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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
31 Aug - 09 Sep 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (tester strains with AT reversion site missing)

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:
adopted in 1983
Deviations:
yes
Remarks:
tester strains with AT reversion site missing (S. typhimurium TA102 or E. coli WP2 were not tested)
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: liquid, yellow
- Analytical purity: 100%
- Lot/batch No.: 44538224
- Expiration date of the lot/batch: August 2000
- Storage condition of test material: at room temperature

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital (intraperitoneally) and beta-Naphthoflavone (orally)
Test concentrations with justification for top dose:
8, 40, 200, 1000 and 5000 µg/plate (2 experiments, each with and without S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controls
Untreated negative controls:
yes
Remarks:
medium
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Remarks:
-S9: sodium azide (2 µg/plate, TA100 and TA1535); 9-aminoacridine (80µg/plate, TA537); 4-nitro-o-phenylendiamine (40 µg/plate, TA98 and TA1538); +S9: 2-aminoanthracene (2.5 or 5 µg/plate, all strains)
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylendiamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: growth reduction of background bacteria
Evaluation criteria:
The test material was considered positive if the following criteria were met:
The plate background of non-converted bacteria did not show any growth reduction versus the respective negative controls.
The spontaneous mutation rates of each tester strain per plate were within the characteristic spontaneous mutation range.
The positive controls showed mutation rates exceeding the control values of TA 100 at least by the factor 2.0 and those of the other tester strains by the factor 3.0.
At more than one dose tested, the test substance caused at least a 2.0-fold increase in comparison with negative controls in the tester strain TA 100, for the other tester strains, an increase in the mutation rates of more than 3.0 above the corresponding negative controls.
Statistics:
Mean values and standard deviation were calculated.

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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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: Results of experiment 1:

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

TA 100

TA1535

TA1537

TA1538

TA98

-

Medium

113

11

11

8

20

-

Vehicle

78

8

7

6

18

-

8

78

8

5

6

16

-

40

80

8

4

6

18

-

200

80

10

5

6

28

-

1000

90

11

8

5

23

-

5000

90

6

5

5

22

Positive

controls

- S9

Name

SA

SA

9AA

4NP

4NP

Concentrations

(μg/plate)

2.0

2.0

80

40

40

Number of colonies/plate

745

518

773

1875

1531

 +

Medium

119

16

8

13

21

+

Vehicle

91

11

6

7

18

+

8

118

17

9

6

21

+

40

110

10

6

10

18

+

200

109

14

5

9

23

+

1000

118

12

4

11

17

+

5000

110

10

9

8

23

Positive

controls

+ S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

5.0

2.5

2.5

5.0

5.0

Number of colonies/plate

318

264

257

2155

1808

SA: sodium azide

9AA : 9-aminoacridine

4NP : 4-nitro-o-phenylenediamine

2-AA: 2 -aminoanthracene

 

 

Table 2: Results of experiment 2:

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates)

TA 100

TA1535

TA1537

TA1538

TA98

-

Medium

100

10

7

8

20

-

Vehicle

98

9

9

10

9

-

8

85

13

11

4

9

-

400

92

10

5

9

11

-

200

84

7

6

11

12

-

1000

77

13

6

8

13

-

5000

99

10

2

9

11

Positive

controls

- S9

Name

SA

SA

9AA

4NP

4NP

Concentrations

(μg/plate)

2.0

2.0

80

40

40

Number of colonies/plate

790

583

922

2031

1631

 +

Medium

99

15

10

13

22

+

Vehicle

80

15

8

17

31

+

8

88

14

8

11

28

+

40

91

16

13

18

27

+

200

110

12

9

16

26

+

1000

83

12

14

17

27

+

5000

103

16

12

15

30

Positive

controls

+ S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

5.0

2.5

2.5

5.0

5.0

Number of colonies/plate

1339

84

215

1192

1069

SA: sodium azide

9AA : 9 -aminoacridine

4NP : 4 -nitro-o-phenylenediamine

2-AA: 2 -aminoanthracene

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative