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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:
28 Jun - 09 Aug 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Umwelt und Verkehr Baden-Württemberg, Stuttgart, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Experiment I:
15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Experiment II:
15, 50, 150, 500 and 1500 µg/plate without metabolic activation
15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn

Statistics:
The X²-test (Mohn and Ellenberger, 1977) was used.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate with and without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate with and without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate with and without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate with and without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate with and without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDY: Based on an initial toxicity test, the test substance was tested in concentrations of 15 to 5000 µg/plate.

Any other information on results incl. tables

Table 1: Test Results of Experiment 1

EXPERIMENT 1

S9-Mix

Without

 

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

NC

35 ± 8

99 ± 10

260 ± 17

33 ± 6

9 ± 2

Solvent control (DMSO)

36 ± 9

113 ± 6

259 ± 15

35 ± 4

12 ± 4

15

-

112 ± 25

-

-

-

50

33 ± 3

100 ± 13

239 ± 13

41 ± 4

9 ± 3

150

41 ± 6

111 ± 10

243 ± 12

45 ± 3

11 ± 6

500

31 ± 4

114 ± 10

244 ± 9

41 ± 5

9 ± 2

1500

30 ± 6

140 ± 12

263 ± 11

40 ± 6

13 ± 2

5000

14 ± 5 (T)

91 ± 19 (T)

109 ± 12 (T)

29 ± 5 (T)

2 ± 1 (T)

NaN3 (0.7)

-

465 ± 41

-

657 ± 71

-

2-NF (2.5)

269 ± 29

-

-

-

-

9-AA (50)

-

-

-

-

364 ± 29

Mitomycin C (0.15)

-

-

581 ± 12

-

-

S9-Mix

With

 

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

 

 

 

 

 

 

NC

40 ± 4

114 ± 4

280 ± 11

20 ± 3

14 ± 3

Solvent control (DMSO)

42 ± 7

114 ± 5

254 ± 26

23 ± 3

13 ± 3

15

-

120 ± 10

-

-

-

50

38 ± 5

103 ± 11

293 ± 16

16 ± 3

19 ± 3

150

36 ± 6

115 ± 8

298 ± 6

22 ± 5

16 ± 3

500

40 ± 4

107 ± 5

305 ± 13

19 ± 3

13 ± 3

1500

47 ± 10

117 ± 10

285 ± 5

20 ± 3

10 ± 2

5000

41 ± 7

70 ± 8 (T)

245 ± 27 (T)

18 ± 2 (T)

4 ± 3 (T)

2-AA (0.7)

1189 ± 70

1092 ± 92

-

-

-

2-AA (1.5)

-

-

725 ± 22

625 ± 38

508 ± 20

NC = Negative Control

T = bacteriotoxic

2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine; 2-AA: 2-aminoanthracene

Table 2: Test Results of Experiment 2

EXPERIMENT 2

S9-Mix

Without

 

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

NC

47 ± 6

118 ± 10

269 ± 11

30 ± 8

16 ± 4

Solvent control (DMSO)

44 ± 12

108 ± 9

262 ± 15

35 ± 4

15 ± 5

15

47 ± 3

133 ± 10

283 ± 12

35 ± 3

14 ± 1

50

33 ± 9

121 ± 13

274 ± 19

29 ± 7

15 ± 5

150

52 ± 5

140 ± 19

265 ± 24

34 ± 8

18 ± 3

500

43 ± 5

132 ± 14

275 ± 8

39 ± 3

12 ± 4

1500

42 ± 11

149 ± 5

262 ± 13

43 ± 3

16 ± 2

NaN3 (0.7)

-

515 ± 9

-

624 ± 39

-

2-NF (2.5)

222 ± 21

-

-

-

-

9-AA (50)

-

-

-

-

212 ± 19

Mitomycin C (0.15)

-

-

820 ± 53

-

-

S9-Mix

With

 

Test item (µg/plate)

TA 98

TA 100

TA 102

TA 1535

TA 1537

 

 

 

 

 

 

NC

48 ± 4

107 ± 7

307 ± 29

22 ± 4

15 ± 4

Solvent control (DMSO)

38 ± 7

91 ± 13

298 ± 9

22 ± 5

19 ± 5

15

-

124 ± 9

322 ± 21

18 ± 4

16 ± 5

50

37 ± 10

124 ± 9

330 ± 7

18 ± 3

17 ± 3

150

40 ± 6

120 ± 9

328 ± 13

21 ± 8

11 ± 3

500

39 ± 3

110 ± 11

296 ± 20

16 ± 3

17 ± 4

1500

45 ± 2

116 ± 8

347 ± 6

17 ± 2

17 ± 5

5000

27 ± 4 (T)

-

-

-

-

2-AA (0.7)

613 ± 33

903 ± 136

-

-

-

2-AA (1.5)

-

-

1106 ± 92

619 ± 51

259 ± 25

NC = Negative Control

T = bacteriotoxic

2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine; 2-AA: 2-aminoanthracene

The test substance induced a slight increase in the mutation frequency in the tester strain TA 1535 and TA 100 in the absence of a metabolic activation system in both experiments. In the presence of S9 mix a slight increase in the frequency of revertants of strain TA102 was observed. However, the estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level, using a X2-test did not reveal a significant effect at any of the test points.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the Ames Assay the substance was not mutagenic in any of the five strains (TA 100, TA 1535, TA 102, TA 98 and TA 1537) tested with and without metabolic activation up to 5000 µg/plate.