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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:
2017-03-08 - 2017-03-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008-05-30
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Content: w(C5H9N) = 89.2 g/100 g water content: 7.8 g/100 g
- Purity: 99.2 area %
- Batch Nr.: 85603856P0
- Physical state, appearance: liquid, colorless, clear
- Date of production: 2016-10-19
- Storage conditions: room temperature

Method

Target gene:
Salmonella typhimurium: All used strains have a defective excision repair system (uvrB). Target genes were his G46 (TA 1535 and TA 100), his C 3076 (TA1537), his D 3052 (TA 98).

Escherichia coli: tryptophan auxotrophy/tryptophan independence
Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 mix from induced rats
Test concentrations with justification for top dose:
Standard Plate Test (SPT): 0; 33;100; 333; 1000; 2800 and 5600 µg/plate
Preincubation Test (PIT): 0; 33;100; 333; 1000; 2800 and 5600 µg/plate

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. In this study, due to the purity of the test substance 5.6 mg/plate was used as top dose in all experiments.

Recommendations of current guidelines: 5 mg/plate or 5 μL/plate (tested even in the case of relatively insoluble test compounds).
Due to the purity of the test substance 5.6 mg/plate was used as top dose in all experiments.
Vehicle:
- Vehicle used: ultrapure water
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene (2-AA)
Details on test system and conditions:
METHOD OF APPLICATION:
- Standard Plate Test (SPT) and Preincubation Test (PIT), both with and without metabolic activation (liver s9 mix from induced rats)

STANDARD PLATE TEST
- Incubation conditions: 37°C, dark conditions
- Incubation period: 48-72 hours
- Number of plates: 3 plates per dose or per control

PREINCUBATION TEST
- Preincubation conditions: 37°C
- Preincubation period: 20 minutes
- Incubation conditions: 37°C, dark conditions
- Incubation period: 48-72 hours
- Number of plates: 3 plates per dose or per control
- Reason: No mutagenicity was observed in the standard plate test

CONTROLS
1) Negative controls:
- Sterility control: with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains
- Vehicle control: only contained the vehicle (ultrapure water) used for the test substance at the same volume for all tester strains (with and wiithout S9 mix)

2) Positive controls:
With S9 mix
• 2-aminoanthracene (2-AA) (Sigma-Aldrich; 96%)
- 2.5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO
- strain: Escherichia coli WP2 uvrA

Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Fluka; 97%)
- 5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD) (Sigma-Aldrich; 98%)
- 10 μg/plate, dissolved in DMSO
- strain: TA 98
• 9-aminoacridine (AAC) (Sigma-Aldrich; 98%)
- 100 μg/plate, dissolved in DMSO
- strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO) (Sigma-Aldrich; 98%)
- 5 μg/plate, dissolved in DMSO
- strain: E. coli WP2 uvrA


The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using Image Analysis System.





Evaluation criteria:
MUTAGENICITY
The mutagenicity of the test substance was detected by individual plate counts and the mean number of revertant colonies per plate at all dose groups as well as for the positive and negative (vehicle) controls in all experiments.
TOXICITY
The toxicity was detected by a decrease in the number of revertants (factor ≤ 0.6) and by clearing or diminution of the background lawn (= reduced his- or trp- background growth). Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

ACCEPTANCE CRITERIA
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain (see Appendix 5).
• The sterility controls revealed no indication of bacterial contamination (see Appendix 3).
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 109 cells per mL were used.

ASSESSMENT CRITERIA
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Standard Plate Test
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Standard Plate Test
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Standard Plate Test
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Standard Plate Test
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Standard Plate Test
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Preincubation Test
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Preincubation Test
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Preincubation Test
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Preincubation Test
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Preincubation Test

Any other information on results incl. tables

Table 1: Results from Standard Plate Test (SPT) without metabolic activation.

