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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:
25 Feb - 01 Jun 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
his/trp operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
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 toxicity test:
- 156, 313, 625, 1250, 2500 and 5000 µg/plate (TA 98 and TA 1535 with and without metabolic activation)
Experiment I+II:
- 313, 625, 1250, 2500 and 5000 µg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle/solvent used: distilled water
- Justification for choice of solvent: Distilled water was used as solvent, since this medium is not suspected of chemical reaction with the test substance and is compatible with the survival of the bacteria and the S9 activity.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9-mix: daunomycin (DM), sodium azide (NaN), ICR 191 acridine (ICR), 4-nitroquinoline-1-oxide (4-NQO); +S9-mix: 2-aminoanthracene (2-AA), benzo(a)pyrene (BaP)
Remarks:
DM (6 µg/plate: TA 98); NaN (1.5 µg/plate: TA 100, TA 1535); ICR (1 µg/plate: TA 1537); 4-NQO (2 µg/plate: E.coli WP2 uvrA); 2-AA (10 µg/plate: TA 98, TA 100, TA 1535, TA 1537; 50 µg/plate: E.coli WP2 uvrA); BaP (20 µg/plate: TA 98, TA 100)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3 plates for each test concentration and control

DETERMINATION OF CYTOTOXICITY
- A preliminary experiment was conducted with the test material in tester strains TA 98 and TA 1535 either in the presence or in the absence of metabolic activation to determine cytotoxicity and solubility of the test substance using concentrations of 156, 313, 625, 1250, 2500 and 5000 µg/plate.
- Method: Cytotoxicity is detected by reduction in the number of revertant colonies
Evaluation criteria:
Means of individual plate counts (triplicates) were calculated for test solutions and controls. In general, a 2 or 2.5-fold increase in the number of revertant colonies per plate over the background (spontaneous revertant frequency) is used as a criterion to distinguish active mutagens from non-mutagenic materials. The presence of dose-response is a further criterion for mutagenic materials.
Statistics:
Mean values and standard deviation were calculated for each test and control concentration.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: 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:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitations were seen in all test material concentrations which did not influence the results of the assay.

RANGE-FINDING/SCREENING STUDIES
As no relevant cytotoxicity was observed in the preliminary test (data not shown), the highest test concentration used in the main test was 5000 µg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY
No signs of cytotoxicity are reported.

COMPARISON WITH HISTORICAL CONTROL DATA
The colony counts of negative and positive controls were in the dimensions of historical and literature data.

Any other information on results incl. tables

Table 1: Test results - Experiment I - Preincubation method

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

TA 100

TA1535

WP2uvrA

TA98

TA1537

0

79 ± 2

8 ± 0

13 ± 2

13 ± 4

8 ± 2

313

87 ± 7

11 ± 2

10 ± 2

12 ± 1

6 ± 1

625

69 ± 8

8 ± 2

13 ± 6

11 ± 4

6 ± 3

1250

74 ± 6

7 ± 3

15 ± 2

11 ± 4

3 ± 1

2500

89 ± 13

7 ± 5

11 ± 3

12 ± 5

4 ± 2

5000

85 ± 8

8 ± 1

11 ± 4

15 ± 6

7 ± 3

Positive controls, –S9

Name

NaN

NaN

4-NQO

DM

ICR

Concentrations

(μg/plate)

1.5

1.5

2

6

1

Mean No. of colonies/plate

(average of

3 ± SD)

499 ± 31

431 ± 26

839 ± 67

435 ± 95

2040 ± 59

+

0

68 ± 11

7 ± 2

10 ± 5

13 ± 3

4 ± 1

+

313

63 ± 6

6 ± 0

12 ± 2

17 ± 3

6 ± 1

+

625

59 ± 16

7 ± 1

12 ± 4

18 ± 4

4 ± 2

+

1250

68 ± 20

8 ± 4

11 ± 2

20 ± 5

5 ± 3

+

2500

70 ± 15

6 ± 2

11 ± 6

16 ± 2

4 ± 2

+

5000

72 ± 10

6 ± 4

10 ± 3

18 ± 2

4 ± 2

Positive controls, +S9 (10%)

