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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:
06 December 2005 - 27 january 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is performed according to internationally accepted guidelines and under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Diformal
Appearance: clear colorless liquidt
chemical name: Bis-(2-chlorethyl)-formal
CAS: 111-91-1
EINECS: 2039202
Purity: 88.7%
Homogenicity: homogenous
Stability: see expiry data
Date of receipt: 29. Nov. 2005
Expiry date: 28. Mai. 2006
Storage: room temperature

Method

Target gene:
histidine operon
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium LT2: strains TA1535, TA97a, TA98, TA100 and TA102
Additional strain / cell type characteristics:
other: TA97a: hisD6610, TA98: hisD3052 TA100 and TA1535 hisG46, TA102: hisG428, TA97a, TA98 und TA100 uvrB; TA97a, TA98, TA100 and TA102 Plasmid pKM 101 and are lipopolysaccharidesidechain deficient (rfa)
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
First experiment: 5019, 1506, 502, 151 or 50 µg/plate
Second experiment: 4987, 2494, 1247 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance is completely soluble in DMSO and it is known that DMSO does not have any effects on the tester strains.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9-mix: NPD 80 µg/Plate: TA97a, 98 und 102. Na-azid 6 µg/Plate: TA100 and 1535. +S9-mix: BaP 40 µg/Plate: TA98. 2-AA 3 µg/Plate, TA97a, 100, 102 and 1535.
Details on test system and experimental conditions:
METHOD OF APPLICATION: First experiment: in agar (plate incorporation). Second experiment: preincubation.

DURATION
- Preincubation period: second experiment: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): histidine
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 4 plates per strains per dose

NUMBER OF CELLS EVALUATED: all colonies were counted manually

DETERMINATION OF CYTOTOXICITY
- Method: the background lawn and number of spontanous reventant colonies was evaluated.


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:
Evaluation criteria:
A positive response is defined as a twofold increase of the number of revertant colonies as compared to the negative control.
Statistics:
None

Results and discussion

Test results
Species / strain:
other: Salmonella typhimurium LT2: strains TA1535, TA97a, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
on the second experiment slight toxicity was observed in the highest concentration tested (5000 µg/plate) in strains TA97a and TA98. Further toxicity was not observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: not observed
- Other confounding effects: no data
RANGE-FINDING/SCREENING STUDIES: no data
COMPARISON WITH HISTORICAL CONTROL DATA: the values for the solvent and positive controls are with the hitorical control values of the laboratory
ADDITIONAL INFORMATION ON CYTOTOXICITY: no data
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

First experiment:

strain

 

TA97a

 

TA98

 

TA100

 

TA102

 

TA1535

 

Metabolic activation

 

-

+

-

+

-

+

-

+

-

+

Water

Average

115

119

 

7

9

148

120

150

168

6

9

 

SD

18.3

23.2

2.9

3.6

21

40

48.5

37.9

1.9

3.0

DMSO

Average

131

136

7

12

143

156

150

186

11

15

 

SD

31.0

11.3

2.2

2.0

26

35

48.0

11.3

3.9

5.1

Positive control

Average

791

673

3.81

831

664

713

446

1101

1001

79

 

SD

244

302

105

341

201

294

19

200

0

18

 

F(I)

6.04

4.95

54.4

69.3

4.49

4.57

2.97

5.92

167

5.27

5019 µg/plate

Average

120

114

13

5

117

141

133

97

22

32

 

SD

33

34

2

3

43

7

44

16

7

6

 

F(I)

0.92

0.84

1.86

0.42

0.82

0.90

0.89

0.52

2.00

2.13

1506 µg/plate

Average

106

95

8

7

89

104

88

113

13

16

 

SD

31

27

8

2

14

9

38

10

4

4

 

F(I)

0.81

0.70

1.14

0.58

0.62

0.67

0.59

0.61

1.18

1.07

502 µg/plate

Average

87

149

10

7

69

134

152

104

9

10

 

SD

7

25

3

4

5

32

8

19

5

3

 

F(I)

0.66

1.10

1.43

0.58

0.48

0.86

1.01

0.56

0.82

0.67

151 µg/plate

Average

129

133

12

5

79

167

123

114

7

9

 

SD

30

42

2

3

44

23

40

33

3

3

 

F(I)

0.98

0.98

1.71

0.42

0.55

1.07

0.82

0.61

0.64

0.60

50 µg/plate

Average

122

92

8

13

168

123

106

83

13

9

 

SD

13

30

3

6

8

37

6

6

3

3

 

F(I)

0.93

0.68

1.14

1.08

1.17

0.79

0.71

0.45

1.18

0.60

SD = standard deviation

F(I) = induction factor

 


 

Second experiment:

strain

 

TA97a

 

TA98

 

TA100

 

TA102

 

TA1535

 

Metabolic activation

 

-

+

-

+

-

+

-

+

-

+

Water

Average

67

138

10

9

141

137

188

180

10

10

 

SD

37.8

36.4

5.0

1.8

10

7

12.7

23.0

4.1

3.1

DMSO

Average

177

116

14

8

134

140

116

144

12

11

 

SD

69.6

96.3

5.7

2.2

14

30

9.7

4.8

2.4

0.0

Positive control

Average

1001

359

995

39

729

504

1019

631

1001

106

 

SD

0

124

13

49

184

111

36

74

0

59

 

F(I)

5.66

3.09

71.1

4.88

5.17

3.60

8.78

4.38

100.

9.64

4987

µg/plate

Average

35

38

6

2

74

123

44

74

13

16

 

SD

40

31

3

1

16

53

11

23

6

7

 

F(I)

0.20

0.33

0.43

0.25

0.55

0.88

0.38

0.51

1.08

1.45

2494 µg/plate

Average

118

96

8

6

131

142

156

167

18

15

 

SD

62

34

2

2

11

14

7

29

2

2

 

F(I)

0.67

0.83

0.57

0.75

0.98

1.01

1.34

1.16

1.50

1.36

1247 µg/plate

Average

227

162

10

6

122

130

159

158

14

18

 

SD

31

39

4

2

15

18

19

26

4

7

 

F(I)

1.28

1.40

0.71

0.75

0.91

0.93

1.37

1.10

1.17

1.64

SD = standard deviation

F(I) = induction factor

 

Comparison with historical control

strain

 

TA97a

 

TA98

 

TA100

 

TA102

 

TA1535

 

Metabolic activation

 

-

+

-

+

-

+

-

+

-

+

Water

Range

72-153

57-162

6-16

6-26

93-179

92-178

91-187

95-239

6-19

5-25

 

Exp 1

115

119

 

7

9

148

120

150

168

6

9

 

Exp 2

67

138

10

9

141

137

188

180

10

10

DMSO

Range

22-176

58-174

4-19

3-24

90-162

73-172

79-199

70-237

5-19

6-17

 

Exp 1

131

136

7

12

143

156

150

186

11

15

 

Exp 2

177

116

14

8

134

140

116

144

12

11

Pos. Control

Range

304-1017

254-1001

223-1001

31-305

389-1170

160-1001

414-1144

118-1001

139-1001

63-359

 

Exp 1

791

673

3.81

831

664

713

446

1101

1001

79

 

Exp 2

1001

359

995

39

729

504

1019

631

1001

106

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

A significant or dose-related increase in the number of revertant colonies was not observed in any of the tested strains with or without metabolic activation. It is concluded that under the conditions of this test the test substance is not mutagenic in the Ames test.
Executive summary:

An ames test was performed accroding to OECD 471 and EC B.13/14 and under GLP. Two independent experiments were performed. The vehicle that was used is DMSO. The strains used were TA97a, TA98, TA100, TA102 and TA1535. The strains were exposed for 48 hours.

In the first experiment 5 concentrations were tested ranging from 50 -5000 µg/plate with and without metabolic activation. No toxicity to the test strains was observed. At the highest concentration a weak increase of revertant colonies was observed for TA1535. Since this was the only effect observed it was not seen as a sign of mutagneicity.

In the second experiment 3 concentartions were tested between 5000 -1250 µg/plate. No signs of mutagenicity were observed and slight toxicity was observed in strain TA97a and TA98 at the highest concentration tested.

In both studies the number of spontaneous revertants in the control group was within the historical control range and controls for confirmation of the genotype of the tester strains and the sterility check were in order. The number of revertant colonies in the positive controls showed a significant mutagenic response with and without S9 -mix.

Under the conditions of this test the test substance is not mutagenic.