Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro gene mutation study in bacteria (OECD 471):

The mutagenic potential of the test substance was assessed in an Ames test 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).

An 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. An 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 mutations in TA100, TA1535, WP2uvrA and TA1537 strains which were used in this study.

 

in vitro chromosome aberration study in mammalian cells (OECD 473):

An in vitro mammalian chromosome aberration study was used to assess clastogenic potential of the test substance. The test was carried out using Chinese Hamster Ovary (CHO-K1) cells in the absence (-S9 mix) and presence (+S9 mix) of metabolic activation system. The test substance did not induce a statistically significant increase in the number of cells with chromosome aberrations at all dose levels when compared with the negative control in the absence of S9 mix (24 hours continuous treatment group and 6 hours treatment and 18 hours recovery group). The test substance did not induce a statistically significant in the number of cells with polyploidy and endoreduplication when compared with negative control group in the absence of S9 mix (24 hours continuous treatment group and 6 hours treatment and 18 hours recovery group).

The test substance was not observed a statistically significant increase in the number of cells with chromosome aberrations at all dose levels when compared with the negative control in the presence of S9 mix (6 hours treatment and 18 hours recovery group). The test substance did not induce a statistically significant in the number of cells with polyploidy and endoreduplication when compared with negative control group in the presence of S9 mix (6 hours treatment and 18 hours recovery group).

Based on the above result, it was decided that the test substance (ESA) did not induce chromosome aberrations in Chinese Hamster Ovary (CHO-K1) cells under the conditions of this study.

 

in vitro gene mutation study in mammalian cells (Mouse Lymphoma assay, OECD 490):This assay is able to detect both mutagenic and clastogenic effects by examination of the size of mutant colonies formed.

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at ten dose levels in duplicate, together with vehicle (R0 medium), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation.

The test item induced dose-related increases in mutant frequency in all three of the exposure groups. The increases in mutant frequency markedly exceeded the GEF at several dose levels, there were marked increases in absolute numbers of mutant colonies, and the increases in mutant frequency were predominantly due to small colonies (indicative of clastogenic activity). The responses observed were therefore considered to be of toxicological significance.

The test item induced toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells that markedly exceeded the GEF, consequently it is considered to be mutagenic in this assay.

Link to relevant study records

Referenceopen allclose all

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
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
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

Target gene:
histidine and tryptophan
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.
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.
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.

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

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation: 30 November 2010, Study termination: 21 March 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Korea
Type of assay:
other: In vitro Mammalian chromosome aberration test
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 %
Storage condition : Recommended temperature: 2-8ºC
Stability : Stable in recommended storage condition
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Obtained from American Type Culture Collection (ATCC).

Justification for selection of cell line: It was well-known that Chinese Hamster Ovary cell (CHO-K1) has the high
susceptibility to chemicals and much baseline data in chromosome aberration studies.

Condition of cultivation:
Culture medium : F-12 Nutrient Mixture (GIBCO, Lot No. 862554) with 10% Fetal Bovine Serum (Hyclone, Lot No. DVC0359).
Culture condition :The cells were incubated at 5% CO2, 37ºC and subcultured every 3-4 days.
Doubling time : about 15 hours
Modal chromosome number : 22
Storage condition : The cells were suspended in culture media with 10% Dimethylsulfoxide (DMSO) and stored in cryovial at -196ºC (liquid nitrogen).
Cytokinesis block (if used):
0.2 µg/ml of colcemid
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat S9 mix
Test concentrations with justification for top dose:
Cytotoxicity test (preliminary range-finding test): 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM
Chromosome aberration test (main test):
Absence metabolic activation system (-S9 mix, 24 hours treatment) :
0.039, 0.078, 0.156 mM
Absence metabolic activation system (-S9 mix, 6 hours treatment and 18 hours recovery) :
0.039, 0.078, 0.156 mM
Presence metabolic activation system (-S9 mix, 6 hours treatment and 18 hours recovery) :
0.039, 0.078, 0.156 mM
Vehicle / solvent:
1,4-Dioxane. The test substance showed high solubility in DMSO but formed crystals as time passes. For this reason, it was judged that DMSO was not suitable for use in the test. As a result of re-solubility 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 as vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
0.04 µg/µl
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
10 µg/µl
Positive control substance:
other: Cytophosphamide
Remarks:
With metaboloc activation
Details on test system and experimental conditions:
Preparation of test substance
The adequate amount of test substance was weighed and dissolved in 1,4-Dioxane and serially diluted with 1,4-Dioxane.

Cytotoxicity test (preliminary range-finding test)
Cytotoxicity test (preliminary range-finding test) was performed to determine the test substance treatment concentration for the chromosome aberration test (main test). 10 mM was selected the highest treatment concentration and then the test substance was treated to 8 concentrations with 2-fold (0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM). The number of viable cells per culture dish was calculated using hemacytometer. Relative cell count (RCC) was calculated according to the following formula :
RCC (Relative Cell Count) = (No. of treated cells / No. of control cells) × 100

Chromosome aberration test (main test)
The chromosome aberration test (main test) was conducted according to cytotoxicity test (preliminary range-finding test) result. Two replicate cultures were used for each concentration level.
1) Absence of metabolic activation system (6 hours treatment and 24 hours
treatment)
2×10^4 ~ 4×10^4 cells/ml were seeded in 60 mm diameter tissue culture dishes and incubated for 3 days. When the test substance (ESA) was treated, the existing culture medium was removed from the culture dish and then 4.95 ml of pre-warmed fresh medium and 0.05 ml of test substance solution were added to each dish which became 5.00 ml. In accordance with the incubating condition, each dish was incubated for 6 hours or 24 hours after treatment.
In case of 6 hours treatment group, the cells were exposed to the test substance for 6 hours and then the treated culture medium was removed and the cells were rinsed with PBS (Ca2+ & Mg2+ free Dulbecco's phosphate buffered saline) at one time. 5 ml of pre-warmed fresh culture medium was added and the cells were incubated for additional 18 hours before metaphase-cells harvest.
2) Presence of metabolic activation system (6 hours treatment)
2×10^4 ~ 4×10^4 cells/ml were seeded in 60 mm diameter tissue culture dishes and incubated for 3 days. When the test substance (ESA) was treated, the existing culture medium was removed from the culture dish and then 4.45 ml of pre-warmed fresh medium, 0.05 ml of test substance solution and 0.50 ml of S9 mix were added to each dish which became 5.00 ml. The cells were exposed to the test substance for 6 hours and then the treated culture medium was removed and the cells were rinsed with PBS (Ca2+ & Mg2+ free Dulbecco's phosphate buffered saline) at one time. 5 ml of pre-warmed fresh culture medium was added and the cells were incubated for additional 18 hours before metaphase-cells harvest.

3) Slide preparation
Approximately 22 hours after treatment, 0.2 µg/ml of colcemid (GIBCO, 798502) was added to each plate which was incubated for additional 2 hours. For the cell harvest, the medium containing mitotic cells was centrifuged at 1,000 rpm for 5 minutes. After discard supernatant, the cell pellets were resuspended in 75 mM potassium chloride (KCl) solution and incubated at 37ºC incubator for 20 minutes. The cells were fixed 3 times with Carnoy's fixative solution (acetic acid: methanol = 1:3) for the slide preparation. The slides were stained with 5% Giemsa (Merck, HX944140) solution for 5 minutes and observed microscopically. Two slides samples were prepared from each plate.
Evaluation criteria:
The number of aberrant metaphases, excluding gaps (according to OECD TG 473), and number of (Polyploid + Endoreduplication) were analyzed. Firstly, The metaphase cells were distinguished between normal metaphase and aberrant metaphase. Then the statistical analysis were performed for the frequency of structural aberrations and the frequency of numerical aberrations.
Statistics:
The statistical analysis were performed with SPSS 12.1K program. The result of statistical evaluation was regarded as significant when the p value was less than 0.05.
1) Comparison between negative control and treated groups : Chi-square test.
2) Linear logistic regression test was performed for dose-response (In case of 1, p<0.05).
3) Comparison between negative and positive control groups : Chi-square test
4) Study results were judged as positive if there was a dose-related and statistically significant increase in the number of aberrant metaphases when compared with negative control group. Also these data should be shown reproducibility.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Cytotoxicity test (preliminary range-finding test)
In order to decide the main test treatment concentration, the cytotoxicity test (preliminary range-finding test) was conducted for 8 dose levels (0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM). After viable cells counting, Relative Cell Count (RCC) was calculated as the number of negative control group cells was set 100 %.
In the absence of metabolic activation system (24 hours treatment group), RCCs were 28.86% and 44.97% for 0.313 mM and 0.156 mM. In the absence of metabolic activation system (6 hours treatment group), RCCs were 29.93% and 45.58% for 0.313 mM and 0.156 mM. Considering RCCs, the highest concentration of main test was selected 0.156 mM in the absence of metabolic activation system (24 hours treatment group and 6 hours treatment and 18 hours recovery group) In the presence of metabolic activation system (6 hours treatment group), RCCs were 31.75% and 47.62% for 0.313 mM and 0.156 mM. Considering RCCs, the highest concentration of main test was selected 0.156 mM in the presence of metabolic activation system (6 hours treatment and 18 hours recovery group).
On the basis of cytotoxicity test (preliminary range-finding test) result, the main test was conducted at the 3 concentration of 2 fold after determination the highest concentration. The details of concentrations are following :
Absence metabolic activation system (-S9 mix, 24 hours treatment) :
0.039, 0.078, 0.156 mM
Absence metabolic activation system (-S9 mix, 6 hours treatment and 18 hours recovery) :
0.039, 0.078, 0.156 mM
Presence metabolic activation system (-S9 mix, 6 hours treatment and 18 hours recovery) :
0.039, 0.078, 0.156 mM

Chromosome aberration (main test)
As a result of main test, the frequency of chromosome aberration is 0.5, 1.0, 2.5 and 2.0 at 0, 0.039, 0.078 and 0.156 mM in the absence of metabolic activation system (24 hours treatment group). In the absence of metabolic activation system (6 hours treatment and 18 hour recovery group), the frequency of chromosome aberration is 0.5, 1.0, 2.0 and 2.5 at 0, 0.039, 0.078 and 0.156 mM. In the presence of metabolic activation system (6 hours treatment and 18 hour recovery group), the frequency of chromosome aberration is 0.5, 1.0, 1.5 and 0.5 at 0, 0.039, 0.078 and 0.156 mM.
In the presence and absence of metabolic activation system, the test substance caused no statistically significant in the number of cells with polyploidy and endoreduplication,
when compared with negative control group. Based on the above result, the test substance caused no statistically significant increase in the number of cells with chromosome aberration at all dose levels when compared with negative control group.

The number of cells with chromosome aberrations in the absence of S9 mix.

Exposure *)

S9 mix

Dose (mM)

No. of total

chromosome

aberrations (Mean)

No. of cells with

chromosome

aberrations (Mean)

PP+ER

(Mean)

(-)Gap

(+)Gap

(-)Gap

(+)Gap

24-0

-

Negative control

0.5

1

0.5

1

0

-

0.039

1

1

1

1

0

-

0.078

2.5

2.5

2.5

2.5

0

-

0.156

2

2

2

2

0

-

MMC (0.04)

32

33.5

29*

30.5

0

6-18

-

Negative control

0.5

0.5

0.5

0.5

0

-

0.039

1

1.5

1

1.5

0

-

0.078

2

2

2

2

0

-

0.156

2.5

2.5

2.5

2.5

0

-

MMC (0.04)

24

25.5

23.5*

25

0

* Significantly different from the negative control at p<0.05

Test substance :ESA

a) Treatment time-recovery time

MMC : Mitomycin C (0.04μg/ml)

PP : Polyploidy

ER : Endoreduplication

 

The number of cells with chromosome aberrations in the presence of S9 mix.

Exposure *)

S9 mix

Dose (mM)

No. of total

chromosome

aberrations (Mean)

No. of cells with

chromosome

aberrations (Mean)

PP+ER

(Mean)

(-)Gap

(+)Gap

(-)Gap

(+)Gap

6-18

+

Negative control

0.5

0.5

0.5

0.5

0

+

0.039

1

1.5

1

1.5

0

+

0.078

1.5

2

1.5

2

0

+

0.156

0.5

0.5

0.5

0.5

0

+

CPA (10)

26.5

27.5

26.5*

27.5

0

* Significantly different from the negative control at p<0.05

Test substance : ESA

a) Treatment time-recovery time

CPA : CyclophosphamideH2O (10μg/ml)

PP : Polyploidy

ER : Endoreduplication

Conclusions:
It is concluded that the test substance ESA is not capable of inducing chromosome aberration in cultured CHO-K1 cells under the condition of this study.
Executive summary:

This study was performed the chromosome aberration test to assess the genotoxicity for ESA using Chinese Hamster Ovary (CHO-K1) cells in the absence (-S9 mix) and presence (+S9 mix) of metabolic activation system. Test substance was dissolved in 1,4-Dioxane and then dilute with 1,4-Dioxane.

The cytotoxicity test (preliminary range-finding test) was conducted for 8 dose levels (0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM) to decide the main test treatment concentration. On the basis of the cytotoxicity test (preliminary range-finding test) result, the treatment times and concentrations were selected for the main test as follows.

Absence metabolic activation system (-S9 mix, 24 hours treatment) :

0.039, 0.078, 0.156 mM

Absence metabolic activation system (-S9 mix, 6 hours treatment and 18 hours recovery) :

0.039, 0.078, 0.156 mM

Presence metabolic activation system (+S9 mix, 6 hours treatment and 18 hours recovery) :

0.039, 0.078, 0.156 mM

As a result of the main test, the test substance did not induce a statistically significant increase in the number of cells with chromosome aberrations at all dose levels when compared with the negative control in the absence of S9 mix (24 hours continuous treatment group and 6 hours treatment and 18 hours recovery group). The test substance did not induce a statistically significant in the number of cells with polyploidy and endoreduplication when compared with negative control group in the absence of S9 mix (24 hours continuous treatment group and 6 hours treatment and 18 hours recovery group).

The test substance was not observed a statistically significant increase in the number of cells with chromosome aberrations at all dose levels when compared with the negative control in the presence of S9 mix (6 hours treatment and 18 hours recovery group). The test substance did not induce a statistically significant in the number of cells with polyploidy and endoreduplication when compared with negative control group in the presence of S9 mix (6 hours treatment and 18 hours recovery group).

Based on the above result, it was decided that the test substance (ESA) did not induce chromosome aberration in Chinese Hamster Ovary (CHO-K1) cells under the condition of this study.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 01 March 2018; Experimental completion date: 05 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Identification: ESA (1,3,2-Dioxathiolane, 2,2-dioxide)
Batch Number: ESA-170926-160
Purity: 100%
Physical State/Appearance: White solid
Expiry Date: 25 June 2018
Storage Conditions: Approximately -20 ºC, in the dark
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e.
THMG without Methotrexate) before being returned to R10 medium.
Metabolic activation:
with and without
Metabolic activation system:
Rat S9
Test concentrations with justification for top dose:
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.

The dose range used in the preliminary toxicity test was 4.84 to 1240 µg/mL for all three of the exposure groups.

The concentrations used in the main experiment were based on the results in the preliminary toxicity test.
Ten dose levels of the test item (1.25 to 40 µg/mL in the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.31 to 30 µg/mL in the 24-hour exposure group in the absence of metabolic activation.
Vehicle / solvent:
Following solubility checks performed in-house, the test item was accurately weighed and formulated in R0 medium prior to serial dilutions being prepared.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
400 µg/mL and 150 µg/mL, respectively, was used as the positive control in the 4-hour and 24-hour exposure groups.
Positive control substance:
ethylmethanesulphonate
Remarks:
absence of metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
1.5 µg/mL
Positive control substance:
cyclophosphamide
Remarks:
presence of metabolic activation
Details on test system and experimental conditions:
Preliminary Toxicity Test

A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 4.84 to 1240 µg/mL for all three of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.