Name

2-AA / BaP

2-AA

2-AA

2-AA / BaP

2-AA

Concentrations

(μg/plate)

10 / 20

10

50

10 / 20

10

Mean No. of colonies/plate

(average of

3 ± SD)

1807 ± 11 / 584 ± 19

56 ± 7

316 ± 62

2068 ± 60 / 290 ± 79

182 ± 25

DM: daunomycin

NaN: sodium azide

ICR: ICR 191 acridine

4-NQO: 4-nitroquinoline-1-oxide

2-AA: 2-aminoanthracene

BaP: benzo(a)pyrene

Table 2: Test results - Experiment II - Plate incorporation method

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

TA 100

TA1535

WP2uvrA

TA98

TA1537

0

70 ± 12

8 ± 3

10 ± 5

29 ± 4

4 ± 1

313

81 ± 6

6 ± 3

12 ± 6

18 ± 8

4 ± 3

625

88 ± 13

8 ± 3

11 ± 1

17 ± 3

5 ± 1

1250

81 ± 9

7 ± 2

13 ± 2

10 ± 3

6 ± 1

2500

97 ± 12

6 ± 3

12 ± 3

17 ± 5

5 ± 2

5000

79 ± 7

6 ± 3

14 ± 2

16 ± 4

4 ± 2

Positive controls, –S9

Name

NaN

NaN

4-NQO

DM

ICR

Concentrations

(μg/plate)

1.5

1.5

2

6

1

Mean No. of colonies/plate

(average of

3 ± SD)

468 ± 37

385 ± 12

1185 ± 291

576 ± 99

82 ± 13

+

0

86 ± 12

4 ± 0

12 ± 6

12 ± 4

5 ± 2

+

313

72 ± 3

6 ± 3

12 ± 5

11 ± 6

4 ± 1

+

625

77 ± 4

7 ± 2

8 ± 3

22 ± 8

5 ± 2

+

1250

73 ± 10

5 ± 2

11 ± 2

19 ± 3

5 ± 1

+

2500

73 ± 16

7 ± 3

13 ± 2

14 ± 3

4 ± 2

+

5000

70 ± 5

5 ± 2

14 ± 1

12 ± 7

4 ± 2

Positive controls, +S9 (10%)

Name

2-AA / BaP

2-AA

2-AA

2-AA / BaP

2-AA

Concentrations

(μg/plate)

10 / 20

10

50

10 / 20

10

Mean No. of colonies/plate

(average of

3 ± SD)

856 ± 154 / 383 ± 9

45 ± 2

378 ± 48

1782 ± 201 / 136 ± 23

175 ± 49

DM: daunomycin

NaN: sodium azide

ICR: ICR 191 acridine

4-NQO: 4-nitroquinoline-1-oxide

2-AA: 2-aminoanthracene

BaP: benzo(a)pyrene

Table 3: Historical control data (negative control)

Strain

MIN

MAX

MEAN

SD

N

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

TA 98

13

21

54

65

29

38

16

19

5

5

TA 100

62

63

246

232

112

108

78

70

5

5

TA 1535

7

8

28

20

14

11

8

5

5

5

TA 1537

3

3

4

6

4

5

n.a.

n.a.

2

2

WP2 uvrA

13

13

17

20

15

18

2

3

4

4

SD: standard deviation

n.a.: not applicable

N: number of studies

Table 4: Historical control data (positive control)

Strain

Chemical

MIN

MAX

N

TA 98

2-Aminoanthracene

135

>1000

5

Daunomycin

198

>500

5

TA 100

2-Aminoanthracene

326

>500

5

Sodium azide

274

>500

5

TA 1535

2-Aminoanthracene

67

>300

5

Sodium azide

83

>300

5

TA 1537

2-Aminoanthracene

107

210

2

ICR 191 Acridine

126

283

2

WP2 uvrA

2-Aminoanthracene

251

407

4

4-Nitroquinoline-1-oxide

756

1532

4

N: number of studies

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative