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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:
28 June - 12 July 2007
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:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only duplicate plating in the absence of scientific justification
Qualifier:
according to
Guideline:
other: Guidelines for genotoxicity studies of drugs by Ministry of Health, Labour and Welfare, Japan (Notification No. 1604, November 1, 1999)
Deviations:
yes
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): VCE
- Substance type: powder
Storage condition of test material: at room temperature in a tightly sealed container protected from light

Method

Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with i.p. injections of phenobarbital (30 and 60 mg/kg bw) and 5,6-benzoflavone (80 mg/kg bw)
Test concentrations with justification for top dose:
Pre-experiment: 312.5, 625, 1250, 2500 and 5000 µg/ plate with and without metabolic activation

Main experiment: 312.5, 625, 1250, 2500 and 5000 µg/ plate with and without metabolic activation
Vehicle / solvent:
- Solvent used: water
- Justification for choice of solvent: The choice of the solvent was based on a preliminary solubility test.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (NaN3), 2-nitrofluorene (2-NF), 2-aminoanthracene (2-AA), 9-aminoacridine (9-AA), 4-nitroquinoline 1-oxide (4-NQO)
Remarks:
+S9: 2-AA (1.0 µg/plate, TA98 and TA100; 2 µg/plate, TA1535, TA1537 and WP2uvrA(pKM101)); -S9: 2-NF (5.0 µg/plate, TA98); 9-AA (80 µg/plate, TA1537); NaN3 (1.5 µg/plate, TA100 and TA1535); 4-NQO (5.0 µg/plate, WP2uvrA(pKM101))
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min

-Number of replicates: duplicate plates for the test substance and control groups in two independent experiments

DETERMINATION OF CYTOTOXICITY
- The background lawn was examined with a microscope.
Evaluation criteria:
Validity of the test result:
Evaluation of the validity of the study was performed based on the following criteria;
- The mean number of revertant colonies for negative and positive controls shall be within the range of historical control data.
- Each test strain should exhibit a remarkable increase in average mutagen induced revertant colonies above the average number of colonies for the negative control, when treated with positive control.
- The results of preliminary dose range-finding test and bacterial reverse mutation test should be reproducible. Otherwise, reproducibility should be clarified by further testing.

Criteria for assessing mutagenic potential:
The test substance was considered positive if the following conditions are met:
- The number of revertant colonies in each dose level treated with the test substance should be increased at least twice as compared with that of the negative control group.
- The number of revertant colonies should be dose dependently increased.
- The results should be reproducible.

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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No deposition was observed on the plates in the absence and presence of metabolic activation in any of the experiments.

Any other information on results incl. tables

Table 1. Test results of the pre-experiment (preincubation).

With or without S9-Mix

Test substance concentration

[μg/plate]

Mean number of revertant colonies per plate

(average of 2 plates ± SD)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2 uvrA (pKM101)

TA98

TA1537

-

0 (water)

116 ± 28

15 ± 1

146 ± 1

20 ± 4

9 ± 4

-

312.5

99 ± 6

18 ± 4

154 ± 3

17 ± 4

11 ± 1

-

625

115 ± 9

13 ± 6

126 ± 1

25 ± 4

10 ± 0

-

1250

109 ± 6

15 ± 4

144 ± 3

17 ± 0

10 ± 1

-

2500

108 ± 2

11 ± 1

144 ± 13

18 ± 10

12 ± 2

-

5000

110 ± 13

15 ± 0

138 ± 8

19 ± 2

8 ± 2

Positive controls, –S9

Name

NaN3

NaN3

4-NQO

2-NF

9-AA

Concentrations

[μg/plate]

1.5

1.5

5.0

5.0

80

Mean No. of colonies/plate

(average of 2 plates ± SD)

435 ± 4

361 ± 23

490 ± 92

426 ± 41

555 ± 50

+

0 (water)

108 ± 4

12 ± 1

114 ± 16

28 ± 3

13 ± 6

+

312.5

130 ± 31

14 ± 7

147 ± 16

22 ± 4

9 ± 3

+

625

119 ± 4

15 ± 6

147 ± 28

33 ± 2

12 ± 1

+

1250

112 ± 11

17 ± 6

155 ± 7

25 ± 3

10 ± 4

+

2500

116 ± 15

17 ± 3

156 ± 19

19 ± 0

15 ± 0

+

5000

132 ± 6

15 ± 7

156 ± 8

31 ± 5

13 ± 1

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

[μg/plate]

1.0

2.0

2.0

1.0

2.0

Mean No. of colonies/plate

(average of 2 plates ± SD)

331 ± 8

192 ± 24

383 ± 38

330 ± 9

131 ± 16

NaN3: sodium azide

2-NF: 2-nitrofluorene

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

4-NQO: 4-nitroquinoline 1-oxide

Table 2. Test results of the main experiment (preincubation).

With or without S9-Mix

Test substance concentration

[μg/plate]

Mean number of revertant colonies per plate

(average of 2 plates ± SD)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2 uvrA (pKM101)

TA98

TA1537

-

0 (water)

110 ± 8

11 ± 1

112 ± 23

33 ± 1

8 ± 4

-

312.5

127 ± 12

12 ± 1

171 ± 30

24 ± 4

7 ± 1

-

625

107 ± 7

17 ± 6

179 ± 4

33 ± 6

10 ± 2

-

1250

113 ± 20

11 ± 1

160 ± 28

34 ± 8

9 ± 2

-

2500

107 ± 4

10 ± 0

150 ± 9

28 ± 2

6 ± 1

-

5000

112 ± 4

10 ± 1

132 ± 15

29 ± 1

10 ± 6

Positive controls, –S9

Name

NaN3

NaN3

4-NQO

2-NF

9-AA

Concentrations

[μg/plate]

1.5

1.5

5.0

5.0

80

Mean No. of colonies/plate

(average of 2 plates ± SD)

524 ± 16

366 ± 8

466 ± 112

616 ± 49

623 ± 155

+

0 (water)

115 ± 15

11 ± 3

136 ± 7

20 ± 2

8 ± 2

+

312.5

113 ± 11

11 ± 4

151 ± 9

31 ± 4

13 ± 2

+

625

99 ± 4

15 ± 4

156 ± 4

19 ± 1

11 ± 1

+

1250

115 ± 11

16 ± 4

164 ± 22

25 ± 7

12 ± 6

+

2500

105 ± 13

11 ± 2

163 ± 23

17 ± 1

14 ± 4

+

5000

117 ± 1

11 ± 2

155 ± 6

21 ± 1

12 ± 0

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

[μg/plate]

1.0

2.0

2.0

1.0

2.0

Mean No. of colonies/plate

(average of 2 plates ± SD)

341 ± 35

125 ± 39

553 ± 100

304 ± 21

127 ± 35

NaN3: sodium azide

2-NF: 2-nitrofluorene

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

4-NQO: 4-nitroquinoline 1-oxide

Applicant's summary and conclusion