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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:
30 Jan - 23 Feb 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21. Jul 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:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Sprague-Dawley rats treated with Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
Experiment 1
15, 50, 150, 500 and 1500 µg/plate with and without metabolic activation for TA100 and TA102
50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation for TA98, TA1535 and TA1537

Experiment 2
50, 150, 500, 1500 and 5000 µg/plate without metabolic activation for TA98, TA100, TA102 and TA1537
500, 1000, 1500, 3000 and 5000 µg/plate without metabolic activation for TA1535
50, 150, 500, 1500 and 5000 µg/plate with metabolic activation for all strains
Vehicle:
- Vehicle/solvent used: DMSO
Controls
Negative controls:
yes
Solvent 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 (2-AA)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertant colonies and/or a diminution of the background lawn
Statistics:
X²-test was used to estimate the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level. Mean values and standard deviation were calculated.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
in TA1535 without S9 mix
Cytotoxicity:
yes
Remarks:
in TA102 at 1500 µg/plate without S9 mix and at 5000 µg/plate with S9 mix
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation occurred up to the highest investigated dose.

Any other information on results incl. tables

Table 1. Test results of Experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

0

99 ± 8

25 ± 2

293 ± 31

31 ± 6

10 ± 5

0 (DMSO)

86 ± 2

24 ± 3

298 ± 19

26 ± 3

12 ± 2

15

89 ± 14

-

305 ± 13

-

-

50

73 ± 8

25 ± 6

296 ± 21

32 ± 3

12 ± 3

150

76 ± 5

32 ± 3

303 ± 14

42 ± 5

12 ± 4

500

83 ± 5

38 ± 7

248 ± 31

33 ± 8

11 ± 5

1500

97 ± 8

43 ± 8

221 ± 6T

39 ± 3

12 ± 4

5000

-

47 ± 3**

-

36 ± 4

10 ± 4

Positive controls, –S9

Name

NaN3

NaN3

MMC

2-NF

9-AA

Concentrations

(μg/plate)

0.7

0.7

0.15

2.5

50

Mean No. of colonies/plate

(average of 3 ± SD)

503 ± 58

905 ± 41

755 ± 51

614 ± 15

606 ± 63

+

0

97 ± 10

10 ± 3

343 ± 28

21 ± 4

17 ± 5

+

0 (DMSO)

88 ± 8

11 ± 3

340 ± 14

21 ± 1

14 ± 3

+

15

88 ± 3

-

349 ± 20

-

-

+

50

84 ± 15

9 ± 2

342 ± 37

25 ± 3

11 ± 4

+

150

81 ± 5

10 ± 3

348 ± 12

22 ± 6

14 ± 5

+

500

81 ± 3

10 ± 2

324 ± 17

25 ± 4

15 ± 3

+

1500

85 ± 8

12 ± 1

305 ± 21

23 ± 4

17 ± 3

+

5000

-

14 ± 3

-

22 ± 3

15 ± 2

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

0.8

0.8

0.9

0.8

1.7

Mean No. of colonies/plate

(average of 3 ± SD)

627 ± 90

216 ± 47

655 ± 61

741 ± 94

374 ± 103

DMSO: Dimethylsulphoxide

NaN3: Sodium azide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

2-NF: 2-nitrofluorene

MMC: Mytomycin C

T: bacteriotoxic

**: Significantly different from control (p < 0.01)

 

Table 2. Test results of Experiment 2 (plate incorporation).

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

0

201 ± 16

19 ± 3

326 ± 20

25 ± 4

8 ± 4

0 (DMSO)

158 ± 6

20 ± 2

292 ± 18

23 ± 5

10 ± 2

50

140 ± 22

40 ± 3**

348 ± 14

23 ± 3

8 ± 4

150

174 ± 21

35 ± 6

304 ± 22

24 ± 3

10 ± 3

500

140 ± 15

44 ± 5**

303 ± 19

26 ± 5

10 ± 3

1500

173 ± 18

43 ± 5**

248 ± 20

21 ± 3

6 ± 3

5000

132 ± 21

38 ± 10

138 ± 8T

24 ± 3

9 ± 4

Positive controls, –S9

Name

NaN3

NaN3

MMC

2-NF

9-AA

Concentrations

(μg/plate)

0.7

0.7

0.15

2.5

50

Mean No. of colonies/plate

(average of 3 ± SD)

471 ± 91

844 ± 12

916 ± 68

772 ± 26

493 ± 174

+

0

171 ± 23

9 ± 6

365 ± 5

29 ± 3

15 ± 6

+

0 (DMSO)

174 ± 6

9 ± 3

360 ± 13

25 ± 3

11 ± 3

+

50

176 ± 23

6 ± 2

353 ± 37

25 ± 3

11 ± 5

+

150

206 ± 14

10 ± 3

369 ± 3

30 ± 1

12 ± 5

+

500

215 ± 15

8 ± 3

328 ± 30

26 ± 3

12 ± 5

+

1500

189 ± 21

8 ± 3

305 ± 48

25 ± 6

13 ± 4

+

5000

210 ± 29

9 ± 4

253 ± 8T

25 ± 4

12 ± 5

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

0.8

0.8

0.9

0.8

1.7

Mean No. of colonies/plate

(average of 3 ± SD)

647 ± 113

129 ± 11

457 ± 23

833 ± 43

248 ± 24

DMSO: Dimethylsulphoxide

NaN3: Sodium azide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

2-NF: 2-nitrofluorene

MMC: Mytomycin C

T: bacteriotoxic

**: Significantly different from control (p < 0.01)

In the concentration range investigated the test substance induced a significant and dose related increase in the mutation frequency of the tester strain TA1535 in the absence of metabolic activation.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this Ames test the test substance was mutagenic in TA1535 without metabolic activation.
Executive summary:

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2001). In two independent experiments, the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were exposed to the test substance using the plate incorporation method. Based on the results of a pre-experiment, test substance concentrations of 15 to 5000 µg/plate were selected for the incubation with and without metabolic activation in the first experiment. In the second experiment, concentrations of 50 to 5000 µg/plate were selected for the incubation with and without metabolic activation. No precipitation occurred up to the highest investigated dose. In the absence and presence of metabolic activation the test substance was bacteriotoxic towards the strain TA102 at 1500 and 5000 µg/plate, respectively. In the concentration range investigated the test substance induced a significant and dose related increase in the mutation frequency of the tester strain TA1535 in the absence of metabolic activation. The results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the full activity of the metabolizing system. Under the conditions of this experiment, the test substance was mutagenic in the S. typhimurium strain TA1535 without metabolic activation.