Registration Dossier

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:
22 Feb - 15 Apr 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
other: Criteria for Mutagenicity Test in Bacterial Systems (Ministry of Labour, Japan, Notification No. 77, September 1, 1988; Notification No. 67, June 2, 1997)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Health, Labor and Welfare; Ministry of Economy, Trade and Industry; and Ministry of the Environment; Heisei 15.11.21 Yakushokuhatsu Notification No. 1121002, Heisei 15.11.13 Seikyoku Notific. No. 2, Kanpokihatsu Notific. No. 031121002
Deviations:
no
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): [trade name given]
- Physical state: white crystalline powder
- Analytical purity: 100%
- Lot/batch No.: 10SC8156928-2-2
- Stability under test conditions: stable for 6 months in a refrigerator

Method

Target gene:
his operon (S. typhimurium strains)
trp operon (E. coli strain)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The S9 used in this study was characterised with mutagen requiring metabolic activation, based on the assay data certificate of S9 batch used.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
The S9 used in this study was characterised with mutagen requiring metabolic activation, based on the assay data certificate of S9 batch used.
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.
Test concentrations with justification for top dose:
Range-finding experiment (all strains): 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 μg/plate with and without metabolic activation
Main experiment (all strains): 156, 313, 625, 1250, 5000 μg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the test substance has a water solubility of 50 mg/mL and is stable at this concentration for 24 h in room temperature
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see 'Remarks' field
Remarks:
2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), Sodium azide (NaN3), 9-aminoacridine (9-AA), 2-Aminoanthracene (2-AA) (see 'Details on test system and conditions' for concentrations)
Details on test system and experimental conditions:
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 2 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: examination of bacterial backgroud lawn under stereomicroscope.

OTHER:
Positive control concentrations: AF-2 (0.01 μg/plate in DMSO, -S9, TA100 and WP2 uvrA; 0.1 μg/plate in DMSO, -S9, TA98); NaN3 (0.5 μg/plate in water, -S9, TA1535); 9-AA (80 μg/plate in DMSO, -S9, TA 1537); 2-AA (1.0 μg/plate in DMSO, +S9, TA100; 2.0 μg/plate in DMSO, +S9, TA1535 and TA1537; 0.5 μg/plate in DMSO, +S9, TA98; 10.0 μg/plate in DMSO, +S9, WP2 uvrA)
Evaluation criteria:
When the test substance shows a dose-dependent increase in the number of revertant colonies to at least twice as many as that of the solvent control, the response is judged to be positive. The study director judges the result with scientific consideration of biological relevance. When the positive response is reproducible, the test substance is judged to have mutagenic potential.
Statistics:
Mean values were calculated at each concentration for the two experiments. For the historical control data, mean values and standard deviation were calculated for each test strain.

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:
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: no precipitation was observed.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
The results of the range-finding study were evaluated together with the main study.

COMPARISON WITH HISTORICAL CONTROL DATA:
Yes, see Table 1 under 'Any other information on materials and methods incl tables'.
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 dose range-finding assay (preincubation method)

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 2 plates)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvrA

TA98

TA1537

Sterilised purified water

104

15

24

37

11

1.22

87

9

22

28

11

-

4.88

92

12

32

32

10

19.5

92

11

21

39

10

-

78.1

97

16

22

39

10

312

88

11

20

44

11

-

1250

84

12

23

39

10

5000

94

14

23

42

12

Positive controls, –S9

Name

AF-2

NaN3

AF-2

AF-2

9-AA

Concentrations

(μg/plate)

0.01

0.5

0.01

0.1

80

Mean No. of colonies/plate

(average of 2 plates)

594

356

102

335

540

+

Sterilised purified water

93

9

20

38

15

+

1.22

93

8

30

40

15

+

4.88

105

8

28

38

12

+

19.5

80

13

20

42

15

+

78.1

85

12

30

39

18

+

313

94

7

22

36

17

+

1250

88

9

23

43

9

+

5000

86

7

23

33

14

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1

2

10

0.5

2

Mean No. of colonies/plate

(average of 2 plates)

760

206

570

214

133

AF-2: 2-(2 -furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: sodium azide

9-AA: 9-aminoacridine

2-AA: 2 -aminoanthracene

Table 2. Results of main assay (preincubation method)

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 2 plates)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2 uvrA

TA98

TA1537

Sterilised purified water

96

10

25

28

13

156

90

7

21

19

11

-

313

98

9

23

26

12

625

94

7

18

27

13

-

1250

90

12

23

26

13

2500

95

9

17

30

9

-

5000

98

11

26

31

10

Positive controls, –S9

Name

AF-2

NaN3

AF-2

AF-2

9-AA

Concentrations

(μg/plate)

0.01

0.5

0.01

0.1

80

Mean No. of colonies/plate

(average of 2 plates)

696

311

110

317

535

+

Sterilised purified water

82

10

25

35

19

+

156

100

10

29

30

21

+

313

83

10

23

28

11

+

625

86

5

18

27

15

+

1250

85

12

29

30

19

+

2500

86

5

31

37

18

+

5000

103

11

29

35

11

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1

2

10

0.5

2

Mean No. of colonies/plate

(average of 2 plates)

729

193

480

216

120

AF-2: 2-(2 -furyl)-3-(5-nitro-2-furyl)acrylamide

NaN3: sodium azide

9-AA: 9-aminoacridine

2-AA: 2 -aminoanthracene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative