Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

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
Reference:
Composition 0
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 information:
Composition 1
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
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:
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
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.
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

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

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1999-11-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Not in full compliance with GLP principles, study conducted according to good scientific practice
Reference:
Composition 0
Qualifier:
no guideline followed
Version / remarks:
No guideline was available until the experiment had been carried out. The test was performed based on the description of Gee, P et al.: Comparison of responses of base-specific Salmonella tester strains with the traditional strains for identifying mutagens: The results of a validation study. Mut. Res., 412, 115 -130 (1998) and on the "Users Manual" prepared by XENOMETRIX, Inc., Colorado, USA (without date).
Principles of method if other than guideline:
PRINCIPLE OF TEST
Mutations were detected by the reversion of mutations present in the amino acid requiring bacterial strains, which Ieads to a restoration of the functional capability of bacteria to synthesize the essential amino acid and thus to the ability to grow in the absence of the amino acid required by the parent strains.

TEST CONDITIONS
Liquid fluctuation test both with and without metabolic activation (Aroclor-induced rat liver S9 -Mix).

ANALYZED PARAMETERS
Mutagenicity detected by reversion of mutations present in amino acid requiring bacterial strains (Salmonella typhimurium TA 98, TA-Mix).
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
- Solubility : no precipitation of the test substance was found
- Date of manufacture: 1999-03-02
- Degree of purity: 91,8% (main component)
- Batch Nr.: Abl.-Nr. 99-0031
- Storage conditions: room temperature (N2 conditions)

Remark: The stability of the test substance throughout the study period has not been verified by reanalysis. The stability of the test substance in the vehicle water has not been determined analytically.
Target gene:
TA 98: his D 3052
TA 7001: hisG 1775
TA 7002: hisC 9138
TA 7003: hisC 9074
TA 7004: hisG 9133
TA 7005: hisG 9130
TA 7006: hisG 9070
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Metabolic activation system:
Exogenous, Aroclor-induced rat liver S-9 mix
Species / strain:
other: TA Mix (Mixed strains TA 7001 - TA 7006)
Metabolic activation:
with and without
Metabolic activation system:
Exogenous, Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
0; 4; 20; 100; 500; 2500 and 5000 µg/mI
Vehicle:
Water
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
other: 2-aminoaanthraacene (2-AA)
Details on test system and conditions:
TEST DESIGN
- Strains: TA 98, TA Mix (TA 7001-7006)
- Type of test: liquid fluctuation test with and without S-9 mix
- Number of microtiter plates: triplicate plates per dose, control chemical or vehicle
- Doses: 0; 4; 20; 100; 500; 2,500 and 5,000 µg/mL



Evaluation criteria:
The test chemical was considered positive if a dose-related and reproducible increase in the number of positive wells by a factor of about 2 (calculated primarily on the basis of baseline data) in at least one tester strain either without S-9 mix or after adding a metabolizing system were met. A test substance was generally considered non-mutagenic in this test if the number of revertant wells for all tester strains were within the historical negative control range under all experimental conditions.

Toxicity was detected by a
- decrease in the number of positive wells
- clearing or diminution of the background lawn (= reduced his- background growth) leading from turbid to non-turbid purple wells
is recorded for all test groups both with and without S-9 mix and indicated in the tables
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
Species / strain:
other: TA-Mix (TA 7001-7006)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: no precipitation of the test substance was found

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, but without confidence interval)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

STERILITY CONTROL
160 µI S-9 mix/ml and 5,000 µg test substance/mI negative (no yellow wells)

TOXICITY
- bacteriotoxic effect (clearing of the background Iawn, decrease in the number of yellow wells): no

Abbreviations: RCSP = Revertant Colony Selection Plate (384 -Well), SD = Standard Seviation, REM = Remarks, MEAN = Mean of Replicates, MEANc= Mean Corrected: < 1 = 1, F = Factor, 1F = Based on MEANc, 2F = Baseline Data/Based on MEANc + SD, 3F = Baseline data/ Based on MEAN + SD of a run, 4 -NQO + 2 -NF = 4 -Nitroquinnoline: 0.0625 pgtml + 2 -nitroflourene 0.25 pgImnl, 2 -AA = 2 -Amninoanthracene: 5.0 pglml.

Strain: TA 98, without S9-Mix.

Dose [µg/ml]

REM

RCSP 1

RCSP 2

RCSP 3

MEAN

MEANc

SD

1F

2F

3F

Water

7

2

4

4.3

4.3

2.05

1.0

0.7

0.9

4

1

2

1

1.3

0.47

0.3

0.2

0.3

20

2

3

6

3.7

1.70

0.8

0.6

0.8

100

1

2

2

1.7

0.47

0.4

0.3

0.4

500

2

2

3

2.3

0.47

0.5

0.4

0.5

2500

2

4

2

2.7

0.94

0.6

0.4

0.6

5000

4

3

0

2.3

1.70

0.5

0.4

0.5

4-NQO + 2-NF

25

27

30

27.3

2.05

6.3

4.3

6.0

Strain: TA 98, with S9-Mix.

