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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD-guideline and GLP-compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his trp
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
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 4-nitro-o-phenylene-diamine, methyl methane sulfonate, 2-aminoanthracene
Details on test system and experimental conditions:
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the minimal agar plates (Experiment I):
100 microl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 microl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 microl Bacteria suspension (cf test system, pre-culture of the strains),
2000 microl Overlay agar
In the pre-incubation assay (Experiment II) 100 µl test solution, 500 µl S9 mix / S9 mix substitution buffer and 100 µl bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45° C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark
Evaluation criteria:
A test item is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced. A test item producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system. A,mutagenic response is described as follows: A test item is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.
Statistics:
No statistical evaluation of the data is required.

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 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

Table 1: Revertants/plate (mean of three plates) in the Ames test with and without metabolic activation

 

Revertants/plate, without S9 mix (mean of three plates)

Strain

TA 1535

TA1537

TA 98

TA 100

WP2 uvra

Experiment

I

II

I

II

I

II

I

II

I

II

Negative control

24

26

8

14

21

19

86

105

54

42

Solvent control

21

22

10

16

19

17

85

78

48

43

Positive control

611

1498

106

52

410

509

1025

1478

802

433

33

20

25

12

10

16

25

89

86

40

37

100

19

25

9

18

15

19

80

75

38

45

333

13

33

9

15

13

14

77

86

42

42

1000

21

23

10

14

16

17

69

73

42

37

2500

18

20

8

8

18

13

86

64

39

30

5000

19

15

4

8

12

13

79

43

29

34

 

Revertants/plate, with S9 mix (mean of three plates)

Negative control

13

11

15

22

23

32

80

100

52

55

Solvent control

10

15

22

13

28

27

85

88

48

50

Positive control

132

109

140

48

314

312

503

510

275

228

33

10

12

13

25

24

29

93

94

48

51

100

10

13

10

15

18

29

85

92

48

45

333

8

12

11

15

25

29

76

89

49

46

1000

8

11

13

15

32

32

78

74

50

41

2500

9

10

12

10

30

23

81

76

45

38

5000

5

11

9

11

20

20

96

71

40

54

Experiment I: plate incorporation method, Experiment II: preincubation method

Applicant's summary and conclusion

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

The test substance was not genotoxic when tested in the Ames test with different strains of Salmonella Typhimurium and the Escherichia coli strain WP2 uvrA according to the OECD 471 (1997).