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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:
2003-10-13 to 2004-01-08
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:
1997-07-21
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000-05-19
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No. of test material: GR31143161
- Expiration date of the batch: 24.04.2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

OTHER SPECIFICS: colourless-yellowish liquid

Method

Target gene:
his
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and ß-Naphthoflavone induced male rat liver.
Test concentrations with justification for top dose:
Experiment I and II ( except for strains TA 1537 and TA 102 without 89 mix): 33, 1 00, 333, 1000, 2500, and 5000 µg/plate
Experiment II (strains TA 1537 and TA 102 without 89 mix): 0.3, 1, 3, 10, 33, 100, 250, and 500 µg/plate
ln the pre-experiment the concentration range of the test item was 3- 5000 µg/plate. The pre-experiment is reported as part of experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration for experiment I.
Vehicle / solvent:
- Solvent used: ethanol
- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix, TA1535, TA100, 10 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
without S9 mix, TA1537, 50 µg/plate; TA98, 10 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix, TA102, 4 µL/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 mix, TA1535, TA1537, TA98, TA100, 2.5 µg/plate; TA102, 10 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in the number of spontaneaus revertants or a clearing of the bacterial background lawn.

Rationale for test conditions:
To establish a dose response effect six dose Ievels with adequately spaced concentrations were tested. The maximum dose Ievel was 5000 µg/plate.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice ( strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent secend experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent contros such an increase is not considered biologically relevant.
Statistics:
A statistical analysis of the data is not required.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
valid
Positive controls validity:
valid
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:
valid
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:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Cytotoxic effects

Strain

Experiment I

Experiment II

 

- S9 mix

+ S9 mix

- S9 mix

+ S9 mix

TA1535

-

-

5000

-

TA1537

2500

5000

100

2500, 5000

TA98

-

-

-

-

TA100

-

-

-

-

TA102

-

-

33 - 500

-

 Table 2: Summary of results, without S9 mix

Concentration [µg/plate]

Revertants/plate [Mean]

TA1535

TA1537

TA98

TA100

TA102

I

II

I

II

I

II

I

II

I

II

Neg. Control

11

19

6

5

25

28

185

17

200

234

Solvent Control

16

22

6

5

25

29

188

183

177

169

Pos. Control #

602

1075

54

73

165

199

557

502

1339

917

0.3

-

-

-

7

-

-

-

-

-

150

1

-

-

-

4

-

-

-

-

-

166

3

-

-

-

6

-

-

-

-

-

172

10

-

-

-

6

-

-

-

-

-

175

33

13

18

6

6

23

20

222

158

152

52

100

11

18

5

8

20

21

225

174

161

32

250

-

-

-

8

-

-

-

-

-

18

333

9

14

4

-

21

17

148

152

159

-

500

-

-

-

4

-

-

-

-

-

0

1000

7

14

3

-

19

14

206

124

152

-

2500

11

16

3

-

19

20

232

154

155

-

5000

9

9

5

-

20

21

208

178

153

-

- Not performed

# Sodium azide (10.0 µg/plate) strains TA 1535 and TA 100; 4-nitro-o-phenylene-diamine strains TA 1537 (50 µg/plate) and TA 98 (10.0 µg/plate), Methylmethanesulfonate (4 1 µL/plate) strain TA 102

Table 3: Summary of results, with S9 mix 

Concentration [µg/plate]

Revertants/plate [Mean]

TA1535

TA1537

TA98

TA100

TA102

I

II

I

II

I

II

I

II

I

II

Neg. Control

11

11

10

14

21

18

179

188

184

216

Solvent Control

19

27

9

13

28

31

202

190

176

134

Pos. Control ##

312

274

265

224

498

472

801

709

1355

1246

33

20

29

10

8

30

29

257

156

156

122

100

18

19

10

12

33

25

211

196

172

108

333

16

17

9

11

32

25

210

167

153

116

1000

12

14

6

6

29

19

198

114

152

98

2500

13

19

6

4

31

26

179

137

162

102

5000

13

18

4

3

30

27

174

142

109

156

## 2-aminoanthracene (2.5 µg/plate) strains TA 1535, TA 1537, TA 98, and TA 100; 2-aminoanthracene (10.0 µg/plate) strain TA 102

Applicant's summary and conclusion