The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiment. Maximum dose levels were selected using the following criteria:

i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB*) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.

ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.

iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival.

* UVCB = Substances of Unknown or Variable composition, Complex reaction products or Biological materials.


This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).


Mutagenicity Test

Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at ten dose levels of the test item (1.25 to 40 µg/mL in the 4-hour exposure groups in both the absence and presence of metabolic activation, and
0.31 to 30 µg/mL in the 24-hour exposure group in the absence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).

The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Assessments

Measurement of Survival, Viability and Mutant Frequency

At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL
5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data, a Relative Total Growth (RTG) value.


Plate Scoring

Microtitre plates were scored using a magnifying mirror box after twelve days incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolized to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.


Calculation of Percentage Relative Suspension Growth (%RSG)

The cell counts obtained immediately post exposure and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.

4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)

24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48 hour cell count/2)

Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count

%RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100


Calculation of Day 2 Viability (%V)

Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.


P(0) = number of negative wells / total wells plated

%V = (- ln P(0) x 100) / number of cells/well


Calculation of Relative Total Growth (RTG)

For each culture, the relative cloning efficiency, RCE, was calculated:

RCE = %V / Mean Solvent Control %V

Finally, for each culture RTG is calculated:

RTG = (RCE x RSG)/100


Calculation of Mutation Frequency (MF)

MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.

The experimental data was analyzed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear-trend events.

Evaluation criteria:
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
Species / strain:
mouse lymphoma L5178Y cells
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
Additional information on results:
Preliminary Cytotoxicity Test
There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all three of the exposure groups when compared to the concurrent vehicle control. No precipitate of the test item was observed in any of the three exposure groups at the end of the exposure periods. Therefore, following the recommendations of the OECD 490 guideline, the maximum dose levels in the Mutagenicity Test were limited by test item-induced toxicity.

Mutagenicity Test
As was seen previously, there was evidence of marked dose-related toxicity in cells treated with the test item in all three of the exposure groups, as indicated by the %RSG and RTG values. Based on the %RSG and / or RTG values observed, optimum levels of toxicity were considered to have been achieved in all three of the exposure groups. There was evidence of reductions in viability (%V) in all three of the exposure groups, indicating that residual toxicity had occurred. No precipitate of the test item was observed in any of the three exposure groups at the end of the exposure periods. The excessive toxicity observed at 40 µg/mL in the 4-hour exposure group in the absence of metabolic activation resulted in this dose level not being plated for viability or 5-TFT resistance. The toxicity observed at 35 µg/mL in the 4-hour exposure group in the absence of metabolic activation, and at and above 35 µg/mL in the 4-hour exposure group in the presence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, these dose levels were excluded from the statistical analysis. Acceptable levels of toxicity were seen with the positive control substances.

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

The test item induced dose-related increases in mutant frequency in all three of the exposure groups. The increases in mutant frequency markedly exceeded the GEF at several dose levels, there were marked increases in absolute numbers of mutant colonies, and the increases in mutant frequency were predominantly due to small colonies (indicative of clastogenic activity). The responses observed were therefore considered to be of toxicological significance.


Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 4.84 to 1240 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

 

Dose(mg/mL)

% RSG (-S9)

4-Hour Exposure

% RSG (+S9)

4-Hour Exposure

% RSG (-S9)

24-Hour Exposure

0

100

100

100

4.84

79

91

80

9.69

56

70

65

19.38

30

29

19

38.75

2

2

0

77.5

1

1

0

155

1

1

0

310

1

1

0

620

2

3

0

1240

6

4

0

Mutagenicity Test

Main Experiment

Concentration(µg/mL)

4-Hours-S9

Concentration(µg/mL)

4-Hours+S9

%RSG

RTG

MF§

%RSG

RTG

MF§

0

 

100

1.00

151.16

0

 

100

1.00

183.21

1.25

Ø

90

 

 

1.25

Ø

102

 

 

2.5

Ø

87

 

 

2.5

Ø

95

 

 

5

 

77

0.69

224.21

5

Ø

87

 

 

10

 

67

0.74

383.96

10

 

76

0.72

228.91

15

 

61

0.55

473.76

15

 

72

0.70

412.24

20

 

45

0.34

971.91

20

 

53

0.42

654.50

25

 

33

0.25

1066.74

25

 

39

0.27

992.93

30

 

24

0.14

1014.28

30

 

31

0.22

1058.31

35

X

13

0.02

3349.01

35

X

20

0.03

1964.31

40

Ø

4

 

 

40

X

8

0.01

2996.90

MF threshold for a positive response = 277.16

MF threshold for a positive response = 309.21

Positive control

Positive control

EMS 400

 

58

 

0.72

 

792.66

CP 1.5

 

74

 

0.48

 

1028.90

 

 

 

Concentration(µg/mL)

24-Hours-S9

%RSG

RTG

MF§

0

 

100

1.00

193.22

0.31

Ø

95

 

 

0.63

Ø

94

 

 

1.25

Ø

91

 

 

2.5

 

102

1.01

187.64

5

 

86

0.78

354.22

10

 

62

0.65

368.57

15

 

53

0.59

701.54

20

 

