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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:
July 26, 2000 - January 10, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
29.Dec.1992
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
September 11, 1989
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
All these strains contain mutations in the histidine operon, thus imposing a requirement for histidine in the growth medium.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Due to migration, the value was transferred to one of the current document's attachments
Test concentrations with justification for top dose:
1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg per plate
2nd series: 5.00, 15.8, 50.0, 158 and 500 µg per plate

The test material concentrations used were selected according to the EEC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (test item and positive controls N-Ethyl-N'-nitro-N-nitrosoguanidine, cumene hydroperoxide, 2-Aminoanthracene, Benzo[a] pyrene) and Ethanol for positive control 9-Aminoanthracene

- Justification for choice of solvent/vehicle: Preferentially distilled water or dimethyl sulfoxide (DMSO), alternatively acetone or ethanol, are used as solvents. Analysis of the historical data of the laboratory and experience of other research groups (Maron et al. 1981) showed that the amounts of the selected solvents used have no influence on the number of spontaneous revertants of any strain. For this reason only the respective solvent control was used as the negative control in this study.

- Justification for percentage of solvent in the final culture medium: Since on the one hand organic solvents may have diverse effects on e.g. gene regulation and, on the other hand, high amounts of water (added as the solvent) will dilute the top agar, usually the maximum amount of solvent is limited to 100 µl per plate for water and 10 µL per plate for DMSO, ethanol, acetone or other organic solvents. Only the highest test material concentration may be plated with either 316 µL water or 31.6 µL organic solvent.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Daunomycin
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
3 parallel plates were used for each concentration step of the test material and the positive controls. Twice as many solvent control plates were used for each bacterial strain.
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Incubation of plates was performed at + 37 °C for 2 to 3 days.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
Evaluation criteria:
A test material is defined as non-mutagenic in this assay if
• "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)
A test material is defined as mutagenic in this assay if
• a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
• "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.
In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test material on the agar plates occurred at concentrations > 500 µg/plate in the 1st test series.

STUDY RESULTS :
- Signs of toxicity :
toxicity to the bacteria was observed at concentrations > 500 or 1580 µg/plate depending upon the experimental conditions used.
- Mean number of revertant colonies per plate and standard deviation : see tables 1-4

Any other information on results incl. tables

Table 1: Results / Series No.: 1

Positive Controls: Individual and Mean Number of Revertant Colonies         

Strain

Positive Control

Concentr. [µg/plate]

+ /-
S9-Mix

#

Mean revertant colonies per plate

SD*

Individual revertant colonies per plate

TA 98

DAUN

2

-

643

196

869

538

521

TA 100

ENNG

5

-

904

41

866

947

899

TA 102

CUM

67

-

396

37

421

414

354

TA 1535

ENNG

10

-

332

12

329

321

345

TA 1537

9-AA

50

-

1683

331

1727

1990

1333

WP2

ENNG

5

-

1327

83

1264

1296

1421

 

 

 

 

 

 

 

 

 

TA 98

2-AA

1

+

322

19

338

301

326

TA 100

2-AA

1

+

537

25

564

532

514

TA 102

B (a) p

10

+

806

3

804

805

809

TA 1535

2-AA

1

+

151

17

152

134

167

TA 1537

2-AA

2

+

92

34

95

124

56

WP2

2-AA

10

+

1459

IB

1477

1441

1459

 

#        Test material incubated in the presence

(+)       or absence

(-)        of S9-Mix

*          Standard deviation

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

9-AA    9-Aminoacridine

2-AA    2-Aminoanthracene

B (a) p Benzo(a)pyrene

 

Table 2: Results/ Series No.: 2

Positive Controls: Individual and Mean Number of Revertant Colonies             

Strain

Positive Control

Concentr. [µg/plate]

+ /-
S9-Mix

#

Mean revertant colonies per plate

SD*

Individual revertant colonies per plate

TA 98

DAUN

2

-

93

18

73

97

108

TA 100

ENNG

5

-

520

39

565

500

494

TA 102

CUM

150

-

661

72

703

702

578

TA 1535

ENNG

10

-

187

11

194

192

174

TA 1537

9-AA

50

-

186

40

144

191

224

WP2

ENNG

5

-

1103

12

1117

1096

1097

 

 

 

 

 

 

 

 

 

TA 98

2-AA

10

+

329

150

434

396

158

TA 100

2-AA

10

+

333

30

302

336

361

TA 102

B (a) p

10

+

775

120

790

887

649

TA 1535

2-AA

2

+

94

11

107

87

649

TA 1537

2-AA

20

+

42

10

17

49

30

WP2

2-AA

10

+

933

81

861

1020

918

 

#        Test material incubated in the presence

(+)       or absence

(-)        of S9-Mix

*          Standard deviation

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

9-AA    9-Aminoacridine

2-AA    2-Aminoanthracene

B (a) p Benzo(a)pyrene

 

Table 3: Results / Series No.: 1

Summary of the Mean Number of Revertant Colonies              

Test material

Concentr. [µg/plate]

+ /-
S9-Mix

 

Mean revertant colonies / plate

TA 98

TA 100

TA 102

TA 1535

TA 1537

WP2

Solvent control

Test item

 

-

14

97

269

12

4

128

5

-

14

91

264

12

3

119

15.8

-

17

96

268

12

6

127

50

-

15

99

249

15

4

122

158

-

16

112

242

12

7

114

500 PE

-

8

73

189

11

2

53

1580 PE

-

0

3

2

2

0

2

5000 PE

-

0

3

0

2

0

2

 

 

 

 

 

 

 

 

 

Solvent control

Test item

 

+

20

120

271

13

7

170

5

+

25

121

277

20

9

164

15.8

+

27

132

288

14

8

180

50

+

26

142

278

13

9

167

158

+

27

143

342

17

8

130

500 PE

+

22

130

214

12

5

81

1580 PE

+

2

4

2

3

0

3

5000 PE

+

0

5

0

3

0

5

 

 

 

 

 

 

 

 

 

Postive controls

Name

-

DAUN

ENNG

CUM

ENNG

9-AA

ENNG

Conc (µg/plate)

2

5

67

10

50

5

Revert. / plate

343

904

396

332

1683

1327

 

 

 

 

 

 

 

 

Name

+

2-AA

2-AA

B (a) p

2-AA

2-AA

2-AA

Conc (µg/plate)

1

1

10

1

2

10

Revert. / plate

322

537

806

151

92

1459

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

2-AA    2-Aminoanthracene

B (a) p Benzo(a)pyrene

9-AA    9-Aminoacridine

Table 4 Results / Series No.: 2

Summary of the Mean Number of Revertant Colonies              

Test material

Concentr. [µg/plate]

+ /-
S9-Mix

 

Mean revertant colonies / plate

TA 98

TA 100

TA 102

TA 1535

TA 1537

WP2

Solvent control

Test item

 

-

15

93

218

11

5

130

5

-

13

87

219

7

7

130

15.8

-

12

92

220

11

8

127

50

-

13

88

222

12

4

122

158

-

16

88

221

15

7

119

500

-

11

79

206

11

6

91

 

 

 

 

 

 

 

 

 

Solvent control

Test item

 

+

25

140

275

18

6

134

5

+

22

139

254

17

5

133

15.8

+

20

153

226

20

7

151

50

+

20

156

259

16

3

144

158

+

26

176

266

18

6

128

500

+

30

153

261

12

6

115

 

 

 

 

 

 

 

 

 

Postive controls

Name

-

DAUN

ENNG

CUM

ENNG

9-AA

ENNG

Conc (µg/plate)

2

5

150

10

50

5

Revert. / plate

93

520

661

187

186

1327

 

 

 

 

 

 

 

 

Name

+

B (a) p

B (a) p

B (a) p

2-AA

B (a) p

2-AA

Conc (µg/plate)

10

10

10

2

20

10

Revert. / plate

329

333

775

94

42

1459

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

B (a) p Benzo(a)pyrene

9-AA    9-Aminoacridine

2-AA    2-Aminoanthracene

 

Applicant's summary and conclusion