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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:
17th - 24th July 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
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): L-2-Chloropropionic acid
- Physical state: Liquid
- Analytical purity: 90.4% w/w
- Purity test date: 30th April 1992
- Lot/batch No.: Bx 0071, ADH 05138
- Expiration date of the lot/batch: Not specified

Method

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
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 Fraction
Test concentrations with justification for top dose:
100, 200, 500, 1000, 2500 and 5000 micrograms/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile deionised water for the test compound, negative control and some positive controls.
DMSO was used for the remaining positive controls.
Controlsopen allclose all
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
WP2P uvrA
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
WP2P strain
Positive controls:
yes
Positive control substance:
other: Acridine Mutagen ICR191
Remarks:
TA1537 strain
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA1535 and TA100
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
All strains
Positive controls:
yes
Positive control substance:
other: Daunomyc in HCl
Remarks:
TA98 Strain
Details on test system and experimental conditions:

METHOD OF APPLICATION: The initial assay was performed using the standard plate incorporation protocol.
The re-test was performed as above using the standard plate incorporation protocol for the -S9 assays but the pre-incubation protocol for the +S9 determinations.

DURATION
- Pre-incubation period: 60 minutes
- Exposure duration: 3 days

NUMBER OF REPLICATIONS: Test compound – 3 plates per dose
Negative controls – 5 plates
Positive controls – 3 dose levels per strain, 2 plates per dose.


DETERMINATION OF CYTOTOXICITY
- Method: Number of revertant colonies
Evaluation criteria:
A positive response in a (valid) individual experiment is achieved, when one or both of the following criteria are met:
a) a statistically significant dose-related increase in the mean number of revertant colonies is obtained;
b) a two-fold or greater increase in the mean number of revertant colonies which is statistically significant, is observed at at least one dose level.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
Species / strain:
E. coli WP2
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
Species / strain:
E. coli WP2 uvr A
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Test data for Experimental Phase 1.

 

Strain

Activation

Dose Levels (micrograms/plate)

No revertants/plate

Plate 1

Plate 2

Plate 3

TA 1535

 

+S9

5000

26

13

13

2500

8

14

25

1000 *

18

22

13

500

12

C

19

200

14

12

16

100

6

10

11

-S9

5000

1

0

0

2500

6

1

5

1000

11

6

8

500

11

6

9

200 *

11

14

16

100

16

8

11

TA 1537

 

+S9

5000

4

3

2

2500

6

2

2

1000

5

2

3

500

3

2

5

200

2

8

3

100

3

4

10

-S9

5000

0

0

0

2500

0

1

1

1000

2

1

3

500

1

1

2

200

1

1

3

100

4

1

1

TA 98

 

+S9

5000 *

30

45

36

2500 *

41

33

38

1000 *

37

36

40

500

37

26

26

200

17

35

30

100

34

37

25

-S9

5000

4

2

0

2500

11

8

6

1000

11

19

13

500

14

16

18

200

16

13

13

100

16

14

12

TA 100

 

+S9

5000 *

113

88

94

2500

78

98

119

1000 **

119

97

103

500

104

92

81

200 *

100

91

94

100

86

81

86

-S9

5000

1

0

0

2500

6

5

5

1000

64

72

65

500

84

80

81

200

86

65

84

100

75

78

85

WP2P

+S9

5000

44

41

42

2500

36

41

35

1000

41

35

41

500

37

38

35

200

38

40

36

100

32

33

34

-S9

5000

19

16

10

2500

27

28

25

1000

30

33

27

500

27

27

22

200

29

24

24

100

28

17

21

WP2P uvrA

+S9

5000 *

159

145

143

2500**

166

162

179

1000 *

140

164

154

500 *

160

C

139

200 *

152

162

135

100

130

155

128

-S9

5000

62

57

51

2500

77

50

75

1000

81

57

73

500

101

85

76

200

81

70

104

100

115

77

80

 

C            denotes contaminated plate

*             Statistically significant

**          Indicative of a possible effect  

 

 

 

Test Data for Experimental Phase 2 (+S9). 

 

Strain

Activation

Dose Levels (micrograms/plate)

No revertants/plate

Plate 1

Plate 2

Plate 3

TA 1535

 

+S9

5000

14

11

17

2500*

18

17

12

1000

13

6

11

500

14

9

8

200

6

8

11

100

8

8

11

TA 1537

 

+S9

5000

1

1

2

2500

4

2

1

1000

3

1

4

500

4

3

2

200

1

4

4

100

2

2

3

TA 98

 

+S9

5000

19

19

5

2500

12

34

29

1000

21

28

29

500

34

24

28

200

34

21

29

100

18

16

19

TA 100

+S9

5000

87

72

81

2500

62

68

76

1000

96

57

79

500

68

80

73

200

73

61

72

100

79

69

82

WP2P

+S9

5000

36

22

22

2500

24

33

27

1000

44

40

30

500

28

34

28

200

36

42

28

100

22

33

34

WP2P uvrA

+S9

5000

119

156

136

2500

132

111

129

1000

136

139

132

500

141

132

130

200

132

106

114

100

146

137

125

*             Statistically significant

 

 

Test Data for Experimental Phase 2 (-S9). 

 

Strain

Activation

Dose Levels (micrograms/plate)

No revertants/plate

Plate 1

Plate 2

Plate 3

TA 1535

 

-S9

5000

0

3

0

2500*

12

9

6

1000

8

6

9

500

6

10

6

200

6

10

8

100

11

5

6

TA 1537

 

-S9

5000

0

0

0

2500

0

0

1

1000

1

1

2

500

2

1

2

200

1

2

2

100

2

2

3

TA 98

 

-S9

5000

0

0

2

2500

11

8

3

1000**

22

13

25

500

12

20

9

200

13

6

9

100**

21

16

17

TA 100

-S9

5000

0

0

0

2500

4

16

18

1000

74

71

76

500

65

72

87

200

72

76

84

100

76

67

79

WP2P

-S9

5000

16

21

16

2500

17

24

20

1000

28

22

19

500

36

44

37

200

36

37

36

100

29

27

34

WP2P uvrA

-S9

5000

64

73

54

2500

70

75

67

1000

120

123

79

500

97

115

106

200

109

128

113

100

99

103

103

**           Statistically significant

**          Indicative of a possible effect  

Applicant's summary and conclusion

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

Under the conditions of this assay, L-2-Chloropropionic acid did not induce any significant, reproducible increases in the number of revertant colonies. Therefore it is concluded that the substance gave a negative i.e. non-mutagenic response in the presence and absence of an auxiliary metabolising system (S9).
Executive summary:

A study was conducted to evaluate the effect of L-2-Chloropropionic acid in a guideline bacterial mutagenicity assay. An initial assay was performed using the standard plate incorporation protocol over a dose range of 5000 – 100 micrograms/plate, both with and without the presence of S9 metabolic activation. Four Salmonella strains (TA1535, TA1537, TA98 and TA100) and two E.coli strains (WP2P and WP2P uvrA) were tested.

The substance was re-tested with all six strains over the same dose range. The +S9 phase of the second assay was conducted using the pre-incubation protocol, the incubation period being 3 days at 37oC.

For each experiment positive controls were tested to validate the bacterial strains and confirm the activity of the S9 mix.

Under the conditions of the assay, L-2-Chloropropionic acid did not induce any significant, reproducible increases in the number of revertant colonies. It was concluded that the substance gave a negative i.e. non-mutagenic response with S. typhimurium strains TA1535, TA2537, TA98 and TA100 and E.coli strains WP2P and WP2P uvrA, in the presence and absence of an auxiliary metabolising system (S9).