used to determine a valid assay:
Negative Control
The frequency of binucleate cells with micronuclei in the vehicle control cultures will normally be within the laboratory historical control data range. The level of spontaneous background micronuclei may be slightly elevated above the normal range and the experiment still considered to be valid.
Positive Control
All the positive control chemicals must induce positive responses (p<0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9 mix.

Statistics:

The frequency of cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analysis may be used if appropriate (Hoffman et aL, 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of cells with aberrations which was reproducible.

HOFFMAN W.P., GARRIOTT MX., LEE C. (2003). In vitro micronucleus test. In: Encyclopaedia of Biopharmaceutical Statistics, 2nd edition. S Chow ed. Marcel Dekker, Inc. New York Press, NY, pp. 463 - 467.

Species / strain:

lymphocytes:

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes:

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

modest reduction in CBPI of 22% at the maximum dose of 1411.2 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes:

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

a modest reduction in the CBPI observed, and that 24% inhibition of cell proliferation was achieved at 1411.2 µg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media
- Effects of osmolality: When the test item was dosed into media the osmolality did not increase by more than 50 mOsm
- Precipitation: No precipitate was observed in either exposure group at the end of the exposure period.

RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 5.51, 11.03, 22.05, 44.1, 88.2, 176.4, 352.8, 705.6 and 1411.2 µg/mL. The maximum dose was the maximum recommended dose level, the 10 mM concentration.
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period, in any of the three exposure groups.

Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present up to 1411.2 µg/mL in all three exposure groups. The CBPI data are presented in Table 1 (see attached to this file). The test item induced no evidence of toxicity in the 4-hour exposure groups in the absence and presence of S9 but demonstrated modest toxicity at the maximum dose of 1411.2 µg/mL in the 24-hour exposure group with a 25% reduction in CBPI. The selection of the maximum dose level was based on the maximum recommended dose level and was 1411.2 µg/mL for all three exposure groups.

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei.

Remarks on result:

other: all strains/cell types tested

Remarks:

Remarks: 4-hour experiment

Experiment 1

The dose levels of the controls and the test item are given in the table below:

Group	Final concentration of test substance (µg/mL)
4-hour without S9	0*, 44.1, 88.2, 176.4, 352.8*, 705.6*, 1411.2*, MMC 0.2*
4-hour with S9 (2%)	0*, 44.1, 88.2, 176.4, 352.8*, 705.6*, 1411.2* CP 5*

* = Dose levels selected for analysis of micronucleus frequency in binucleate cells;

MMC = Mitomycin С;

CP = Cyclophosphamide.

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the maximum dose level of test item, 1411.2 µg/mL in the presence and absence of S9. No precipitate was observed in either exposure group at the end of the exposure period.

The CBPI data are given in Table 2 (see attached to this file) for the without and with S9 groups. They confirm the qualitative observations in that there was no marked toxicity in the presence of S9. In the absence of S9 there was a modest reduction in CBPI of 22% at the maximum dose of 1411.2 µg/mL.

The maximum dose level selected for analysis of binucleate cells was the maximum recommended dose level, the 10 mM concentration (1411.2 µg/mL).

The micronucleus data are given in Table 4 and Table 5 (see attached to this file). The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, either in the absence or presence of metabolic activation.

 

Experiment 2

The dose levels of the controls and the test item are given in the table below:

Group	Final concentration of test substance (µg/mL)
24-hour without S9	0*, 44.1, 88.2, 176.4, 352.8*, 705.6*, 1411.2*, DC 0.075*

* = Dose levels selected for analysis of micronucleus frequency in binucleate cells

DC = Demecolcine

The qualitative assessment of the slides determined that there were binucleate cells suitable for scoring at the maximum test item dose level of 1411.2 µg/mL.

No precipitate was observed at the end of the exposure period.

The CBPI data are given in Table 3 (see attached to this file) for the 24-hour exposure group. They confirm the qualitative observations in that there was a modest reduction in the CBPI observed, and that 24% inhibition of cell proliferation was achieved at 1411.2 µg/mL. The maximum dose level selected for binucleate cell analysis was the maximum recommended dose level (1411.2 µg/mL).

The micronucleus data are given in Table 6. The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, either in the absence or presence of metabolic activation.

 

Conclusions:

The test item did not induce any statistically significant increases in the frequency of cells with micronuclei in either the absence or presence of a metabolizing system, in either of two separate experiments. The test item was therefore considered to be non-clastogenic and non aneugenic to human lymphocytes in vitro.

Executive summary:

An in vitro study for the detection of the clastogenic and aneugenic potential of N-Butylpyrrolidone on the nuclei of normal human lymphocytes was conducted (Harlan Laboratories, 2014; Report No. 41303962). Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions were used for the study. Experiment 1 used a 4-hour exposure in the presence and absence of a standard metabolizing system (S9, at a 2% final concentration). Experiment 2, used a 24-hour exposure in the absence of metabolic activation and was performed concurrently with the exposure groups of Experiment 1. At the end of the exposure period, the cell cultures were washed and then incubated for a further 28 hours in the presence of Cytochalasin B. The dose levels used in the main experiments (4 hour with and without S9 mix and in 24 -hour without S9 mix) were selected using data from the preliminary toxicity test and were 44.1, 88.2, 176.4, 352.8, 705.6, and 411.2 µg/mL.

All vehicle (solvent) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments, using a dose range that included a dose level that was the maximum recommended dose level.

In conclusion, N-Butylpyrrolidone was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-12-04 to 2014-02-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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: Japanese MITI/MHW guidelines for testing of new chemical substances: "Каnроаn No. 287 - - Environment Protection Agency"; "Eisei No. 127 - - Ministry of Health and Welfare"; "Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin В (2.5 µg/mL) and 10 % donor horse serum (giving R10 media). RPMI 1640 with 20 % donor horse serum (R20) and without serum (R0) are used during the course of the study.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes. Master stocks of cells were tested and found to be free of mycoplasma.
- Periodically checked for karyotype stability: not reported
- Periodically "cleansed" against high spontaneous background: yes. The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 |ug/mL), Hypoxanthine (15 |ug/mL), Methotrexate (0.3 ^ig/mL) and Glycine (22.5 ^ig/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Additional strain / cell type characteristics:

other: -3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

The S9 microsomal fraction prepared from Sprague Dawley male rats induced with Phenobarbital/p-Naphthoflavone at 80/100 mg/kg/day (2% of final concentration in the Preliminary Experiment and Experiment 1; 1% of final concentration in the Experiment 2).

Test concentrations with justification for top dose:

- Preliminary Cytotoxicity Test: 0, 5.51, 11.03, 22.05, 44.1, 88.2, 176.4, 352.8, 705.6 and 1411.2 µg/mL;
- Main Test (Experiments 1 and 2): 0, 44.1, 88.2, 176.4, 352.8, 705.6, 1411.2

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium without serum (R0)
- Justification for choice of solvent/vehicle: the test item was soluble in culture media at 1411.2 µg/mL in solubility checks performed in-house.

Untreated negative controls:

yes

Remarks:

solvent controls

Negative solvent / vehicle controls:

yes

Remarks:

RPMI 1640 medium without serum (R0)

True negative controls:

no

Positive controls:

yes

Remarks:

in the absence of S9 mix

Positive control substance:

ethylmethanesulphonate

Remarks:

at 400 µg/mL and 150 µg/mL for Experiment 1 and Experiment 2, respectively.

Positive controls:

yes

Remarks:

in the presence of metabolic activation in both experiments

Positive control substance:

cyclophosphamide

Remarks:

at 2 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours (with and without of S9 mix in the Preliminary Experiment and in the Experiment 1 and with S9 mix in the Experiment 2); 24 hours (without S9 mix in the Experiment 2).
- Expression time (cells in growth medium): 48 hours (all experiments)
- Selection time (if incubation with a selection agent): 24 hours (on Day 2 of the experiments)
- Fixation time (start of exposure up to fixation or harvest of cells): Microtitre plates were scored using a magnifying mirror box after ten to fourteen days' incubation at 37 °С with 5% CO2 in air.

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF CELLS EVALUATED: for mutant frequency 2000 cells /well in selective medium and for viability 2 cells/ well in non-selective medium (96-well microtitre plates were used).

