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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:
03 May 1996 - 06 May 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
(Lack on test material details and no TA102 or E. coli strain was included in the test.)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
lack on test material details, no TA102 or E. coli strain was included.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA1538, TA 98 and TA 100
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:
10, 100, 1000 and 10000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
The material under examination was used following dilution in distilled water in an amount of 100 mg/mL.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (5 µg/plate, -S9, TA1535 and TA100); 9-aminoacridine (40 µg/plate, -S9, TA1537); 2-nitrofluorene (10 µg/plate, -S9, TA 1538 and TA98); 2-acetylaminoflurorene (1 µg/plate, +S9, all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 24 h
- Exposure duration: 48-72 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48-72 h

NUMBER OF REPLICATIONS: Three repetitions were carried out for the assay sample and two for the positive controls.

DETERMINATION OF CYTOTOXICITY
- Method: The toxicity of the assay sample for the mutant strains of Salmonella typhimurium was evaluated by planting the bacteria in the minimum medium and determining any reduction in the background lawn and in the number of spontaneous revertants caused by the assay sample at the maximum concentration tested.

Evaluation criteria:
CRITERIA FOR THE VALIDITY OF THE ASSAY
The S9 Mix must not be contaminated by more than two colonies per 0.5 mL in the sterility assay carried out following conclusion of the experiment on plates containing the full medium.
In the sterility assay carried out following conclusion of the experiment with the greatest concentration used, the assay sample must not be contaminated by more than two colonies per plate.
On average, the positive control must induce the development of revertant colonies in an amount at least three times greater than the negative control.
The number of spontaneous revertant colonies in the negative controls must lie within the following thresholds:

Strain: TA-1535
Threshold: 20 ± 15

Strain: TA-1538
Threshold: 20 ± 15

Strain: TA-1537
Threshold: 15 ± 10

Strain: TA-98
Threshold: 40 ± 25

Strain: TA-100
Threshold: 150 ± 90

Statistics:
Mean and standard deviation were calculated.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
at 10000 µg/plate with and without metabolic activation
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
10000 µg/plate (with and without 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. Plate test without metabolic activation.

Strain

Plate

No. of revertant colonies/plate

Positive controls µg/plate

ASSAY SAMPLE µg/plate

TA 1535

 

C

10

102

103

104

Na azide

5

1

24

24

22

22

0

NC

2

20

22

12

12

0

NC

3

21

25

19

19

0

/

average

21.7

23.7

17.7

17.7

/

/

s.d.

2.08

1.53

5.13

5.13

/

/

TA 1537

 

 

 

 

 

 

9-AA

40

1

5

13

10

9

0

222

2

10

16

11

4

0

192

3

12

13

15

10

0

/

average

9

14

12

7.7

/

207

s.d.

3.60

1.73

2.64

3.21

/

21.2

TA 1538

 

 

 

 

 

 

2-NF

10

1

22

24

23

26

0

73

2

21

40

34

24

0

97

3

19

22

31

20

0

/

average

20.7

21.7

29.3

23.3

/

85

s.d.

1.53

9.86

5.67

3.05

/

16.97

TA 98

 

 

 

 

 

 

2-NF

10

1

16

16

24

20

0

106

2

23

22

23

10

0

107

3

15

17

19

17

0

/

average

18

18.3

22

15.7

/

106.5

s.d.

4.36

3.21

2.64

5.13

/

0.71

TA 100

 

 

 

 

 

 

Na azide

5

1

129

112

141

118

0

NC

2

137

136

123

125

0

NC

3

130

130

129

122

0

/

average

132

126

131

122

/

/

s.d.

4.36

12.48

9.16

3.51

/

/

Table 2. Plate test with metabolic activation. 

Strain

Plate

No. of revertant colonies/plate

Positive controls µg/plate

ASSAY SAMPLE µg/plate

TA 1535

 

C-

10

102

103

104

2-AA

1

1

10

10

28

15

3

160

2

16

19

25

14

6

180

3

18

20

19

9

7

 

average

14.7

16.3

24

12.7

5.3

 

s.d.

4.17

5.51

4.58

3.21

2.08

 

TA 1537

 

 

 

 

 

 

2-AA

1

1

16

13

8

17

0

90

2

18

18

15

10

0

110

3

23

17

10

17

0

/

average

19

16

11

14.7

/

100

s.d.

3.60

2.64

3.60

4.04

/

14.14

TA 1538

 

 

 

 

 

 

2-AA

1

1

17

29

8

9

0

NC

2

13

15

14

12

0

NC

3

14

17

17

20

0

/

average

14.7

18.3

13

13.7

/

/

s.d.

2.08

4.16

4.58

5.67

/

/

TA 98

 

 

 

 

 

 

2-AA

1

1

15

12

5

10

2

154

2

20

10

10

12

3

150

3

19

15

16

15

4

/

average

18

12.3

10.3

12.3

/

152

s.d.

2.64

2.52

5.51

2.52

30

2.83

TA 100

 

 

 

 

 

 

2-AA

1

1

240

125

175

137

1

NC

2

200

156

174

360

0

NC

3

176

168

174

180

0

/

average

205.3

149.7

174.3

225.7

0.33

/

s.d.

32.33

22.19

0.58

118.3

0.58

/

N.C.: no explanation given in the study report. However, all positive controls resulted in a significant increase in the number of revertants.

Applicant's summary and conclusion