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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:
weight of evidence
Study period:
From 2010-02-25 to 2010-03-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in Jul 1997
Deviations:
no
Qualifier:
according to guideline
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
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

Constituent 1
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Purity: 100%
- Lot No.: 10SC8156928-2-2

Method

Target gene:
his operon (S. typhimurium strains)
trp operon (E. coli strain)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
- Selection reason: because they have high sensitivity to detect mutagens and are widely used in the reverse mutation test
-Source and: The Salmonella strains were obtained from Dr. B. N. Ames (University of California, Berkeley, U.S.A.). The Escherichia strain was obtained from Dr. T. Matsushima (Institute of Medical Science, the University of Tokyo, Tokyo, Japan).
- Preparation: A culture of the test strain was mixed with DMSO using a ratio of 8 parts cell culture stock and 0.7 parts DMSO by volume and frozen in an acetone-dry ice bath. These frozen stocks were kept in a deep freezer at -80℃ until use.
The stock cultures of each strain were checked for their phenotypic characteristics [the amino acid requirement, UV sensitivity and presence of rfa and R factors]. The results confirmed that all strains retained their own phenotypic characteristics. These strains also yielded solvent and positive control substances-induced revertants within the frequency ranges expected from the historical control data. Each bacterial frozen stock culture was thawed and 0.1 mL of bacterial suspension was added to 60 mL of nutrient broth medium in a flask of 300 mL volume and cultured for 10 hours at 37℃ with shaking.
- Composition of nutrient broth medium (per 1000 mL of purified water):
Nutrient broth (Bacto Nutrient Broth): 8g; NaCl: 5g
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan)
- method of preparation of S9 mix: cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and 5,6-benzoflavone.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The S9 used in this study was characterised with mutagen requiring metabolic activation, based on the assay data certificate of S9 batch used.
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
Since neither precipitation of the test substance nor cytotoxicity to bacteria was observed in the dose-finding assay, the dose of 5000 μg/plate was selected as the highest dose in the main assay as specified in the guidelines.
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

METHOD OF TREATMENT/ EXPOSURE:
Test substance solution (0.1 mL), either 100 mM Na-phosphate buffer (0.5 mL, pH 7.4) or S9 mix (0.5 mL), and bacterial suspension (0.1 mL) were mixed in a small test tube and incubated for 20 minutes at 37℃ with shaking. After addition of 2.0 mL of the top agar solution, the mixture was poured onto a minimal glucose agar plate, spread evenly and solidified at room temperature. All plates were incubated for 48 hours at 37℃.


METHODS FOR MEASUREMENTS OF GENOTOXICIY
The number of revertant colonies on a plate was counted using an automatic colony counter.

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. Any statistical analysis was not applied to evaluate the test results.

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
True 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
True 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:
Neither precipitation of the test substance nor cytotoxicity to bacteria was observed. 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'.

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:
The test item is not mutagenic under the test conditions.
Executive summary:

The test item was evaluated for its mutagenic potential by a reverse mutation test with four strains of Salmonella typhimurium (TA100, TA98, TA1535 and TA1537) and one strain of Escherichia coli (WP2 uvrA) based on OECD 471. The test was conducted in the presence and the absence of a rat liver drug metabolizing enzyme system (S9 mix).

In a dose-finding assay, the highest dose of test item was set at 5000 μg/plate, and the test was conducted at dose levels ranging from 5000 to 1.22 μg/plate in duplicate plating. Neither precipitation of the test substance nor cytotoxicity to bacteria was observed.

In the main assay, the test item was tested at doses ranging from 5000 to 156 μg/plate for all five test strains with and without S9 mix in duplicate plating based on the results of the dose-finding assay.

Throughout the tests, the test item did not show any statistically significant dose-dependent increase in the number of revertant colonies to at least twice as many as that of the solvent control with or without S9 mix in any of the strains. Positive control substances showed marked increases in the number of revertant colonies.

Based on these results, it is concluded that test item is not mutagenic under the test conditions.