1-butylpyrrolidin-2-one

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

1-butylpyrrolidin-2-one

EC Number:

222-437-8

EC Name:

1-butylpyrrolidin-2-one

Cas Number:

3470-98-2

Molecular formula:

C8H15NO

IUPAC Name:

1-butylpyrrolidin-2-one

Test material form:

other: liquid

Details on test material:

- Substance type: organic
- Physical state: colourless liquid
- Purity test date: 2012-09-13
- Expiration date of the lot/batch: September 13, 2032
- Storage condition of test material: at +10 °C to +25 °C, in a tightly closed container in a dry place

Method

Target gene:

his-

Species / strain

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

Controls

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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)

 

Applicant's summary and conclusion

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.