Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - October 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliable without restrictions; Guideline study (OECD 471) according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Acetoguanamine
- Physical state: white powder
- Lot./batch-no.:10/08/89 AG
- Purity: 99.2 %
- Storage condition of test material: In the dark at room temperature

Method

Target gene:
- genes involved in histidine synthesis
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: histidine requiring
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: histidine requiring
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: histidine requiring
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: histidine requiring
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: histidine requiring
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Concentrations of Treatment Solution (mg/mL):
Toxicity range-finder experiment: 0.016, 0.080, 0.400, 2.000, 10.00 mg Acetoguanamine /ml.
Mutation experiment 1: 0.016, 0.080, 0.400, 2.000, 10.00 mg Acetoguanamine /ml.
Mutation experiment 2: 2.0, 4.0, 6.0, 8.0, 10.0 mg Acetoguanamine /ml.

Final Concentration (µg/plate):
Toxicity range-finder experiment: 8, 40, 200, 1000, 5000 µg Acetoguanamine /plate.
Mutation experiment 1: 8, 40, 200, 1000, 5000 µg Acetoguanamine /plate.
Mutation experiment 2: 1000, 2000, 3000, 4000, 5000 µg Acetoguanamine /plate.
Vehicle / solvent:
Test chemical solutions:
- prepared by dissolving Acetoguanamine in distilled water.

Stock solution of positive control chemicals:
- 2-nitrofluorene, 9-aminoacridine and 2-aminoanthracene were dissolved in DMSO
- sodium azide was dissolved in distilled water
Controlsopen allclose all
Positive controls:
yes
Remarks:
500 µg/ml (stock concentration), 50.0 µg/plate (final concentration); for strains TA98 and TA1538; induced in quintuplicate without S-9 mix
Positive control substance:
2-nitrofluorene
Positive controls:
yes
Remarks:
20 µg/ml (stock concentration), 2.0 µg/plate (final concentration); for strains TA100 and TA1535; induced in quintuplicate without S-9 mix
Positive control substance:
sodium azide
Positive controls:
yes
Remarks:
500 µg/ml (stock concentration), 50.0 µg/plate (final concentration); for strain TA1537; induced in quintuplicate without S-9 mix
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: 500µ
Positive controls:
yes
Remarks:
50 µg/ml (stock concentration), 5.0 µg/plate (final concentration); for strains TA98, TA1538, TA100, TA1535, TA1537; induced in quintuplicate with S-9 mix
Positive control substance:
other: 2-aminoanthracene
Untreated negative controls:
yes
Remarks:
sterile distilled water, included in quintuplicate without and with S-9 mix
Evaluation criteria:
EVALUATION CRITERIA:
A test compound was considered to be mutagenic if:
i) the assay was valid (see acceptance criteria)
ii) two or three-fold increase (depent on strain: TA98 and TA100 two-fold increase; TA1535, TA1537 and TA1538 three-fold increase) in revertant numbers, were accompanied by significant F-statistics and dose response correlations
iii) the positive resposes described in (ii) were reproducible

ACCEPTANCE CRITERIA:
The assay was considered valid if the following criteria were met:
i) the mean negative control counts fell within the normal range (compared to historical data)
ii) the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation.
iii) no more than 5% of the plates were lost through contamination or some other unforseen event.
Statistics:
no data

Results and discussion

Test results
Species / strain:
other: TA98, TA100, TA1535, TA1537 and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- carried out in TA100 only
- using final concentrations of Acetoguanamine at 8, 40, 200, 1000 and 5000 µg/plate plus a solvent and positive control
- no toxicity was observed following these treatments
- same dose range was therefore used for treatment of the remaining test strains in experiment 1
- for experiment 2 a concentration of 5000 µg/plate was retained as a maximum test dose, but with a narrowed dose range utilised in order to investigate more closely those concentrations of Acetoguanamine most likely to exhibit a mutagenic effect.

Any other information on results incl. tables

Table 1: Results of the AMES Test with the test substance Acetoguamine.

Sample

Dose µg/plate

 

TA98

TA100

TA1535

TA1537

TA1538

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Mutation Experiment 1

Acetoguanamine

0

 

 

 

 

 

 

mean

sd

29

21

23

26

23

 

24.4

3.1

35

34

31

28

32

 

32.0

2.7

77

89

86

105

82

 

87.8

10.6

71

85

70

87

94

 

81.4

10.5

44

27

36

29

36

 

34.4

6.7

25

33

13

28

26

 

25.0

7.4

5

5

9

8

3

 

6.0

2.4

6

11

9

14

13

 

10.6

3.2

12

8

12

6

11

 

9.8

2.7

36

32

21

28

25

 

28.4

5.9

Acetoguanamine

8

 

 

 

 

mean

sd

18

16

26

 

20.0

5.3

29

35

31

 

31.7

3.1

78

72

80

 

76.7

4.2

99

116

122

 

112.3

11.9

20

28

31

 

26.3

5.7

32

29

C

 

30.5

 

9

8

3

 

6.7

3.2

17

10

17

 

14.7

4.0

9

12

13

 

11.3

2.1

25

23

29

 

25.7

3.1

Acetoguanamine

40

 

 

 

 

mean

sd

19

28

24

 

23.7

4.5

33

24

39

 

32.0

7.5

69

102

88

 

86.3

16.6

84

82

104

 

90.0

12.2

23

27

29

 

26.3

3.1

27

29

C

 

28.0

 

5

2

2

 

3.0

1.7

17

20

16

 

17.7

2.1

16

11

9

 

12.0

3.6

25

41

33

 

33.0

8.0

Acetoguanamine

200

 

 

 

 

mean sd

14

19

26

 

19.7

6.0

47

40

41

 

42.7

3.8

84

82

95

 

87.0

7.0

80

108

107

 

98.3

15.9

43

34

34

 

37.0

5.2

19

21

28

 

22.7

4.7

8

11

9

 

9.3

1.5

20

14

18

 

17.3

3.1

11

19

11

 

13.7

4.6

23

28

18

 

23.0

5.0

Acetoguanamine

1000

 

 

 

 

mean sd

23

18

20

 

20.3

2.5

39

34

33

 

35.3

3.2

80

92

86

 

86.0

6.0

123

112

104

 

113.0

9.5

33

32

36

 

33.7

2.1

23

19

28

 

23.3

4.5

4

3

3

 

3.3

0.6

12

23

12

 

15.7

6.4

12

5

14

 

10.3

4.7

28

19

28

 

25.0

5.2

Acetoguanamine

5000

 

 

 

 

mean sd

20

21

18

 

23.0

7.0

26

25

26

 

25.7

0.6

94

87

67

 

82.7

14.0

84

99

104

 

95.7

10.4

31

31

C

 

31.0

C

18

23

31

 

24.0

6.6

10

2

8

 

6.7

4.2

11

10

12

 

11.0

1.0

9

10

17

 

12.0

4.4

C

20

24

 

22.0

 

Positive Controls (Mutation Experiment 1)

2-nitrofluorene

50.0

 

 

 

 

 

 

mean

sd

1152

1021

969

1293

1144

 

1115.8

126.5

-

-

-

-

-

-

-

219

268

246

177

170

 

216.0

42.6

-

Sodium azide

2.0

 

 

 

 

 

 

mean

sd

-

-

453

427

441

439

444

 

440.8

9.4

-

305

345

332

343

339

 

332.8

16.3

-

-

-

-

-

9-aminoacridine

50.0

 

 

 

 

 

 

mean

sd

-

-

-

-

-

-

1127

1145

887

899

926

 

996.8

128.0

-

-

-

2-aminoanthracene

5.0

 

 

 

 

 

 

mean

sd

-

339

360

365

360

369

 

358.6

11.6

-

1042

886

781

834

819

 

872.4

102.0

-

194

157

156

183

154

 

168.8

18.4

-

65

50

61

59

43

 

55.6

8.9

-

227

210

202

209

192

 

208.0

12.8

 

 

 

 

 

 

 

 

 

 

 

 

 

Mutation Experiment 2

Acetoguanamine

0

 

 

 

 

 

 

mean

sd

25

35

32

29

18

 

27.8

6.6

43

39

48

35

44

 

41.8

5.0

110

107

122

108

86

 

106.6

13.0

130

150

138

107

115

 

128.0

17.3

47

37

44

46

41

 

43.0

4.1

28

31

33

23

21

 

27.2

5.1

11

8

11

14

6

 

10.0

3.1

23

25

29

23

13

 

22.6

5.9

14

13

12

19

17

 

15.0

2.9

20

29

32

33

37

 

30.2

6.4

Acetoguanamine

1000

 

 

 

 

mean

sd

29

29

25

27.7

2.3

44

37

36

 

39.0

4.4

102

116

115

 

111.0

7.8

138

127

130

 

131.7

5.7

28

25

20

 

24.3

4.0

16

28

31

 

25.0

7.9

9

5

10

 

8.0

2.6

9

9

13

 

10.3

2.3

13

13

25

 

