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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:
Study initiation: 22 November 2010, Final report: 22 February 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Remarks:
Korea
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:
Chemical name : 1,3,2-Dioxathiolane, 2,2-dioxide
Product name: ESA
Lot No. : Not available
Received date : 18 November, 2010
Appearance : Lemon yellow solid
Purity : 99.1 %
Stability : Stable in recommended storage condition

Method

Target gene:
histidine and tryptophan
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Liver of Sprague-Dawley male rat induced by Aroclor-1254, 500 mg/kg i.p.
Test concentrations with justification for top dose:
•In the absence of metabolic activation system (S9 mix(-))
TA100, TA1537, WP2uvrA
0, 5, 10, 20, 39, 78, 156, 313 µg/plate
TA1535
0, 39, 78, 156, 313, 625, 1250, 2500 µg/plate
TA98
0, 0.6, 1.25, 2.5, 5, 10, 20, 39 µg/plate
•In the presence of metabolic activation system (S9 mix(+))
0, 5, 10, 20, 39, 78, 156, 313 µg/plate
Vehicle / solvent:
The test substance showed high solubility in DMSO but crystals were formed after. DMSO was not suitable for use in the test. The test substance showed high solubility in 1,4-Dioxane and did not react with the 1,4-Dioxane. Therefore, 1,4-Dioxane was selected to negative control.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1,4-Dioxane
True negative controls:
no
Positive controls:
yes
Remarks:
0.5 µg/plate for strain TA1535
Positive control substance:
sodium azide
Remarks:
Without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1,4-Dioxane
True negative controls:
no
Positive controls:
yes
Remarks:
80 µg/plate for strain TA1537
Positive control substance:
other: 9-Aminoacridine hydrochloride hydrate
Remarks:
Without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1,4-Dioxane
True negative controls:
no
Positive controls:
yes
Remarks:
0.01 µg/plate for strains TA100 and WP2uvrA, 0.1 µg/plate for strain TA98
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
Remarks:
Without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
1,4-Dioxane
True negative controls:
no
Positive controls:
yes
Remarks:
0.5 µg/plate for strain TA98, 1.0 µg/plate for strain TA100, 2.0 µg/plate for strains TA1535 and TA1537, 10 µg/plate for strain WP2uvrA
Positive control substance:
other: 2-Aminoanthracene
Remarks:
With S9 mix
Details on test system and experimental conditions:
Condition of pre-cultivation
Nutrient broth: No. 2, Oxoid, 588285
Cultivation time: 10 hours (Stop incubation in the early stationary phase)
Shaking incubator: Model: SHKE-5000-1CE
Incubation method: Shaking type: rotation (RPM: 180 /min) Rotation diameter: 2.54 cm
Culture vessel: Erlenmeyer flask Capacity: 50 ml
Volume of culture medium: 15 ml
Volume of inoculum: 30 ml

Preparation of test substance
The test substance was dissolved in 1,4-dioxane and then, serially diluted (two-fold) from highest concentration.

Method
This study was conducted using preincubation method with and without metabolic activation system (S9).

Test condition
Preincubation: 37 ºC, 20 min
Incubation: 37 ºC, 48 hours

Experiments with test item and controls were performed in triplicate

Method of observation, measurement and analysis

Colony counting
The number of revertants were counted only inside area of plate (diameter : 90 mm) by manual measurement using electronic register (Model 570, SUNTEX, Taiwan).
Test method of growth inhibition
All plate was observed with the naked eye.

Measurement of bacterial concentration
It was performed by step-dilution method according to the Standard Operation Procedure, KCL.

Sterility test
0.1 ml of S9 mix and test substance were mixed with top agar respectively, and then, it was added onto minimum glucose agar plate. The plate was incubated at 37ºC for 48 hours. Since the bacteria did not grow after incubation, it was confirmed that the test was performed aseptically.
Evaluation criteria:
The test result was recorded experimental value, average value and standard deviation for the number of revertant colonies per plate. The result was judged as ‘Positive’, if there is a dose-dependent increase and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system. Also, the number of revertant colony should increase more than 2 times than the negative control group.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Increase in the number of revertant colonies was seen in TA100, TA1535 and WP2uvrA strains compared with negative control groups in the absence of metabolic activation system. Increase in the number of revertant colonies was seen in TA100, TA1535, WP2uvrA and TA1537 strains compared with negative control groups in the presence of metabolic activation system.

Preliminary range-finding test was performed to determine dose levels at the main test.
Preliminary range-finding test was carried out on 313, 625, 1250, 2500, 5000 µg/plate (two-fold serial dilutions). As a result of preliminary range-finding test, cytotoxicity was observed in TA98, TA100, TA1537 and WP2uvrA strains more than 313 µg/plate dose levels regardless metabolic activation system. In the case of TA1535 strain, cytotoxicity was observed at a concentration of 2500 µg/plate or higher in the absence of metabolic activation system, and cytotoxicity was observed at a concentration of 313 µg/plate or higher in the presence of metabolic activation system. In the case of TA98 strain, main test (II) was performed on 0~39 µg/plate because cytotoxicity was observed at TA98 strain more than 39 µg/plate dose levels in absence of metabolic activation system.

As a result of main test, cytotoxicity was observed in TA100, TA1537 strains more than 156 µg/plate dose levels at absence of metabolic activation system. At the concentration of 2500 µg/plate for TA1535, 313 µg/plate for WP2uvrA and 39 µg/plate and above for TA98, cytotoxicity was observed in the absence of metabolic activation system. At the concentration of 156 µg/plate and above for TA100, 313 µg/plate for TA98, TA1535, TA1537 and WP2uvrA, cytotoxicity was observed in the presence of metabolic activation system.
When compared to the negative control, significant increases in the number of revertant colonies were observed in TA100, TA1535 and WP2uvrA strains regardless presence of metabolic activation system. In the case of TA1537 strain, significant increases in the number of revertant colonies were observed in presence of metabolic activation system.

Sterility of the solvent and S9 mix were certified by the sterility test. It was counted that the number of revertant colonies of positive control groups and negative control groups were within or close to the range of the historical data. Therefore, this test was performed properly.

As the results, ESA did induce reverse mutation in this test condition.

Any other information on results incl. tables

Test results (Main study)

Metabolic activation

Dose (µg/plate)

Number of colony/plate

Base-pair substitution type

Frameshift type

TA100

Dose (µg/plate)

TA1535

Dose (µg/plate)

WP2uvrA

Dose (µg/plate)

TA98

Dose (µg/plate)

TA1537

S9 Mix (-)

0

70

64

61

0

9

6

4

0

45

43

46

0

13

13

15

0

3

6

5

Mean ± SD

66 ± 4.6

Mean ± SD

6 ± 2.5

Mean ± SD

45 ± 1.5

Mean ± SD

14 ± 1.2

Mean ± SD

5 ± 1.5

5

107

138

89

39

315

286

272

5

750

636

688

5

11

14

5

5

3

8

7

Mean ± SD

111 ± 24.8

Mean ± SD

291 ± 21.9

Mean ± SD

691 ± 57.1

Mean ± SD

10 ± 4.6

Mean ± SD

6 ± 2.6

10

73

76

109

78

418

456

350

10

854

938

1104

10

6

14

9

10

5

5

3

Mean ± SD

86 ± 20.0

Mean ± SD

408 ± 53.7

Mean ± SD

965 ± 127.2

Mean ± SD

10 ± 4.0

Mean ± SD

4 ± 1.2

20

144

126

150

156

764

794

752

20

1824

1680

1542

20*

8

13

7

20

3

5

2

Mean ± SD

140 ± 12.5

Mean ± SD

770 ± 21.6

Mean ± SD

1682 ± 141.0

Mean ± SD

9 ± 3.2

Mean ± SD

3 ± 1.5

39

175

200

232

313

924

996

984

39

2200

1836

2068

39*

P/C

P/C

P/C

39

8

2

7

Mean ± SD

202 ± 28.6

Mean ± SD

968 ± 38.6

Mean ± SD

2035 ±184.3

Mean ± SD

- ± -

Mean ± SD

6 ± 3.2

78

150

107

134

625

870

1176

1300

78

2306

2280

2784

78*

P/C

P/C

P/C

78

5

6

4

Mean ± SD

130 ± 21.7

Mean ± SD

1115 ± 221.3

Mean ± SD

2457 ± 283.8

Mean ± SD

- ± -

Mean ± SD

5 ± 1.0

156*

P/C

P/C

P/C

1250

788

1332

996

156

2894

2502

2778

156*

P/C

P/C

P/C

156*

P/C

P/C

P/C

Mean ± SD

- ± -

Mean ± SD

1039 ± 274.5

Mean ± SD

2725 ± 201.4

Mean ± SD

- ± -

Mean ± SD

- ± -

313*

P/C

P/C

P/C

2500*

0

0

0

313*

P/C

P/C

P/C

313*

0

0

0

313*

P/C

P/C

P/C

Mean ± SD

- ± -

Mean ± SD

0 ± 0

Mean ± SD

- ± -

Mean ± SD

0 ± 0

Mean ± SD

- ± -

S9 Mix (+)

0

100

124

137

0

8

7

8

0

75

85

70

0

27

22

38

0

4

7

3

Mean ± SD

120 ± 18.8

Mean ± SD

8 ± 0.6

Mean ± SD

77 ± 7.6

Mean ± SD

29 ± 8.2

Mean ± SD

5 ± 2.1

5

324

354

370

5

812

930

960

5

326

327

328

5

26

33

38

5

5

7

7

Mean ± SD

349 ± 23.4

Mean ± SD

901 ± 78.2

Mean ± SD

327 ± 1.0

Mean ± SD

32 ± 6.0

Mean ± SD

6 ± 1.2

10

620

498

712

10

1106

1118

1110

10

1308

1340

1284

10

37

41

45

10

3

6

3

Mean ± SD

610 ± 107.3

Mean ± SD

1111 ± 6.1

Mean ± SD

1311 ± 28.1

Mean ± SD

41 ± 4.0

Mean ± SD

4 ± 1.7

20

888

734

688

20

1792

1728

1420

20

1960

2272

2040

20

48

61

71

20

7

4

7

Mean ± SD

770 ± 104.7

Mean ± SD

1647 ± 198.9

Mean ± SD

2091 ± 162.1

Mean ± SD

60 ± 11.5

Mean ± SD

6 ± 1.7

39

852

860

854

39

2104

2000

1740

39

3044

3624

3048

39

45

58

62

39

15

15

10

Mean ± SD

855 ± 4.2

Mean ± SD

1948 ±187.5

Mean ± SD

3238 ± 333.7

Mean ± SD

55 ± 8.9

Mean ± SD

13 ± 2.9

78

407

329

443

78

1680

1792

1816

78

2576

3112

2880

78

43

56

47

78

18

13

20

Mean ± SD

393 ± 58.3

Mean ± SD

1763 ± 72.6

Mean ± SD

2856 ± 268.8

Mean ± SD

49 ± 6.7

Mean ± SD

17 ± 3.6

156*

P/C

P/C

P/C

156

644

1116

1324

156

3144

2884

3196

156*

5

5

7

156

5

3

6

Mean ± SD

- ± -

Mean ± SD

1028 ± 348.4

Mean ± SD

3075 ±167.2

Mean ± SD

6 ± 1.2

Mean ± SD

5 ± 1.5

313*

P/C

P/C

P/C

313*

P/C

P/C

P/C

313*

P/C

P/C

P/C

313*

P/C

P/C

P/C

313*

0

0

0

Mean ± SD

- ± -

Mean ± SD

- ± -

Mean ± SD

- ± -

Mean ± SD

- ± -

Mean ± SD

0 ± 0

Positive controls

S9 Mix (-)

Positive controls

AF-2

Positive controls

NaN3

Positive controls

AF-2

Positive controls

AF-2

Positive controls

9-AA

Dose (µg/plate)

0.01

Dose (µg/plate)

0.5

Dose (µg/plate)

0.01

Dose (µg/plate)

0.1

Dose (µg/plate)

80

Number of colony

499

401

410

Number of colony

237

198

225

Number of colony

280

228

223

Number of colony

501

345

396

Number of colony

2464

2260

2584

Mean ± SD

437 ± 54.2

Mean ± SD

220 ±20.0

Mean ± SD

244 ± 31.6

Mean ± SD

414 ± 79.5

Mean ± SD

2436 ± 163.8

S9 Mix (+)

Positive controls

2-AA

Positive controls

2-AA

Positive controls

2-AA

Positive controls

2-AA

Positive controls

2-AA

Dose (µg/plate)

1

Dose (µg/plate)

2

Dose (µg/plate)

10

Dose (µg/plate)

1

Dose (µg/plate)

2

Number of colony

448

592

626

Number of colony

207

201

251

Number of colony

280

261

264

Number of colony

246

234

259

Number of colony

191

155

167

Mean ± SD

555 ± 94.5

Mean ± SD

220 ± 27.3

Mean ± SD

268 ± 10.2

Mean ± SD

246 ± 12.5

Mean ± SD

171 ± 18.3

* cytotoxicity

Test results (Main study Ⅱ)

Metabolic activation

Dose (µg/plate)

 

 

Number of colony/plate

Base-pair substitution type

Frameshift type

 

TA100

TA1535

WP2uvrA

TA98

TA1537

 

S9 Mix (-)

0

 

 

 

28

22

24

 

 

Mean ± SD

 

 

 

25 ± 3.1

 

 

0.6

 

 

 

26

27

24

 

 

Mean ± SD

 

 

 

26 ± 1.5

 

 

1.25

 

 

 

23

29

22

 

 

Mean ± SD

 

 

 

25 ± 3.8

 

 

2.5

 

 

 

27

24

30

 

 

Mean ± SD

 

 

 

27 ± 3.0

 

 

5

 

 

 

28

28

34

 

 

Mean ± SD

 

 

 

30 ± 3.5

 

 

10

 

 

 

30

33

33

 

 

Mean ± SD

 

 

 

32 ± 1.7

 

 

20

 

 

 

21

25

30

 

 

Mean ± SD

 

 

 

25 ± 4.5

 

 

39*

 

 

 

08

9

8

 

 

Mean ± SD

 

 

 

8 ± 0.6

 

 

S9 Mix (+)

0

 

 

 

24

27

21

 

 

Mean ± SD

 

 

 

24 ± 3.0

 

 

5

 

 

 

28

27

37

 

 

Mean ± SD

 

 

 

31 ± 5.5

 

 

10

 

 

 

25

32

33

 

 

Mean ± SD

 

 

 

30 ± 4.4

 

 

20

 

 

 

40

43

33

 

 

Mean ± SD

 

 

 

36 ± 5.1

 

 

39

 

 

 

45

37

46

 

 

Mean ± SD

 

 

 

43 ± 4.9

 

 

78

 

 

 

46

36

57

 

 

Mean ± SD

 

 

 

46 ± 10.5

 

 

156

 

 

 

30

22

20

 

 

Mean ± SD

 

 

 

24 ± 5.3

 

 

313*

 

 

 

P/C

P/C

P/C

 

 

Mean ± SD

 

 

 

- ± -

 

 

Positive controls

S9 Mix (-)

Positive controls

AF-2

NaN3

AF-2

AF-2

9-AA

 

Dose (µg/plate)

0.01

0.5

0.01

0.1

80

 

Number of colony

 

 

 

490

466

452

 

 

Mean ± SD

 

 

 

469 ± 19.2

 

 

S9 Mix (+)

Positive controls

2-AA

2-AA

2-AA

2-AA

2-AA

 

Dose (µg/plate)

1

2

10

1

2

 

Number of colony

 

 

 

564

540

572

 

 

Mean ± SD

 

 

 

559 ± 16.7

 

 

* cytotoxicity

Applicant's summary and conclusion

Conclusions:
When compared to the negative control, significant increases in the number of revertant colonies were observed in TA100, TA1535 and WP2uvrA strains regardless of presence of metabolic activation system. In the case of TA1537 strain, significant increases in the number of revertant colonies were observed in presence of metabolic activation system.
ESA did induce reverse mutation in under the conditions of this test.
Executive summary:

Summary

To evaluate mutagenic potential of ESA in bacteria, bacterial reverse mutation study was performed with preincubation method using four histidine-requiring strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and one tryptophan-requiring strain of Escherichia coli (WP2uvrA) in the presence and absence of metabolic activation system (S9 mix). The test substance was dissolved 1,4-dioxane and then it was applied to the test strains at the dose levels of followings.

•In the absence of metabolic activation system (S9 mix(-))

TA100, TA1537, WP2uvrA: 0, 5, 10, 20, 39, 78, 156, 313 ㎍/plate

TA1535: 0, 39, 78, 156, 313, 625, 1250, 2500 ㎍/plate

TA98: 0, 0.6, 1.25, 2.5, 5, 10, 20, 39 ㎍/plate

•In the presence of metabolic activation system (S9 mix(+))

0, 5, 10, 20, 39, 78, 156, 313 ㎍/plate

Increase in the number of revertant colonies was seen in TA100, TA1535 and WP2uvrA strains compared with negative control groups in the absence of metabolic activation system. Increase in the number of revertant colonies was seen in TA100, TA1535, WP2uvrA and TA1537 strains compared with negative control groups in the presence of metabolic activation system.

Considering these findings, it is concluded that the test substance ESA does induce reverse mutation in TA100, TA1535, WP2uvrA and TA1537 strains which were used in this study.