46

0.52

1119.24

25

 

29

0.36

1675.25

30

 

14

0.22

2212.67

MF threshold for a positive response = 319.22

Positive control

EMS

150

 

38

 

0.36

 

2131.98

Cell and 96-Well Plate Counts: Mutagenicity Test (-S9) 4-HourExposure

Concentration(µg/mL)

 

 

 

0h

Cell counts $

 

 

24h         48h

Viability § after day 2

2 cells/well

Resistant mutants § after day 2

2000 cells/well

0

A

9.65

7.58

7.50

72

68

76

78

21

17

21

19

 

B

9.06

7.54

7.45

78

71

78

76

17

17

19

25

1.25

A

9.69

8.13

6.12

NP

NP

 

 

NP

NP

 

 

 

B

8.42

7.96

6.95

NP

NP

 

 

NP

NP

 

 

2.5

A

9.29

6.69

7.55

NP

NP

 

 

NP

NP

 

 

 

B

9.21

6.72

7.34

NP

NP

 

 

NP

NP

 

 

5

A

9.08

6.21

7.75

65

66

 

 

21

21

 

 

 

B

8.37

7.28

6.16

76

77

 

 

32

26

 

 

10

A

7.93

7.18

6.47

78

78

 

 

45

30

 

 

 

B

8.43

6.20

6.54

76

79

 

 

53

53

 

 

15

A

7.63

6.87

6.50

72

74

 

 

48

47

 

 

 

B

7.86

5.75

6.64

71

67

 

 

45

41

 

 

20

A

8.11

5.51

5.71

63

69

 

 

66

58

 

 

 

B

7.94

4.57

6.13

64

64

 

 

69

63

 

 

25

A

7.48

4.03

5.42

71

67

 

 

65

65

 

 

 

B

7.80

3.50

6.63

60

63

 

 

75

65

 

 

30

A

7.27

3.02

5.54

68

65

 

 

78

70

 

 

 

B

7.75

2.93

5.60(2.93)

58

58

 

 

48

55

 

 

X  35

A

6.64

2.58

4.50(2.58)

25

19

 

 

63

62

 

 

 

B

7.45

2.41

3.42(2.41)

29

17

 

 

56

46

 

 

40

A

6.99

1.60

2.23(1.60)

NP

NP

 

 

NP

NP

 

 

 

B

7.43

1.40

1.96(1.40)

NP

NP

 

 

NP

NP

 

 

Positive control EMS (µg/mL)

400

A

8.65

5.65

5.85

86

81

 

 

75

76

 

 

 

B

9.16

5.10

6.99

80

78

 

 

75

71

 

 

Summary Analysis: Mutagenicity Test (-S9) 4-HourExposure 

Concentration(µg/mL)

SG

%RSG

%V

RTG

MF§

0

 

14.13

100

75.11

1.00

151.16

1.25

Ø

13.14

90

 

 

 

2.5

Ø

12.48

87

 

 

 

5

 

11.73

77

67.27

0.69

224.21

10

 

10.88

67

83.01

0.74

383.96

15

 

10.36

61

67.27

0.55

473.76

20

 

7.46

45

56.52

0.34

971.91

25

 

5.67

33

56.92

0.25

1066.74

30

 

4.14

24

52.27

0.14

1014.28

35

X

2.47

13

13.35

0.02

3349.01

40

Ø

0.79

4

 

 

 

Positive control EMS

Concentration(µg/mL)

SG

%RSG

%V

RTG

MF§

400

8.63

58

93.66

0.72

792.66

 

GEF =126, therefore MF threshold for a positive response = 277.16

Large and Small Colonies Analysis: Mutagenicity Test(-S9)4-HourExposure

Concentration(µg/mL)

Viability # after day 2

Small colonies # after day 2

Large colonies # after day 2

0

A

72

68

76

78

10

8

11

9

11

9

10

10

 

B

78

71

78

76

9

7

10

10

8

10

9

15

5

A

65

66

 

 

10

12

 

 

11

9

 

 

 

B

76

77

 

 

14

19

 

 

18

7

 

 

10

A

78

78

 

 

28

16

 

 

17

14

 

 

 

B

76

79

 

 

38

35

 

 

15

18

 

 

15

A

72

74

 

 

34

35

 

 

14

12

 

 

 

B

71

67

 

 

32

26

 

 

13

15

 

 

20

A

63

69

 

 

44

42

 

 

22

16

 

 

 

B

64

64

 

 

43

42

 

 

26

21

 

 

25

A

71

67

 

 

49

48

 

 

16

17

 

 

 

B

60

63

 

 

47

48

 

 

28

17

 

 

30

A

68

65

 

 

60

55

 

 

18

15

 

 

 

B

58

58

 

 

38

43

 

 

10

12

 

 

X  35

A

25

19

 

 

50

50

 

 

13

12

 

 

 

B

29

17

 

 

43

40

 

 

13

6

 

 

400 EMS

A

86

81

 

 

38

41

 

 

37

35

 

 

 

B

80

78

 

 

37

36

 

 

38

35

 

 

Mutation frequencies

Concentration(µg/mL)

 

 

 

 

Viable

Yv         Nv

Small colonies

Large colonies

Proportionsmall colony mutants

Mutants Ym        Nm

 

MF§

Mutants Ym        Nm

 

MF§

0

171

768

694

768

67.4

686

768

75.2

0.47

5

100

384

329

384

114.9

339

384

92.6

0.55

10

73

384

267

384

218.9

320

384

109.8

0.65

15

100

384

257

384

298.5

330

384

112.6

0.70

20

124

384

213

384

521.4

299

384

221.3

0.67

25

123

384

192

384

608.8

306

384

199.4

0.71

30

135

384

188

384

683.2

329

384

147.9

0.78

35

294

384

201

384

2423.9

340

384

455.7

0.81

400 EMS

59

384

232

384

269.0

239

384

253.2

0.51

Cell and 96-Well Plate Counts: Mutagenicity Test(+S9)4-HourExposure 

Concentration(µg/mL)

 

 

 

0h

Cell counts $

 

 

24h            48h

Viability § after day 2

2 cells/well

Resistant mutants § after day 2

2000 cells/well

0

A

8.67

7.78

7.28

73

70

77

71

27

18

24

23

 

B

8.60

7.13

8.82

65

72

73

71

22

17

17

22

1.25

A

9.50

7.07

7.98

NP

NP

 

 

NP

NP

 

 

 

B

8.69

6.90

8.66

NP

NP

 

 

NP

NP

 

 

2.5

A

8.23

7.70

7.06

NP

NP

 

 

NP

NP

 

 

 

B

8.76

7.57

8.12

NP

NP

 

 

NP

NP

 

 

5

A

8.61

7.38

7.54

NP

NP

 

 

NP

NP

 

 

 

B

8.65

6.33

7.62

NP

NP

 

 

NP

NP

 

 

10

A

7.69

7.12

6.74

71

70

 

 

22

34

 

 

 

B

7.47

7.20

7.77

69

68

 

 

21

21

 

 

15

A

8.64

5.68

6.98

75

68

 

 

35

30

 

 

 

B

8.20

5.88

8.41

70

66

 

 

45

49

 

 

20

A

7.95

5.07

7.46

65

64

 

 

49

35

 

 

 

B

6.41

5.62

6.97

59

66

 

 

54

57

 

 

25

A

7.23

4.51

5.75

61

58

 

 

59

67

 

 

 

B

7.37

3.97

7.22

61

55

 

 

58

50

 

 

30

A

7.34

3.56

5.35

56

60

 

 

61

56

 

 

 

B

7.66

3.67

6.67

55

63

 

 

69

56

 

 

X  35

A

7.81

2.29

4.76(2.29)

20

19

 

 

36

55

 

 

 

B

7.43

2.72

6.08(2.72)

27

17

 

 

23

32

 

 

X  40

A

6.35

1.71

3.47(1.71)

11

10

 

 

27

24

 

 

 

B

8.31

1.54

3.12(1.54)

7

10

 

 

28

24

 

 

Positive control CP (µg/mL)

1.5

A

8.40

6.09

7.11

52

58

 

 

57

63

 

 

 

B

9.56

6.17

6.83

60

55

 

 

56

53

 

 


 

Summary Analysis: Mutagenicity Test (+S9) 4-HourExposure

Concentration(µg/mL)

SG

%RSG

%V

RTG

MF§

0

 

15.00

100

68.28

1.00

183.21

1.25

Ø

14.53

102

 

 

 

2.5

Ø

14.49

95

 

 

 

5

Ø

12.99

87

 

 

 

10

 

12.99

76

64.36

0.72

228.91

15

 

11.12

72

64.83

0.70

412.24

20

 

9.64

53

54.16

0.42

654.50

25

 

6.87

39

47.33

0.27

992.93

30

 

5.43

31

47.00

0.22

1058.31

35

X

3.39

20

12.18

0.03

1964.31

40

X

1.34

8

5.21

0.01

2996.90

Positive control CP

Concentration(µg/mL)

SG

%RSG

%V

RTG

MF§

1.5

10.68

74

44.09

0.48

1028.90

 

GEF =126, therefore MF threshold for a positive response = 309.21


 

Large and Small Colonies Analysis: Mutagenicity Test(+S9)4-HourExposure

Concentration(µg/mL)

Viability # after day 2

Small colonies # after day 2

Large colonies # after day 2

0

A

73

70

77

71

13

7

11

12

14

11

13

11

 

B

65

72

73

71

12

8

8

10

10

9

9

12

10

A

71

70

 

 

14

17

 

 

8

17

 

 

 

B

69

68

 

 

12

9

 

 

9

12

 

 

15

A

75

68

 

 

19

18

 

 

16

12

 

 

 

B

70

66

 

 

27

36

 

 

18

13

 

 

20

A

65

64

 

 

35

31

 

 

14

4

 

 

 

B

59

66

 

 

35

39

 

 

19

18

 

 

25

A

61

58

 

 

47

49

 

 

12

18

 

 

 

B

61

55

 

 

37

29

 

 

21

21

 

 

30

A

56

60

 

 

47

37

 

 

14

19

 

 

 

B

55

63

 

 

48

37

 

 

21

19

 

 

X  35

A

20

19

 

 

28

40

 

 

8

15

 

 

 

B

27

17

 

 

15

17

 

 

8

15

 

 

X  40

A

11

10

 

 

24

19

 

 

3

5

 

 

 

B

7

10

 

 

25

18

 

 

3

6

 

 

1.5 CP

A

52

58

 

 

36

46

 

 

21

17

 

 

 

B

60

55

 

 

38

38

 

 

18

15

 

 

Mutation frequencies

Concentration(µg/mL)

 

 

 

 

Viable

Yv         Nv

Small colonies

Large colonies

Proportionsmall colony mutants

Mutants Ym        Nm

 

MF§

Mutants Ym        Nm

 

MF§

0

196

768

687

768

81.6

679

768

90.2

0.48

10

106

384

332

384

113.0

338

384

99.1

0.53

15

105

384

284

384

232.6

325

384

128.6

0.63

20

130

384

244

384

418.7

329

384

142.7

0.72

25

149

384

222

384

578.8

312

384

219.3

0.69

30

150

384

215

384

617.0

311

384

224.3

0.70

35

301

384

284

384

1238.7

338

384

523.9

0.68

40

346

384

298

384

2433.2

367

384

434.5

0.83

1.5 CP

159

384

226

384

601.2

313

384

231.9

0.69


 

Cell and 96-Well Plate Counts: Mutagenicity Test(-S9)24-HourExposure 

Concentration(µg/mL)

Cell counts $

 

 

0h         24h         48h

Viability § after day 2

2 cells/well

Resistant mutants § after day 2

2000 cells/well

0

A

9.80

8.05

6.67

78

75

71

73

26

19

24

27

 

B

9.75

9.20

6.01

72

72

74

75

19

24

25

25

0.31

A

9.52

9.71

5.41

NP

NP

 

 

NP

NP

 

 

 

B

10.03

8.67

5.84

NP

NP

 

 

NP

NP

 

 

0.63

A

10.03

9.26

4.92

NP

NP

 

 

NP

NP

 

 

 

B

9.88

8.59

6.23

NP

NP

 

 

NP

NP

 

 

1.25

A

10.03

8.41

5.62

NP

NP

 

 

NP

NP

 

 

 

B

9.62

8.26

6.14

NP

NP

 

 

NP

NP

 

 

2.5

A

10.26

8.64

5.44

73

78

 

 

24

24

 

 

 

B

9.93

8.59

6.70

72

75

 

 

22

24

 

 

5

A

10.67

7.05

5.77

70

75

 

 

32

35

 

 

 

B

9.86

7.99

5.57

72

71

 

 

42

40

 

 

10

A

9.13

7.32

5.40

75

68

 

 

35

32

 

 

 

B

8.41

8.34

5.38

70

72

 

 

42

42

 

 

15

A

8.62

6.78

5.29

74

73

 

 

63

54

 

 

 

B

8.29

7.20

5.79

72

72

 

 

65

60

 

 

20

A

7.87

7.48

5.45

68

68

 

 

74

65

 

 

 

B

7.43

7.23

5.73

76

68

 

 

79

77

 

 

25

A

5.46

8.21

5.67

64

59

 

 

75

75

 

 

 

B

6.21

6.78

6.41

67

61

 

 

88

81

 

 

30

A

4.56

6.85

5.26

61

64

 

 

89

86

 

 

 

B

4.13

8.06

4.85

59

65

 

 

86

85

 

 

Positive control EMS (µg/mL)

150

A

6.88

7.67

5.48

57

57

 

 

87

84

 

 

 

B

7.57

7.96

4.16

67

64

 

 

87

82

 

 


 

Summary Analysis: Mutagenicity Test (-S9) 24-HourExposure

Concentration(µg/mL)

SG

%RSG

%V

RTG

MF§

0

 

89.09

100

73.10

1.00

193.22

0.31

Ø

84.22

95

 

 

 

0.63

Ø

82.55

94

 

 

 

1.25

Ø

80.25

91

 

 

 

2.5

 

87.98

102

74.81

1.01

187.64

5

 

72.95

86

69.31

0.78

354.22

10

 

61.69

62

67.78

0.65

368.57

15

 

54.57

53

70.90

0.59

701.54

20

 

52.42

46

65.31

0.52

1119.24

25

 

44.02

29

53.01

0.36

1675.25

30

 

27.29

14

52.27

0.22

2212.67

Positive control EMS

Concentration(µg/mL)

SG

%RSG

%V

RTG

MF§

150

45.36

38

50.81

0.36

2131.98

 

GEF =126, therefore MF threshold for a positive response = 319.22


 

Large and Small Colonies Analysis: Mutagenicity Test(-S9)24-HourExposure 

Concentration(µg/mL)

Viability # after day 2

Small colonies # after day 2

Large colonies # after day 2

0

A

78

75

71

73

11

8

9

11

15

11

15

16

 

B

72

72

74

75

15

13

11

11

4

11

14

14

2.5

A

73

78

 

 

16

9

 

 

8

15

 

 

 

B

72

75

 

 

7

9

 

 

15

15

 

 

5

A

70

75

 

 

13

16

 

 

19

19

 

 

 

B

72

71

 

 

27

24

 

 

15

16

 

 

10

A

75

68

 

 

16

15

 

 

19

17

 

 

 

B

70

72

 

 

21

19

 

 

21

23

 

 

15

A

74

73

 

 

42

34

 

 

21

20

 

 

 

B

72

72

 

 

48

40

 

 

17

20

 

 

20

A

68

68

 

 

53

47

 

 

21

18

 

 

 

B

76

68

 

 

67

58

 

 

12

19

 

 

25

A

64

59

 

 

52

47

 

 

23

28

 

 

 

B

67

61

 

 

61

60

 

 

27

21

 

 