Dose [µg/ml]

REM

RCSP 1

RCSP 2

RCSP 3

MEAN

MEANc

SD

1F

2F

3F

Water

7

4

2

4.3

4.3

2.05

1.0

0.7

0.8

4

4

4

2

3.3

0.94

0.8

0.5

0.6

20

0

3

2

1.7

1.25

0.4

0.3

0.3

100

9

6

3

6.0

2.45

1.4

0.9

1.1

500

2

2

2

2.0

0.00

0.5

0.3

0.4

2500

2

2

2

2.0

0.00

0.5

0.3

0.4

5000

1

1

3

1.7

0.94

0.4

0.3

0.3

2-AA

48

48

48

48.0

0.00

11.1

7.5

9.2

Strain: TA-Mix, without S9-Mix.

Dose [µg/ml]

REM

RCSP 1

RCSP 2

RCSP 3

MEAN

MEANc

SD

1F

2F

3F

Water

0

0

1

0.3

1.0

0.47

1.0

0.7

0.6

4

2

2

1

1.7

0.47

1.7

1.1

1.0

20

2

1

1

1.3

0.47

1.3

0.9

0.8

100

0

1

0

0.3

0.47

0.3

0.2

0.2

500

0

0

0

0.0

0.00

0.0

0.0

0.0

2500

1

0

1

0.7

0.47

0.7

0.5

0.4

5000

0

0

1

0.3

0.47

0.3

0.2

0.2

4-NQO + 2-NF

22

25

22

23.0

1.41

23.0

15.6

14.4

Strain: TA-Mix, with S-9 -Mix

Dose [µg/ml]

REM

RCSP 1

RCSP 2

RCSP 3

MEAN

MEANc

SD

1F

2F

3F

Water

0

1

0

0.3

1.0

0.47

1.0

0.7

0.7

4

0

0

1

0.3

0.47

0.3

0.2

0.2

20

0

0

1

0.3

0.47

0.3

0.2

0.2

100

0

0

1

0.3

0.47

0.3

0.2

0.2

500

0

1

0

0.3

0.47

0.3

0.2

0.2

2500

1

3

1

1.7

0.94

1.7

1.1

1.1

5000

0

1

0

0.3

0.47

0.3

0.2

0.2

4-NQO + 2-NF

45

47

43

45.0

1.63

45.0

30.6

30.2

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic Toxicity In Vitro

Key information

The test substance was assessed for mutagenicity in the Salmonella typhimurium / Escherichia coli reverse mutation assay in the Standard Plate Test and in the preincubation test with and without the addition of a metabolizing system (S9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA (BASF SE, 2017). The study was conducted according to the guidelines OECD TG 471, EU Method B 13/14 and US EPA OPPTS 870.5100. The dose range chosen was 0; 33; 100; 333; 1000; 2800 and 5600 µg/plate (vehicle = ultrapure water), whereby three test plates per dose or per control were used. No test substance precipitation was found with and without S9 mix. In both tests negative controls (sterility control with S9 mix, vehicle control with and without S9 mix) and positive controls (with and without S9 mix) were performed. After incubation at 37°C for 48-72 hours in the dark, the bacterial colonies of his+ (Salmonella typhimurium) respectively trp+ revertants (Escherichia coli) were counted. In the Preincubation Test, 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were pre-incubated beforehand at 37°C for the duration of about 20 minutes using a shaker.

In result, the test substance did not lead to a relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). The results of the negative as well as the positive controls performed in parallel indicated the validity of this study. Under the experimental conditions chosen, it is concluded that the test substance is not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Supporting Information

The substance 1,1-Dimethylpropin-2-ylamin was tested for its mutagenic potential based on the ability to induce point mutations in selected Ioci of several strains of Salmonella typhimurium (TA 98, TA-Mix) in a modified version of the traditional Ames test, i.e. the Ames II Assay (microtiter Version), both with and without the addition of a metabolizing system (S-9 mix) obtained from rat liver (BASF SE, 1999).

In result, an increase in the number of positive wells (his+ revertants) was not observed either without S-9 mix or after the addition of a metabolizing system. Furthermore, no bacteriotoxic effect was observed. Regarding solubility, no precipitation of the test substance was found. All established validity criteria were met. Therefore it can be concluded that the test substance is not mutagenic in the Ames II Assay (Salmonella typhimurium reverse mutation assay) under the experimental conditions chosen. According to the results of the study, the test substance is not mutagenic in the Ames II Assay (Salmonella typhimurium reverse mutation assay) under the experimental conditions chosen.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data is reliable and suitable for classification purposes. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.