Strain

Test group

Dose (μg/plate)

Mean revertants per plate

Standard deviation

Factor

 Individual revertant colony counts

 

 

 

 

 

 

 

TA 1535

Water Test item

-

10.7

2.1

-

9, 13, 10

 

 

 

 

 

 

 

 

 

33

11.3

3.8

1.1

13, 14, 7

 

 

100

12.3

2.5

1.2

10, 12, 15

 

 

333

13.0

3.5

1.2

15, 9, 15

 

 

1000

11.7

3.2

1.1

14, 13, 8

 

 

2800

11.3

3.8

1.1

7, 14, 13

 

 

5600

7.7

1.2

0.7

7, 9, 7

 

MNNG

5.0

5811.0

752.0

544.8

6560, 5056, 5817

 

 

 

 

 

 

 

TA 100

Water Test item

-

109.0

16.5

-

128, 101, 98

 

 

33

117.3

5.5

1.1

120, 121, 111

 

 

100

121.3

6.5

1.1

121, 115, 128

 

 

333

123.0

12.8

1.1

126, 134, 109

 

 

1000

110.0

10.0

1.0

120, 110, 100

 

 

2800

130.0

5.2

1.2

127, 127, 136

 

 

5600

120.3

20.5

1.1

120, 100, 141

 

MNNG

5.0

3971.7

673.0

36.4

4318, 3196, 4401

 

 

 

 

 

 

 

TA 1537

Water Test item

-

9.3

2.3

-

12, 8, 8

33

11.0

2.6

1.2

9, 14, 10

 

 

100

8.7

6.7

0.9

3, 16, 7

 

 

333

6.7

3.2

0.7

8, 9, 3

 

 

1000

8.7

5.1

0.9

10, 13, 3

 

 

2800

10.0

1.7

1.1

9, 12, 9

 

 

5600

9.0

3.6

1.0

13, 8, 6

 

 

 

AAC

100

1299.3

176.2

139.2

1271, 1139, 1488

 

 

 

 

 

 

 

TA 98

Water Test item

-

20.7

4.0

-

23, 16, 23

 

 

33

20.0

5.6

1.0

15, 19, 26

 

 

100

22.3

4.0

1.1

26, 18, 23

 

 

333

14.0

5.6

0.7

15, 8, 19

 

 

1000

20.0

1.0

1.0

19, 21, 20

 

 

2800

21.3

9.0

1.0

30, 12, 22

 

 

5600

18.7

4.5

0.9

14, 19, 23

 

NOPD

10

555.3

19.3

26.9

540, 549, 577

 

 

 

 

 

 

 

E. coli

 Water Test item

-

22.7

3.1

-

20, 22, 26

 

 

33

27.7

0.6

1.2

 28, 28, 27

 

 

100

26.7

8.0

1.2

 26, 19, 35

 

 

333

20.3

9.1

0.9

 30, 19, 12

 

 

1000

16.0

3.0

0.7

 13, 16, 19

 

 

2800

24.3

9.1

1.1

 16, 34, 23

 

 

5600

19.0

3.0

0.8

16, 22, 19

 

4-NQO

5

1018.7

16.6

44.9

1017, 1003, 1036

Table 2: Results from Standard Plate Test (SPT) with metabolic activation.

Strain

Test group

Dose (μg/plate)

Mean revertants per plate

Standard deviation

Factor

Individual revertant colony counts

 

 

 

 

 

 

 

TA 1535

Water Test item

-

9.0

2.0

-

11, 7, 9

 

 

33

11.3

4.2

1.3

8, 16, 10

 

 

100

13.3

1.5

1.5

15, 12, 13

 

333

7.3

4.2

0.8

6, 12, 4

 

 

 

1000

6.7

4.0

0.7

 9, 9, 2

 

 

2800

9.7

3.5

1.1

6, 13, 10

 

 

5600

12.0

3.6

1.3

13, 8, 15

 

2-AA

2.5

300.3

49.1

33.4

282, 356, 263

 

 

 

 

 

 

 

TA 100

Water Test item

-

116.3

13.7

-

104, 114, 131

 

 

33

114.3

11.2

1.0

106, 127, 110

 

 

100

120.0

16.1

1.0

105, 137, 118

 

 

333

123.3

9.7

1.1

121, 115, 134

 

 

1000

139.3

19.9

1.2

161, 135, 122

 

 

2800

127.3

3.8

1.1

123, 130, 129

 

 

5600

118.3

5.7

1.0

112, 120, 123

 

2-AA

2.5

2987.3

220.9

25.7

3241, 2837, 2884

 

 

 

 

 

 

 

TA 1537

Water Test item

 -

 6.7

 3.1

 -

6, 4, 10

 

 

33

6.3

3.1

1.0

7, 3, 9

 

 

100

7.7

2.5

1.2

5, 8, 10

 

 

333

8.7

2.3

1.3

6, 10, 10

 

 

1000

5.3

2.1

0.8

3, 7, 6

 

 

2800

8.7

3.2

1.3

11, 10, 5

 

 

5600

6.7

2.9

1.0

5, 5, 10

 

2-AA

2.5

270.0

32.4

40.5

234, 279, 297

 

 

 

 

 

 

 

TA 98

Water Test item

-

26.7

1.5

-

27, 28, 25

 

 

33

24.7

6.1

0.9

30, 18, 26

 

 

100

25.7

3.1

1.0

29, 23, 25

 

 

333

24.7

6.7

0.9

23, 32, 19

 

 

1000

25.3

3.2

1.0

24, 29, 23

 

 

2800

25.3

3.2

1.0

23, 29, 24

 

 

5600

30.0

4.0

1.1

30, 34, 26

 

2-AA

2.5

1398.7

236.8

52.5

1672, 1270, 1254

 

 

 

 

 

 

 

E. coli

Water Test item

-

22.7

9.9

-

18, 34, 16

 

 

33

24.7

4.5

1.1

29, 25, 20

 

 

100

21.0

2.6

0.9

22, 23, 18

 

 

333

21.7

0.6

1.0

21, 22, 22

 

 

1000

22.3

2.5

1.0

20, 25, 22

 

 

2800

21.0

7.8

0.9

16, 30, 17

 

 

5600

18.7

7.6

0.8

10, 24, 22

 

2-AA

60

127.7

5.5

5.6

122, 128, 133

 

Table 3: Results of Preincubation Test (PIT) without metabolic activation.

Strain

Test group

Dose (μg/plate)

Mean revertants per plate

Standard deviation

Factor

 Individual revertant colony counts

 

 

 

 

 

 

 

TA 1535

 Water Test item

-

11.0

1.0

-

12, 11, 10

 

 

33

10.3

3.1

0.9

11, 13, 7

 

 

100

10.0

1.0

0.9

10, 11, 9

 

 

333

10.0

1.7

0.9

9, 9, 12

 

 

1000

8.7

4.6

0.8

14, 6, 6

 

 

2800

9.0

2.0

0.8

9, 11, 7

 

 

5600

8.0

2.6

0.7

5, 9, 10

 

MNNG

5.0

6627.3

155.7

602.5

6637, 6778, 6467

 

 

 

 

 

 

 

TA 100

Water Test item

-

140.7

4.7

-

137, 146, 139

 

 

33

130.3

5.0

0.9

135, 131, 125

 

 

100

132.3

5.9

0.9

130, 139, 128

 

 

333

126.3

6.4

0.9

119, 131, 129

 

 

1000

121.7

8.5

0.9

112, 125, 128

 

 

2800

115.3

11.2

0.8

103, 118, 125

 

 

5600

127.0

24.0

0.9

100, 135, 146

 

MNNG

5.0

4926.0

242.6

35.0

4646, 5058, 5074

 

 

 

 

 

 

 

TA 1537

Water Test item

-

12.0

1.7

-

10, 13, 13

 

 

33

9.3

3.1

0.8

10, 12, 6

 

 

100

9.0

2.6

0.8

10, 6, 11

 

 

333

10.0

4.6

0.8

14, 5, 11

 

 

1000

14.0

3.6

1.2

15, 17, 10

 

 

2800

9.7

4.0

0.8

14, 6, 9

 

 

5600

10.7

4.7

0.9

16, 9, 7

 

AAC

100

777.7

39.7

64.8

749, 761, 823

 

 

 

 

 

 

 

TA 98

Water Test item

-

24.7

7.2

-

21, 20, 33

 

 

33

25.0

2.6

1.0

28, 23, 24

 

 

100

31.0

6.1

1.3

35, 34, 24

 

 

333

26.0

2.0

1.1

26, 28, 24

 

 

1000

28.0

2.0

1.1

30, 28, 26

 

 

2800

27.0

4.4

1.1

24, 25, 32

 

 

5600

25.7

6.7

1.0

29, 18, 30

 

NOPD

10

790.0

174.5

32.0

702, 677, 991

 

 

 

 

 

 

 

E. coli

Water Test item

-

26.3

3.1

-

29, 27, 23

 

 

33

23.3

4.9

0.9

20, 29, 21

 

 

100

24.3

5.8

0.9

21, 31, 21

 

 

333

21.0

1.0

0.8

21, 20, 22

 

 

1000

30.0

2.0

1.1

30, 28, 32

 

 

2800

24.0

4.6

0.9

19, 28, 25

 

 

5600

22.0

2.0

0.8

24, 22, 20

 

4-NQO

5

757.3

69.5

28.8

688, 757, 827

Table 4: Results from Preincubation Test (PIT) with metabolic activation.

