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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 74-7718
- Test substance No.: 89/91

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Method

Target gene:
his
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:
Aroclor 1254
Test concentrations with justification for top dose:
- first test: 20, 100, 500, 2500 and 5000 µg/plate (standard plate test; all test strains)
- second test: 200, 400, 600, 800 and 1000 µg/plate (standard plate test; test strain TA98)
- third test: 20, 100, 500, 2500 and 5000 µg/plate (preincubation test; all test strains)
- fourth test: 4, 20, 100, 500 and 2500 µg/plate (preincubation test; test strain TA98)
Vehicle / solvent:
aqua dest.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9: aminoanthracene (2-AA): all test strains; + S9: methyl-N-nitro-N-nitrosoguanidine (MNNG): TA100 and TA1535, 4-nitro-o-phenylendiamine (NPD): TA98 and 9-aminoacridine chloride monohydrate (AAC): TA1537
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
Plate incorcoration method:
- Exposure duration: ca. 48-72 hours at 37 °C in the dark
Preincubation method:
- Preincubation period: 20 minutes at 37 °C
- Exposure duration: ca. 48-72 hours at 37 °C in the dark

NUMBER OF REPLICATIONS:
- 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Decrease in the number of revertants
- Reduced his- or trp- background growth
- Reduction in the titer
Evaluation criteria:
In order to characterized a substance as positive in the Ames test it has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control);
- dose-response relationship;
- reproducibility of the results.

Results and discussion

Test results
Key result
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
Additional information on results:
Toxicity:
No bacteriotoxic effect (reduced his- background growth) was observed.

Solubility:
Complete solubility of test substance in aqua dest.

Any other information on results incl. tables

Table 1: Standard Plate Test (TA 98)

Dose

[µg/plate]

TA 98

Mean ± SD

Without S9 mix

Negative control

(aqua dest)

28 ± 5

200

33 ± 12

400

26 ± 4

600

24 ± 5

800

20 ± 2

1000

26 ± 4

10 NPD

1118 ± 125

With S9 mix

Negative control

(aqua dest)

 44 ± 4

200

38 ± 4

400

40 ± 3

600

 36 ± 1

800

 39 ± 3

1000

 30 ± 11

10 2-AA

 1850 ± 26

Table 2: Standard Plate Test (TA 98, TA 100, TA 1535, TA 1537)

Dose

[µg/plate]

TA 98

Mean ± SD

TA 100

Mean ± SD

TA 1535

Mean ± SD

TA1537

Mean ± SD

Without S9 mix

Negative control

(aqua dest)

28 ± 2

140 ± 5

19 ± 6

15 ± 4

20

33 ± 5

129 ± 8

24 ± 3

10 ± 4

100

33 ± 3

131 ± 27

19 ± 1

15 ± 4

500

38 ± 11

99 ± 8

22 ± 5

13 ± 5

2500

26 ± 6

114 ± 6

22 ± 4

10 ± 5

5000

23 ± 7

139 ± 5

23 ± 4

10 ± 3

10 NPD

922 ± 88

 

 

 

100 AAC

 

 

 

430 ± 91

5 MNNG

 

1347 ± 43

1349 ± 273

 

With S9 mix

Negative control

(aqua dest)

41 ± 5

122 ± 13

20 ± 11

14 ± 2

20

57 ± 5

139 ± 6

21 ± 4

15 ± 5

100

54 ± 12

126 ± 4

18 ± 2

15 ± 7

500

62 ± 23

137 ± 16

20 ± 4

21 ± 1

2500

36 ± 9

140 ± 45

19 ± 4

20 ± 8

5000

36 ± 10

117 ± 30

18 ± 1

16 ± 3

10 2-AA

785 ± 62

334 ± 47

379 ± 53

96 ± 15

Table 3: Preincubation Test (TA 98)

Dose

[µg/plate]

TA 98

Mean ± SD

Without S9 mix

Negative control

(aqua dest)

26 ± 2

4

23 ± 2

20

26 ± 2

100

21 ± 2

500

27 ± 1

2500

26 ± 2

10 NPD

1120 ± 98

With S9 mix

Negative control

(aqua dest)

41 ± 4

4

39 ± 6

20

34 ± 13

100

35 ± 13

500

38 ± 6

2500

33 ± 4

10 2-AA

1247 ± 87

Table 4: Preincubation Test (TA 98, TA 100, TA 1535, TA 1537)

Dose

[µg/plate]

TA 98

Mean ± SD

TA 100

Mean ± SD

TA 1535

Mean ± SD

TA 1537

Mean ± SD

Without S9 mix

Negative control

(aqua dest)

24 ± 4

104 ± 11

14 ± 3

12 ± 2

20

39 ± 2

117 ± 16

15 ± 3

12 ± 2

100

36 ± 3

95 ± 15

16 ± 2

14 ± 1

500

38 ± 6

95 ± 5

16 ± 6

9 ± 4

2500

39 ± 9

115 ± 13

19 ± 3

13 ± 2

5000

31 ± 2

118 ± 11

14 ± 2

11 ± 2

10 NPD

1320 ± 131

 

 

 

100 AAC

 

 

 

313 ± 36

5 MNNG

 

1343 ± 81

1317 ± 6

 

With S9 mix

Negative control

(aqua dest)

35 ± 3

113 ± 19

17 ± 6

12 ± 1

20

59 ± 20

126 ± 12

18 ± 2

14 ± 2

100

51 ± 10

148 ± 18

19 ± 1

11 ± 1

500

49 ± 4

129 ± 18

14 ± 2

14 ± 5

2500

48 ± 5

126 ± 21

17 ± 2

20 ± 3

5000

46 ± 3

135 ± 10

17 ± 2

19 ± 1

10 2-AA

865 ± 241

886 ± 63

167 ± 11

141 ± 22

2 -AA: 2 -aminoanthracene

MNNG: N-methyl-N-nitro-N-nitroso-guanidine

NPD: 4 -nitro-o-phenylendiamine

AAC: 9 -aminoacridine chloride monohydrate

Applicant's summary and conclusion