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Toxicological information

Genetic toxicity: in vitro

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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:
From 16 October 2003 to 26 March 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD guideline compliant.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
: 2-anthamine was used as the sole positive control for the efficacy of S9- mix, whereas an other positive control (e.g BaP should have been use in addition to 2-anthramine)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Vératrole

Method

Target gene:
Histidine gene
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:
S9 mix induced with Aroclor 1254
Test concentrations with justification for top dose:
Pre-test: 10, 100, 500, 1000, 2500, 5000 µg/plate with and without metabolic activation.
Main test:   312.5, 625, 1250, 2500, 5000 µg/plate with and without metabolic activation.
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO 0.1 ml/2ml overlay agar
- Justification for choice of solvent/vehicle: substance freely soluble in DMSO
-Vehicle controls tested: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-anthramine
Details on test system and experimental conditions:
METHOD OF APPLICATION:
-without activation, and first experiment with activation: in agar (plate incorporation);
- with activation, second experiment: preincubation

DURATION
- Preincubation period: 60 minutes

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY - Method: other: reduction in number of revertant colonies per plate and/or a thinning of the bacterial lawn.
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result.
Statistics:
The number of revertants per plate was scored for each strain and concentration. The mean number of revertants, with standard deviation and ratio of mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle were determined.

The Reviewer considers the analyses used to be appropriate.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity testing with strains TA 98, TA 100 and TA 102 did not demonstrate toxicity up to 5000 µg per plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
The results obtained for solvent and positive controls were comparable to historic values

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was observed at the two highest concentrations with metabolic activation in the preincubation assay
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/1: Number of revertants per plate (mean of 3 plates), First experiment: Direct plate incorporation method

 

 

TA 1535

TA 1537

TA 98

Conc.
[unit]

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

 0*

16

15

No

8

9

No

24

35

No

 312.5

12

11

No

9

6

No

28

21

No

 625

11

15

No

6

11

No

21

32

No

 1250

13

13

No

7

8

No

33

26

No

 2500

7

11

No

6

6

No

35

25

No

 5000

11

9

no

5

5

No

20

16

No

Positive control

420

168

no

371

102

no

190

2054

no

*solvent control with DMSO

S.D. Standard deviation

MA: metabolic activation

Table 7.6.1/2: Number of revertants per plate (mean of 3 plates), First experiment: Direct plate incorporation method

 

TA 100

TA 102

Conc.
[unit]

- MA

S.D.

+ MA

S.D.

Cytotoxic
(yes/no)

- MA

S.D.

+ MA

S.D.

Cytotoxic
(yes/no)

0*

137

75

75

21

 No

511

108

452

102

 No

 312.5

88

5

77

9

 No

346

17

297

115

 No

 625

145

96

119

47

 No

435

79

340

21

 No

 1250

86

8

74

20

 No

321

17

201

33

 No

 2500

77

15

109

38

 No

280

12

293

40

 No

 5000

89

25

65

9

 no

185

22

193

13

 No

Positive control

386

142

637

166

 no

1202

49

4021

203

 no

*solvent control with DMSO

S.D. Standard deviation

MA: metabolic activation

Table 7.6.1/3: Number of revertants per plate (mean of 3 plates), Second experiment:

Direct plate incorporation method (without S9 mix) and preincubation method (with S9 mix)

 

TA 1535

TA 1537

TA 98

Conc.
[unit]

- MA

S.D.

+ MA

S.D.

Cytotoxic
(yes/no)

- MA

S.D.

+ MA

S.D.

Cytotoxic
(yes/no)

- MA

S.D.

+ MA

S.D.

Cytotoxic
(yes/no)

0*

10

3

16

6

 No

9

4

8

3

 No

28

3

33

6

 No

 312.5

12

3

17

6

 No

6

1

7

1

 No

40

20

24

5

 No

 625

13

3

15

4

 No

9

3

11

3

 No

24

4

25

5

 No

 1250

9

5

13

3

 no

8

3

12

4

 No

34

10

30

11

 No

 2500

16

6

12

2

No

9

4

9

3

 Yes

22

6

26

7

 yes

 5000

15

4

8

3

 yes

5

3

1

1

 yes

25

12

10

8

 yes

Positive control

518

46

73

12

 

262

165

75

11

 

172

14

1009

16

 

*solvent control with DMSO

S.D. Standard deviation

MA: metabolic activation

Table 7.6.1/4: Number of revertants per plate (mean of 3 plates), Second experiment:

Direct plate incorporation method (without S9 mix) and preincubation method (with S9 mix)

 

TA 100

TA 102

Conc.
[unit]

- MA

S.D.

+ MA

S.D.

Cytotoxic
(yes/no)

- MA

S.D.

+ MA

S.D.

Cytotoxic
(yes/no)

0*

88

12

101

11

 No

365

38

489

47

 No

 312.5

125

42

120

32

 No

363

27

538

134

 No

 625

94

7

96

18

 No

362

4

504

44

 No

 1250

144

76

122

38

 No

368

49

545

137

 No

 2500

125

9

89

14

yes

319

27

263

53

 yes

 5000

141

50

0

0

yes

230

29

28

49

 yes

Positive control

604

18

492

74

 

1846

98

3484

350

 

*solvent control with DMSO

MA: metabolic activation

Applicant's summary and conclusion

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

Veratrole was considered to be non-mutagenic in bacteria under the conditions of the test.
Executive summary:

The objective of this study was to evaluate the potential of the test item Veratrole to induce reverse mutation in Salmonella typhimurium. The study was performed according to the international guidelines (OECD 471, Commission Directive No. B13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.

A preliminary toxicity test was performed to define the dose-levels of Vératrole to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item Veratrole was dissolved in dimethylsulfoxide (DMSO).

Five known mutagens (Sodium azide; 9 -Aminoacridine; 2 -nitrofluorene; Mitomycine C and 2 -Anthramine) were used to check the sensitivity of the test system. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 μg/plate, for both mutagenicity experiments with and without S9 mix.

No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.

A moderate to marked toxicity was noted towards all the strains used, generally at dose-levels ≥ 2500 μg/plate, with S9 mix using the preincubation method.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Under these experimental conditions, the test item Veratrole did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium with and without metabolic activation.