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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:
07 Mar-21 Mar 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study with acceptable restrictions. Only 4 S. typhimurium strains tested, no TA102 or E. coli strain was included.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 S. typhimurium strains tested, no TA102 or E. coli strain included
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
only 4 S. typhimurium strains tested, no TA102 or E. coli strain included
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): only trade name given
- Physical state: yellow liquid
- Analytical purity: no data
- Lot/batch No.: 8779
- Storage condition of test material: at room temperature in the dark

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Preliminary experiment: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate with and without metabolic activation
First experiment: 100, 333, 1000, 3330 and 5000 µg/plate with and without metabolic activation
Second experiment: 100, 333, 1000, 3330 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle used: DMSO (0.1 mL per plate)
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: 9-aminoacridine (9AC; 60 µg/plate in saline, TA1537); sodium azide (SA; 1 µg/plate in saline, TA1535); daunomycine (DM; 4 µg/plate in saline, TA98); methylmethanesulfonate (MMS; 650 µg/plate in DMSO, TA100)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S9: 2-amino-anthracene (2-AA; 0.5 or 5 µg/plate in DMSO for all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of bacterial background lawn

Evaluation criteria:
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with of without metabolic activation. However, any mean plate count of less than 20 revertants is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
Statistics:
Mean values and standard deviation were calculated.

Results and discussion

Test results
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes, at concentrations of 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
Strain TA100 was used for testing up to 5000 µg/plate with and without S9-mix. The results of the dose range finding experiment were included into the first experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No reduction of the bacterial background lawn and no decrease in the number of revertants was observed. Therefore, the test substance was tested up to a concentration of 5000 µg/plate in the absence or presence of S9-mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Ames Test Results of Experiment 1.

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

Frameshift type

 

TA 100

TA1535

TA98

TA1537

0

63 ± 6

15 ± 2

13 ± 1

7 ± 3

3

59 ± 1

 

 

 

10

70 ± 6

 

 

 

33

73 ± 11

 

 

 

100

65 ± 1

13 ± 5

15 ± 5

7 ± 3

333

57 ± 8

15 ± 3

18 ± 4

7 ± 1

1000

53 ± 12

15 ± 4

15 ± 3

9 ± 2

3330

54 ± 2

12 ± 2

16 ± 4

8 ± 1

5000*

54 ± 5

15 ± 3

17 ± 5

7 ± 4

Positive controls, –S9

Name

MMS

SA

DM

9AC

Concentrations (μg/plate)

650

1

4

60

Mean No. of colonies/plate (average of 3 ± SD)

804 ± 25

6142 ± 5

241; 186**

238 ± 113

 +

0

76 ± 7

13 ± 6

18 ± 6

7 ± 1

 +

3

66 ± 18

 

 

 

 +

10

71 ± 6

 

 

 

+

33

67 ± 11

 

 

 

+

100

54 ± 9

17 ± 1

18 ± 7

8 ± 2

+

333

63 ± 13

11 ± 2

22 ± 2

4 ± 1

+

1000

59 ± 16

15 ± 2

23 ± 1

6 ± 4

+

3330

66 ± 15

12 ± 6

20 ± 4

6 ± 3

+

5000*

58 ± 6

9 ± 3

18 ± 6

7 ± 2

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

Concentrations (μg/plate)

0.5

5

0.5

5

Mean No. of colonies/plate (average of 3 ± SD)

647 ± 69

246 ± 14

471 ± 20

572 ± 39

* Test subtance precipitated slightly on the plates

**One plate infected with other bacteria

SA = Sodium azide

9AC = 9 Aminoacridine

DM = Daunomycine

MMS = Methylmethanesulfonate

2AA = 2 -Aminoanthracene

Table 2. Ames Test Results of Experiment 2.

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

Frameshift type

 

TA 100

TA1535

TA98

TA1537

0

60 ± 1

6 ± 3

14 ± 3

4 ± 1

100

56 ± 8

7 ± 2

15 ± 6

5 ± 1

333

68± 3

9 ± 2

14 ± 5

5 ± 1

1000

68 ± 9

6 ± 1

13 ± 1

7 ± 1

3330

71 ± 6

9 ± 3

15 ± 3

6 ± 2

5000*

58 ± 10

11 ± 3

14 ± 1

9 ± 3

Positive controls,

–S9

Name

MMS

SA

DM

9AC

Concentrations (μg/plate)

650

1

4

60

Mean No. of colonies/plate (average of 3 ± SD)

1047 ± +65

178 ± 9

194 ± 11

204 ± 43

+

0

73 ± 3

10 ± 3

20 ± 2

7 ± 3

+

100

70 ± 15

6 ± 2

17 ± 4

5 ± 1

+

333

72 ± 13

11 ± 1

22 ± 9

6 ± 2

+

1000

95 ± 9

13 ± 2

24 ± 2

8 ± 1

+

3330

89 ± 15

7 ± 1

23 ± 6

7 ± 1

+

5000*

81 ± 4

9 ± 4

19 ± 3

7 ± 2

+

Name

2AA

2AA

2AA

2AA

+

Concentrations (μg/plate)

0.5

5

0.5

5

+

Positive controls, +S9

Mean No. of colonies/plate (average of 3 ± SD)

1169 ± 87

475 ± 12

593 ± 6

669 ± 93

SA = Sodium azide

9AC = 9 -Aminoacridine

DM = Daunomycine

MMS = Methylmethanesulfonate

2AA = 2 -Aminoanthracene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative