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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:
1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
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:
Histidine-requiring gene in four strains of S. typhimurium, and tryptophan requiring gene in E. coli WP2.
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):
CELLS USED
- Source of cells: National Institute of Public Health (obtained December 19, 1994)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital (PB) and 5,6-benzoflavone (BF) induced rat liver S9.
Test concentrations with justification for top dose:
- Preliminary Toxicity Test: 100 - 5000 μg/plate; no bacterial toxicity up to highest dose level.
- Main tests 1 and 2: 0, 312.5, 625, 1250, 2500 and 5000 μg / plate.
- Justification: In order to determine the appropriate concentration of the test substance in this test, tests were conducted by the same experimental method as this test using six concentrations of 100, 200, 500, 1000, 2000 and 5000 μg/plate for all indicator strains. The test was performed on one plate of each concentration. As a result, regardless of the presence or absence of metabolic activation, no growth inhibition was observed at any concentration in any strain.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was reported to be soluble in DMSO, slightly soluble in water.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
furylfuramide
other: 2-aminoanthracene (2-AA)
Remarks:
see table 4 in 'Any other information on materials and methods'
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Plate incorporation method, with and without metabolic activation. 0.1 mL of the solvent, test item solution or positive control substance solution to were put in a test tube; then, 0.5 ml of phosphate buffer solution (or 0.5 ml of S9 mixture solution in metabolic activation test) and 0.1 ml of assay bacterial solution were mixed in, and cultured with shaking at 37ºC for 20 minutes. Next, a mixture containing 2 ml of top agar (kept at 45ºC) was overlaid on the plate. After solidification, plates were incubated at 37ºC for 48 hours. After the incubation period, the number of revertant colonies was counted, and at the same time, the presence or absence of growth inhibition of the indicator strain was observed using a stereomicroscope.
- Cell density at seeding (if applicable): see table 3.

DURATION
- Preincubation period: no.
- Exposure duration: 48h

SELECTION AGENT (mutation assays): No specific selection agent was used. The lack of amino-acid in the medium allowed only mutants to grow because of their ability to synthesize the essential amino acid.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth.
Evaluation criteria:
The results were judged as positive if the following three criteria were satisfied based on the average number of revertant colonies on the plate at each concentration.
1) In the test substance treatment group, the number of revertant colonies was increased more than twice the solvent control value.
2) The number of revertant colonies increases with increasing test substance concentration (concentration dependence).
3) The repeatability of the increase in the number of revertant colonies is observed from the results of the two tests.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: none reported

RANGE-FINDING/SCREENING STUDIES: in the preliminary test, performed in the range of 100 to 5000 μg/plate, no growth inhibition by the test substance was observed in any of the strains regardless of the presence or absence of metabolic activation. Therefore, the top dose for the main tests was 5000 μg/plate, and the next doses were 2500, 1250, 625 and 312.5 μg/plate (common ratio 2).

HISTORICAL CONTROL DATA: not specified.

Any other information on results incl. tables

Table 1. Results of reverse mutation test of o-TSA on bacteria (1st trial) without metabolic activation (direct method).

Test substance

concentration

(µg/plate)

Number of revertant colonies per plate (Mean ± S.D.)

TA100

TA1535

WP2 uvrA

TA98

TA1537

0

124

136

126

8

8

7

10

17

7

16

19

24

9

11

14

(129 ± 6)

(8 ± 1)

(11 ± 5)

(20 ± 4)

(11 ± 3)

312.5

116

127

145

7

8

18

15

19

13

23

15

26

12

13

8

(129 ± 15)

(11 ± 6)

(16 ± 3)

(21 ± 6)

(11 ± 3)

625

144

138

105

9

6

9

10

13

14

21

22

24

10

3

10

(129 ± 21)

(8 ± 2)

(12 ± 2)

(22 ± 7)

(11 ± 2)

1250

137

121

103

10

5

10

18

16

6

13

17

19

7

10

7

(120 ± 17)

(8 ± 3)

(13 ± 6)

(16 ± 3)

(8 ± 2)

2500

114

125

101

11

7

7

12

14

18

21

21

22

5

9

8

(113 ± 12)

(8 ± 2)

(15 ± 3)

(21 ± 1)

(7 ± 2)

5000

119

111

130

7

8

7

15

14

10

17

22

15

14

7

11

(120 ± 10)

(7 ± 1)

(13 ± 3)

(18 ± 4)

(11 ± 4)

Posit.

control

803

847

873

340

313

321

1125

1177

1106

292

298

308

978

906

641

(841 ± 35)

(325 ± 14)

(1136 ± 37)

(299 ± 8)

(842 ± 177)

Table 2. Results of reverse mutation test of o-TSA on bacteria (1st trial) with metabolic activation (S9 mix).

Test substance

concentration

(µg/plate)

Number of revertant colonies per plate (Mean ± S.D.)

TA100

TA1535

WP2 uvrA

TA98

TA1537

0

120

152

123

8

11

8

11

20

25

32

46

37

13

10

13

(132 ± 18)

(0 ± 2)

(19 ± 7)

(38 ± 7)

(12 ± 2)

312.5

154

128

138

11

10

10

15

8

19

37

34

37

15

8

0

(140 ± 13)

(10 ± 1)

(14 ± 6)

(36 ± 2)

(11 ± 4)

625

136

129

142

13

11

11

14

17

21

51

45

44

14

18

0

(145 ± 11)

(9 ± 2)

(13 ± 2)

(41 ± 7)

(13 ± 4)

1250

157

135

143

8

11

8

15

11

13

35

49

38

11

10

17

(132 ± 11)

(11 ± 4)

(17 ± 5)

(37 ± 7)

(16 ± 4)

2500

139

137

119

8

15

11

21

17

38

30

44

 

16

13

20

(132 ± 11)

(11 ± 4)

(17 ± 5)

(37 ± 7)

(16 ± 4)

5000

120

130

126

16

12

9

16

18

12

36

43

37

18

14

19

(125 ± 5)

(12 ± 4)

(15 ± 3)

(39 ± 4)

(17 ± 3)

Posit.

control

548

589

604

127

128

120

331

1251

1307

324

337

316

108

94

105

(580 ± 29)

(125 ± 4)

(1297 ± 42)

(326 ± 11)

(102 ± 7)

Table 3. Results of reverse mutation test of o-TSA on bacteria (2nd trial) without metabolic activation (direct method).

Test substance

concentration

(µg/plate)

 

Number of revertant colonies per plate (Mean ± S.D.)

 

TA100

TA1535

WP2 uvrA

TA9S

TA1537

0

117

134

114

10

10

11

9

13

11

16

20

22

7

6

3

(122 ± 11)

(10 ± 1)

(11 ± 2)

(19 ± 3)

( 5 ± 2)

312.5

139

119

139

6

10

9

23

14

14

31

20

28

5

6

3

(132 ± 12)

( S ± 2)

(17 ± 5)

(26 ± 6)

( 5 ± 2)

625

107

121

126

6

7

7

14

10

11

27

14

19

7

8

3

(118 ± 10)

( 7 ± 1)

(12 ± 2)

(20 ± 7)

( 6 ± 3)

1250

105

107

103

10

6

6

15

S

9

13

20

19

7

5

7

(105 ± 2)

( 7 ± 2)

(11 ± 4)

(17 ± 4)

( 6 ± 1)

2500

125

93

100

12

4

12

19

12

14

20

19

14

3

6

12

(103 ± 17)

( 9 ± 5)

(15 ± 4)

(18 ± 3)

( 7 ± 5)

5000

102

91

101

7

7

6

15

12

16

17

16

19

5

8

5

( 99 ± 4)

( 7 ± 1)

(14 ± 2)

(17 ± 2)

( 6 ± 2)

Positive

control

797

793

795

301

311

293

961

1109

993

264

262

305

606

654

610

(795 ± 2)

(303 ± 9)

(1028 ± 71)

(277 ± 2-1)

(621 ± 26)

Table 4. Results of reverse mutation test of o-TSA on bacteria (2nd trial) with metabolic activation (S9 mix).

Test substance

concentration

(µg/plate)

 

Number of revertant colonies per plate (Mean ± S.D.)

 

TA100

TA1535

WP2 uvrA

TA9S

TA1537

0

154

144

136

8

6

15

20

20

25

35

42

39

11

13

9

(145 ± 9)

(10 ± 5)

(22 ± 3)

(39 ± 4)

(11 ± 2)

312.5

165

157

156

7

11

10

22

20

13

31

41

41

6

7

9

(159 ± 5)

(9 ± 2)

(18 ± 5)

(38 ± 6)

(7 ± 2)

625

157

155

144

13

9

18

12

23

11

37

37

41

13

10

8

(152 ± 7)

(13 ± 5)

(15 ± 7)

(38 ± 2)

(10 ± 3)

1250

158

131

120

12

7

13

23

16

10

42

30

43

9

12

10

(136 ± 20)

(11 ± 3)

(16 ± 7)

(38 ± 7)

(10 ± 2)

2500

122

111

123

11

7

7

17

18

11

46

39

43

12

7

17

(119 ± 7)

(8 ± 2)

(15 ± 4)

(43 ± 3)

(12 ± 5)

5000

134

125

110

8

8

14

10

13

18

38

27

32

15

10

8

( 123 ± 12)

(10 ± 3)

(14 ± 4)

(32 ± 6)

(11 ± 4)

Positive

control

504

499

472

127

160

132

1207

1170

1290

357

373

341

106

136

101

(492 ± 17)

(140 ± 18)

(1222 ± 61)

(357 ± 16)

(114 ± 19)

 

Applicant's summary and conclusion

Conclusions:
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. Therefore, the test item was not mutagenic under test conditions.
Executive summary:

The ability of the test item to induce mutation was studied in a bacterial reverse mutation test (Ames test) according to OECD Guidelines No. 471 and the Guidelines for Screening Mutagenicity Testing of Chemicals (Japan) (GLP study). Four histidine requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and tryptophan requiring Escherichia coli WP2 uvrA were tested in triplicate at six concentrations of test item in up to 5000 μg/plate, with and without metabolic activation (S9 mix), based on the results of a preliminary range-finding test. The plate incorporation method was used. Vehicle (DMSO) and positive controls were run in parallel. No cytotoxicity was observed at any dose tested, the vehicle control plates gave counts of revertant colonies within the normal range, and all of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. All validity criteria were met. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. Therefore, the test item was not mutagenic under test conditions.