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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
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
- Analytical purity: 99.3%
- Purity test date: 2012-09-13
- Lot/batch No.: 120913_639
- 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
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and 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
Remarks:
Migrated from field 'Test system'.

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.