17.0

6.9

39

36

26

 

33.7

6.8

Acetoguanamine

2000

 

 

 

 

 

mean

sd

29

13

10

 

20.7

9.7

49

36

27

 

37.3

11.1

92

93

90

 

91.7

1.5

143

140

117

 

133.3

14.2

24

28

25

 

25.7

2.1

28

34

32

 

31.3

3.1

13

5

6

 

8.0

4.4

16

16

16

 

16.0

0.0

12

18

9

 

13.0

4.6

32

29

33

 

31.3

2.1

Acetoguanamine

3000

 

 

 

 

 

mean

sd

27

19

13

 

19.7

7.0

36

37

37

 

36.7

0.6

118

89

120

 

109.0

17.3

128

141

158

 

142.3

15.0

31

25

24

 

26.7

3.8

29

48

27

 

34.7

11.6

13

8

6

 

9.0

3.6

35

13

11

 

19.7

13.3

18

9

10

 

12.3

4.9

26

20

28

 

24.7

4.2

Acetoguanamine

4000

 

 

 

 

 

mean

sd

31

25

13

 

23.0

9.2

36

25

34

 

31.7

5.9

122

130

102

 

118.0

14.4

112

142

127

 

127.0

15.0

31

26

37

 

31.3

5.5

34

36

24

 

31.3

6.4

4

12

9

 

8.3

4.0

23

16

18

 

19.0

3.6

11

17

21

 

16.3

5.0

32

35

35

 

34.0

1.7

Acetoguanamine

5000

 

 

 

 

mean sd

29

12

28

 

23.0

9.5

41

29

46

 

38.7

8.7

103

112

111

 

108.7

4.9

138

107

161

 

135.3

27.1

28

35

19

 

27.3

8.0

32

26

37

 

31.7

5.5

11

4

13

 

9.3

4.7

20

19

19

 

19.3

0.6

14

16

21

 

17.0

3.6

37

18

31

 

28.7

9.7

Positive Controls (Mutation Experiment 2)

2-nitrofluorene

50.0

 

 

 

 

 

 

mean

sd

1321

1285

1224

1297

1108

 

1247.0

85.5

 -

 -

 -

289

309

268

280

276

 

284.4

15.7

Sodium azide

2.0

 

 

 

 

 

 

 

mean

sd

 -

 -

926

776

875

907

864

 

869.6

57.9

 -

831

776

833

758

739

 

787.4

42.8

 -

 -

 -

9-aminoacridine

50.0

 

 

 

 

 

 

mean

sd

 -

 -

 -

1076

1183

1310

1022

1035

 

1125.2

121.1

2-aminoanthracene

5.0

 

 

 

 

 

 

mean

sd

 -

301

348

355

316

318

 

327.6

22.9

 -

781

853

715

683

735

 

753.4

66.1

203

158

158

143

125

 

157.4

28.9

 -

65

78

69

84

55

 

70.2

11.3

 -

275

263

224

444

242

 

289.6

88.5

 S9: liver homogenate from rats treated with Arcolor

mean: average number of revertants per plate

sd: standard deviation

C: Contaminated plate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

From the findings of this study it is concluded that Acetoguanamine was unable to induce mutation in five strains of Salmonella Typhimurium when tested up to 5000 µg/plate in the absence and presence of a rat liver metabolic activation system.
Executive summary:

Acetoguanamine was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA1538) of Samonella typhimurium, both in the absence and presence of metabolic activation by an Arcolor 1254 induced rat liver post-mitochondrial fraction (S-9), in two seperate experiments. The study was carried out according to OECD 471.

An initial toxicity range-finder experiment was carried ot in TA100 only, using final concentrations of Acetoguanamine at 8, 40, 200, 1000 and 5000 µg/plate plus solvent and positive control. No toxicity was observed following these treatments and the same dose range was therefore used for treatment of the remaining test strains in experiment 1 (again no toxicity was observed). For experiment 2, 5000 µg/plate was retained as the maximum test dose, but with a narrowed dose range utilised in order to investigate more closely those concentrations of Acetoguanamine most likely to exhibit a mutagenic effect.

Negative (solvent) and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies on negative control plates all fell within acceptable ranges, and were significantly elevated by positive control treatments.

No Acetoguanamine treatment of any of the test strains, either in the absence or presence of S-9, resulted in increases in revertant numbers sufficient to be considered evidence of mutation iduction.

It is concluded that Acetogunamine was unable to induce mutation in five strains of Salmonella typhimurium when tested up to 5000 µg/plate in the absence and presence of a rat liver metabolic activation system.