Strain

Test group

Dose (μg/plate)

Mean revertants per plate

Standard deviation

Factor

 Individual revertant colony counts

 

 

 

 

 

 

 

TA 1535

Water Test item

-

11.0

1.0

-

10, 11, 12

 

 

33

9.0

1.0

0.8

8, 10, 9

 

 

100

8.7

0.6

0.8

9, 8, 9

 

 

333

9.0

2.6

0.8

11, 6, 10

 

 

1000

12.7

2.5

1.2

10, 13, 15

 

 

2800

10.3

0.6

0.9

10, 11, 10

 

 

5600

11.0

3.6

1.0

7, 12, 14

 

2-AA

2.5

275.7

43.8

25.1

285, 314, 228

 

 

 

 

 

 

 

TA 100

Water Test item

-

150.0

12.3

-

141, 145, 164

 

 

33

128.3

5.1

0.9

127, 134, 124

 

 

100

133.7

18.9

0.9

155, 127, 119

 

 

333

139.3

10.1

0.9

138, 150, 130

 

 

1000

133.0

12.8

0.9

130, 147, 122

 

 

2800

118.0

3.0

0.8

121, 115, 118

 

 

5600

136.0

13.5

0.9

122, 137, 149

 

2-AA

2.5

2766.0

290.7

18.4

2755, 3062, 2481

 

 

 

 

 

 

 

TA 1537

Water Test item

-

9.0

1.0

-

8, 10, 9

 

 

33

8.3

3.2

0.9

7, 6, 12

 

 

100

8.3

2.1

0.9

9, 10, 6

 

 

333

7.3

2.5

0.8

5, 7, 10

 

 

1000

8.0

4.4

0.9

13, 6, 5

 

 

2800

9.7

4.0

1.1

6, 9, 14

 

 

5600

7.7

2.9

0.9

6, 11, 6

 

2-AA

2.5

280.3

32.1

31.1

317, 257, 267

 

 

 

 

 

 

 

TA 98

Water Test item

-

26.0

2.6

-

24, 25, 29

 

 

33

24.7

6.7

0.9

23, 32, 19

 

 

100

23.0

1.7

0.9

21, 24, 24

 

 

333

28.0

4.6

1.1

23, 32, 29

 

 

1000

21.0

2.0

0.8

21, 23, 19

 

 

2800

23.0

1.7

0.9

25, 22, 22

 

 

5600

26.3

2.5

1.0

24, 26, 29

 

2-AA

2.5

2258.3

140.0

86.9

2356, 2321, 2098

 

 

 

 

 

 

 

E. coli

Water Test item

-

24.3

3.2

-

23, 28, 22

 

 

33

26.7

7.6

1.1

32, 18, 30

 

 

100

19.0

2.6

0.8

22, 18, 17

 

 

333

26.3

8.1

1.1

30, 32, 17

 

 

1000

25.3

4.0

1.0

26, 29, 21

 

 

2800

33.7

8.3

1.4

31, 27, 43

 

 

5600

30.7

4.6

1.3

28, 36, 28

 

2-AA

60

116.3

14.6

4.8

100, 128, 121

Table 5: Results from sterility control, 1st experiment.

Components

Results

2.0 mL soft agar

No growth

0.5 mL S9 mix

No growth

0.5 mL buffer

No growth

0.5 mL vehicle

No growth

5600μg test substance

No growth

Table 6: Results from sterility control, 2nd experiment.

Components

Results

2.0 mL soft agar

No growth

0.5 mL S9 mix

No growth

0.5 mL buffer

No growth

0.5 mL vehicle

No growth

5600μg test substance

No growth

Applicant's summary and conclusion