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; other: Relative Suspension Growth (% RSG)

Evaluation criteria:

Acceptability of the Assay
A mutation assay is considered acceptable if it meets the following criteria (the current recommendations of the IWGT will be considered):
1. The majority of the plates, for either viability (%V) or TFT resistance are analyzable for each experiment.
2. The absolute viability (%V) at the time of mutant selection of the solvent controls is 65 to 120 %.
3. The total suspension growth of the solvent control following 4 hour treatment, calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold increase in cell number, should be in the range of 8 to 32. Following 24 hour treatment the total suspension growth should be in the range of 32 to 180.
4. The vehicle control mutant frequency should be in the range of 50 - 170 x 10E6 cells. Experiments where the vehicle control values are markedly greater than 200 x 10E6 mutant frequency per survivor are not acceptable and will be repeated.
5. Positive control chemicals (EMS and CP) should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control. The positive controls should ideally yield an absolute increase in total MF, that is an increase above spontaneous background MF (an induced MF [IMF]), of at least 300 x 10E6 cells.
6. The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures i.e. the relative total growth (RTG) and percentage relative suspension growth (%RSG) should be greater than 10 % of the concurrent selective control group.
7. The highest concentration of the test item should be 10 mM or 5000 µg/mL, unless limited by toxicity or solubility of the test item. If toxicity occurrs, the highest concentration should lower the relative total growth (RTG) and/or %RSG to approximately 10 to 20 % of survival. If precipitation is noted, the highest analyzed concentration should be the lowest concentration where precipitation is observed by the naked eye.

Statistics:

The experimental data was analyzed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W.D. et al., 1989):
ROBINSON, W.D. et al (1989) Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University Press Report part III, pp 102-140.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

as indicated by the % RSG and RTG values

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media
- Effects of osmolality: When the test item was dosed into media the osmolality did not increase by more than 50 mOsm.
- Precipitation: No precipitate of the test item was observed at any dose level in all experiments.

RANGE-FINDING/SCREENING STUDIES:
In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was no evidence of marked reductions in the Relative Suspension Growth (% RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was evidence of marked reductions of % RSG values of cells treated with test item. No precipitate of the test item was observed at any dose level. In the subsequent mutagenicity experiments the maximum dose was the 10 mM Maximum Recommended Dose level of 1411.2 (µg/mL).

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Remarks: Experiment 1

Preliminary Toxicity Test

The results for the Relative Suspension Growth (%RSG) were as follows:

Dose	% RSG (-S9)	% RSG (+S9)	% RSG (-S9)
(µg/mL)	4-Hour Exposure	4-Hour Exposure	24-Hour Exposure
0	100	100	100
5.51	101	95	89
11.03	92	95	82
22.05	108	103	83
44.1	85	98	74
88.2	88	88	82
176.4	94	100	82
352.8	93	111	57
705.6	85	89	55
1411.2	101	96	26

Main Test (summary of results)

Experiment 1
4-Hour (-S-9)				4-Hour (+S-9)
Treatment (µg/mL)	%RSG	RTG	MF§	Treatment (µg/mL)	%RSG	RTG	MF§
0	100	1.00	148.90	0	100	1.00	157.72
44.1	88	0.82	140.74	44.1	97	0.92	152.08
88.2	91	0.85	146.43	88.2	103	1.10	117.37
176.4	90	0.93	126.27	176.4	99	0.99	152.74
352.8	99	0.89	147.36	352.8	99	0.96	140.91
705.6	98	0.91	154.40	705.6	89	0.99	137.59
1411.2	93	0.98	146.56	1411.2	86	0.95	127.00
Linear trend			NS	Linear trend			NS
EMS				CP
400	67	0.59	1138.00	2	43	0.31	985.90
Experiment2
24-Hour (-S-9)				4-Hour (+S-9)
Treatment (µg/mL)	%RSG	RTG	MF§	Treatment (µg/mL)	%RSG	RTG	MF§
0	100	1.00	140.34	0	100	1.00	152.64
44.1	82	0.83	117.10	44.1	103	1.02	169.92
88.2	77	0.66	125.12	88.2	109	1.34	128.74
176.4	80	0.86	124.91	176.4	98	1.07	129.14
352.8	76	0.75	128.57	352.8	101	1.13	137.65
705.6	57	0.68	110.75	705.6	109	1.21	139.82
1411.2	22	0.36	103.68	1411.2	99	1.01	136.24
Linear trend			NS	Linear trend			NS
EMS				CP
150	34	0.30	1283.20	2	69	0.45	1028.86

MF§ = 5-TFT resistant mutants/ 10E6 viable cells 2 days after treatment

NS = Not significant

EMS = Ethylmethanesulphonate

CP Cyclophosphamide

Experiment 1

There was no evidence of toxicity following exposure to the test item in either the absence or presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of any marked reductions in viability (% V), therefore indicating that no residual toxicity occurred, in either the absence or presence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10E6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10E6 per viable cell in either the presence or absence of metabolic activation. No precipitate of test item was observed.

