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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:
09 Jan - 09 Mar 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: The guideline of the Act on the Evaluation of Chemical Substances and Regulation of Their Manufacture, etc. (Kanpogyo No. 700, Yakuhatsu No. 1039, 61 Kikyoku No. 1014, 1986)
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon; trp operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
First experiment: 50, 100, 500, 1000, 2000 and 5000 µg/plate with and without metabolic activation
Second experiment: 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Solvent used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
furylfuramide
other: 2-Aminoanthracene: +S9: 2 or 20 µg/plate for TA1535 and WP2uvrA
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Duplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Reduction of bacterial growth
Evaluation criteria:
From these results, the mutagenicity of the test substance was evaluated as negative:
- The number of colonies with reverse mutation of test bacterial strains induced by the test substance was within the range of naturally induced reverse mutation found in the bacterial strains of each test.
- The test substance showed antibacterial activity against the test bacterial strains.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: ≥ 500 µg/plate with and without metabolic activation; Experiment II: ≥ 500 µg/plate without metabolic activation and ≥ 250 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: ≥ 500 µg/plate with and without metabolic activation; Experiment II: ≥ 250 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: ≥ 500 µg/plate with and without metabolic activation; Experiment II: ≥ 250 µg/plate without metabolic activation and ≥ 500 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: ≥ 500 µg/plate with and without metabolic activation; Experiment II: ≥ 250 µg/plate without metabolic activation and ≥ 500 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: ≥ 500 µg/plate with and without metabolic activation; Experiment II: ≥ 500 µg/plate without metabolic activation and at 1000 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Results of Experiment I

Presence of metabolic activation system

Concentration of test substance (μg/plate)

Number of reverse mutation (colonies/plate)

Base substitution

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

- S9

Solvent control

159

174

(167)

9

11

(10)

24

17

(21)

20

26

(23)

10

10

(10)

50

187

149

(168)

11

16

(14)

29

21

(25)

23

16

(20)

9

9

(9)

100

152

146

(149)

17

11

(14)

28

18

(23)

20

12

(16)

7

4

(6)

500

0*

0*

10

15

(13)*

1

0

(1)*

0*

1000

0*

0*

0*

0*

0*

2000

0*

0*

0*

0*

0*

5000

0*C

0*C

0*C

0*C

0*C

+ S9

Solvent control

163

212

(188)

16

15

(16)

26

24

(25)

33

37

(35)

13

5

(9)

50

209

192

(201)

18

13

(16)

37

28

(33)

37

48

(43)

9

8

(9)

100

178

166

(172)

17

26

(22)

21

15

(18)

41

55

(48)

7

6

(7)

500

79

65

(72)*

9

7

(8)*

16

19

(18)*

32

9

(21)*

0*

1000

7

12

(10)*

3

1

(2)*

5

6

(6)*

0*

0*

2000

0*

0*

0*

0*

0*

5000

0*C

0*C

0*C

0*C

0*C

Positive control

- S9

Name

AF-2

SA

ENNG

AF-2

9-AA

Concentration (μg/plate)

0.01

0.5

2

0.1

80

Colonies/plate

606

543

(575)

294

284

(289)

669

653

(661)

344

354

(349)

423

565

(497)

+ S9

Name

BP

2-AA

2-AA

BP

BP

Concentration (μg/plate)

5

2

20

5

5

Colonies/plate

1105

1155

(1130)

169

210

(190)

999

1062

(1031)

413

421

(417)

174

214

(194)

 ( ): The mean colony number is given in parentheses

*: Bacterial growth suppression was observed

C: Precipitation (crystals) was observed

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (furfurylamide)

SA: Sodium azide

ENNG: N-ethyl-N-nitroso-N’-nitroguanidine (ENNG)

9-AA: 9-Aminoacridine

BP: Benzo[a]pyrene

2-AA: 2-Aminoanthracene

Table 2: Results of Experiment II

Presence of metabolic activation system

Concentration of test substance (μg/plate)

Number of reverse mutation (colonies/plate)

Base substitution

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

- S9

Solvent control

143

176

(160)

13

9

(11)

23

13

(18)

33

23

(28)

8

7

(8)

15.6

161

146

(154)

10

14

(12)

22

18

(20)

23

13

(18)

6

3

(5)

31.3

138

167

(153)

15

17

(16)

11

21

(16)

22

12

(17)

5

10

(8)

62.5

175

148

(162)

21

13

(17)

14

12

(13)

24

25

(25)

11

5

(8)

125

148

162

(155)

16

11

(14)

9

18

(14)

16

21

(19)

5

4

(5)

250

38

10

(24)*

11

13

(12)

16

13

(15)

12

9

(11)*

0*

500

0*

2

2

(2)*

4

5

(5)*

0*

0*

1000

 

 

0*

 

 

+ S9

Solvent control

185

139

(162)

19

15

(17)

25

18

(22)

48

46

(47)

7

11

(9)

15.6

190

157

(174)

11

13

(12)

17

16

(22)

45

60

(53)

9

6

(8)

31.3

164

162

(163)

16

16

(16)

16

17

(17)

49

45

(47)

13

20

(17)

62.5

176

171

(174)

17

20

(19)

17

28

(23)

62

54

(58)

9

15

(12)

125

176

195

(186)

20

16

(18)

22

20

(21)

57

54

(56)

5

13

(9)

250

165

131

(148)

0*

20

21

(21)

34

37

(36)

10

14

(12)*

500

69

85

(77)*

0*

10

17

(14)

32

31

(32)*

0*

1000

 

 

7

2

(5)*

 

 

Positive control

- S9

Name

AF-2

SA

ENNG

AF-2

9-AA

Concentration (μg/plate)

0.01

0.5

2

0.1

80

Colonies/plate

516

597

(557)

204

188

(196)

239

296

(268)

320

393

(357)

780

432

(606)

+ S9

Name

BP

2-AA

2-AA

BP

BP

Concentration (μg/plate)

5

2

20

5

5

Colonies/plate

1357

1371

(1364)

318

354

(336)

1286

1229

(1258)

367

447

(407)

215

210

(213)

( ): The mean colony number is given in parentheses

*: Bacterial growth suppression was observed

C: Precipitation (crystals) was observed

AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (furfurylamide)

SA: Sodium azide

ENNG: N-ethyl-N-nitroso-N’-nitroguanidine (ENNG)

9-AA: 9-Aminoacridine

BP: Benzo[a]pyrene

2-AA: 2-Aminoanthracene

Applicant's summary and conclusion