Experiment 2

There was evidence of toxicity following exposure to the test item in the absence of metabolic activation only, as indicated by the % RSG and RTG values. There was no evidence of any marked reductions in viability (% V), therefore indicating that no residual toxicity occurred, in either the absence or presence of metabolic activation. Near to optimum levels of toxicity were observed in the 24-hour exposure group.

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a marked effect on the toxicity of the test item. Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10E6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional. The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency in either the absence or presence of metabolic activation. No precipitate of test item was observed.

Conclusions:

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenicity of N-Butylpyrrolidone on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line (OECD 476). Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels (44.1, 88.2, 176.4, 352.8, 705.6, 1411.2 µg/mL), in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2 % S9). In Experiment 2, the cells were treated with the test item at the same six dose levels using a 4-hour exposure group in the presence of metabolic activation (1 % S9) and a 24 hour exposure group in the absence of metabolic activation.

The maximum dose level used in the main test was the 10 mM Maximum Recommended Dose level. No precipitate of test item was observed. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

N-Butylpyrrolidone did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at the TK +/- locus in L5178Y cells at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment. Therefore, N-Butylpyrrolidone is considered to be non-mutagenic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test (OECD 471)

N-Butylpyrrolidone was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver; LTP, 2013; Report No. 29175). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. N-Butylpyrrolidone was completely dissolved in acetone. The vehicle served as the negative control. The test item was examined in a preliminary cytotoxicity test without metabolic activation in test strain TA 100 employing a plate incorporation test. Ten concentrations ranging from 0.316 to 5000 μg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 μg/plate. Hence, 5000 μg test item /plate were chosen as top concentration for the main study in both tests.
In the main study, six concentrations ranging from 31.6 to 5000 μg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. No signs of cytotoxicity were noted in both tests without and with metabolic activation up to the top concentration of 5000 μg test item/plate in all test strains. No increase in revertant colony numbers as compared with control counts was observed for the test item, tested up to a concentration of 5000 μg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test). 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 conditions of the test, N-Butylpyrrolidone tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Micronucleus Test in vitro (OECD 487)

An in vitro study for the detection of the clastogenic and aneugenic potential of N-Butylpyrrolidone on the nuclei of normal human lymphocytes was conducted (Harlan Laboratories, 2014d; Report No. 41303962). Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions were used for the study. Experiment 1 used a 4-hour exposure in the presence and absence of a standard metabolizing system (S9, at a 2 % final concentration). Experiment 2, used a 24-hour exposure in the absence of metabolic activation and was performed concurrently with the exposure groups of Experiment 1. At the end of the exposure period, the cell cultures were washed and then incubated for a further 28 hours in the presence of Cytochalasin B. The dose levels used in the main experiments (4 hour with and without S9 mix and in 24 -hour without S9 mix) were selected using data from the preliminary toxicity test and were 44.1, 88.2, 176.4, 352.8, 705.6, and 411.2 µg/mL.

All vehicle (solvent) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments, using a dose range that included a dose level that was the maximum recommended dose level.

In conclusion, N-Butylpyrrolidone was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Mouse Lymphoma Test (OECD 476)

The study was conducted according to a method that was designed to assess the potential mutagenicity of N-Butylpyrrolidone on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line (OECD 476). Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels (44.1, 88.2, 176.4, 352.8, 705.6, 1411.2 µg/mL), in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2 % S9). In Experiment 2, the cells were treated with the test item at the same six dose levels using a 4-hour exposure group in the presence of metabolic activation (1 % S9) and a 24 hour exposure group in the absence of metabolic activation.

The maximum dose level used in the main test was the 10 mM Maximum Recommended Dose level. No precipitate of test item was observed. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

N-Butylpyrrolidone did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at the TK +/- locus in L5178Y cells at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment. Therefore, N-Butylpyrrolidone is considered to be non-mutagenic under the conditions of the test.

Justification for classification or non-classification

N-Butylpyrrolidone did not induce genetic toxicity in all available tests. No mutagenic activity was observed in bacterial strains of Salmonella typhimurium (Ames Test; OECD 471) and in mouse lymphoma cell line L5178Y (OECD 476). The substance was non-clastogenic and non-aneugenic to human lymphocytes in vitro (OECD 487). Therefore, according to Regulation (EC) 1272/2008, the substance does not need to be classified and labelled for this endpoint.