30

A

61

64

 

 

63

62

 

 

26

24

 

 

 

B

59

65

 

 

63

71

 

 

23

14

 

 

150 EMS

A

57

57

 

 

51

51

 

 

36

33

 

 

 

B

67

64

 

 

54

44

 

 

33

38

 

 

Mutation frequencies

Concentration(µg/mL)

 

 

 

 

Viable

Yv         Nv

Small colonies

Large colonies

Proportionsmall colony mutants

Mutants Ym        Nm

 

MF§

Mutants Ym        Nm

 

MF§

0

178

768

679

768

84.2

668

768

95.4

0.47

2.5

86

384

343

384

75.5

331

384

99.3

0.44

5

96

384

304

384

168.5

315

384

142.9

0.54

10

99

384

313

384

150.8

304

384

172.3

0.47

15

93

384

220

384

392.8

306

384

160.1

0.68

20

104

384

159

384

675.0

314

384

154.1

0.76

25

133

384

164

384

802.4

285

384

281.2

0.69

30

135

384

125

384

1073.6

297

384

245.8

0.75

150 EMS

139

384

184

384

724.0

244

384

446.3

0.59

$

=

Cell counts (x105cells/mL). Set up on previous day to 2 x 105cells/mL unless

 

 

otherwise stated in parenthesis.

%RSG

=

Relative Suspension Growth

RTG

=

Relative Total Growth

%V

=

Viability Day 2

§ or #

=

Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis

A,B

=

Replicate cultures

CP

=

Cyclophosphamide

EMS

=

Ethylmethanesulphonate

MF§

=

5-TFT resistant mutants/106viable cells 2 days after exposure

Nv

=

Number of wells scored, viability plates

Yv

=

Number of wells without colonies, viability plates

Ym

=

Number of wells without colonies, mutation plates

Nm

=

Number of wells scored, mutation plates

Ø

=

Not plated, due to toxicity or surplus to requirements

NP

=

Not plated for viability or 5-TFT resistance

X

=

Treatment excluded from test statistics due to toxicity

Conclusions:
The test item induced toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells that markedly exceeded the GEF, consequently it is considered to be mutagenic in this assay.
Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In VitroMammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

 

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at ten dose levels in duplicate, together with vehicle (R0 medium), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

 

Mutagenicity Test

 

Group

Concentration of ESA (1,3,2-Dioxathiolane, 2,2-dioxide) (µg/mL) plated for viability and mutant frequency

4-hour without S9

5, 10, 15, 20, 25, 30, 35

4-hour with S9 (2%)

10, 15, 20, 25, 30, 35, 40

24-hour without S9

2.5, 5, 10, 15, 20, 25, 30

 

Results

The maximum dose levels in the Mutagenicity Test were limited by test item-induced toxicity in all three of the exposure groups, as recommended by the OECD 490 guideline. The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

 

The test item induced dose-related increases in mutant frequency in all three of the exposure groups. The increases in mutant frequency markedly exceeded the GEF at several dose levels, there were marked increases in absolute numbers of mutant colonies, and the increases in mutant frequency were predominantly due to small colonies (indicative of clastogenic activity). The responses observed were therefore considered to be of toxicological significance.


 

Conclusion

The test item induced toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells that markedly exceeded the GEF, consequently it is considered to be mutagenic in this assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

in vivo mammalian somatic cell study: erythrocyte micronucleus

The substances potential to induce structural and numerical chromosome aberrations was assessed in an in vivo mammalian erythrocyte micronucleus test in mice.

The test substance was intraperitoneally administered to the mice twice within a 24-hour interval, at three dose levels (16 mg/kg, 32 mg/kg, 63 mg/kg) and then frequencies of micronucleated polychromatic erythrocytes (MNPCEs) and cytotoxicity (polychromatic erythrocyte (PCE) ratio in the erythrocytes) were evaluated by collecting bone marrow at 18~24 hours after administration of test substance. As a results of counting the frequency of MNPCEs of 2000 PCEs, there was no statistically significant increase at any dose groups compared to negative control group. There was a statistically significant increase in positive control group compared to negative control group (p<0.01). The PCEs ratio of 200 erythrocytes (PCE/(PCE+NCE)), as an index of cytotoxicity, did not significantly decrease in PCE/(PCE+NCE) ratio compared to negative control group.

It was concluded that, under the condition of this study, test substance ESA did not induce micronucleus in mice bone marrow.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation: 15 March 2011; Submission of final report: 18 July 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Korea
Type of assay:
mammalian erythrocyte micronucleus test
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 %
Storage condition : Recommended temperature: 2-8ºC
Stability : Stable in recommended storage condition
Species:
mouse
Strain:
ICR
Details on species / strain selection:
Reason for animal selection: ICR mice has been widely used in mammalian erythrocyte micronucleus test. It has been known for suitable experimental animal for general toxicity. Moreover, sufficient fundamental data have been accumulated in toxicological study using ICR mice. Such data will be useful for interpretation and evaluation of test results.
Sex:
male
Details on test animals and environmental conditions:
Species and strains: SPF (Specific Pathogenic Free), CrljOri:CD1 (ICR) mice
Supplier: Orient Bio Co., Ltd.
Number of animals at the time of arrival:
Preliminary dose-range finding study: 33 male mice
Main study: 33 male mice
Age at the time of arrival: 7 weeks old
Period of quarantine and acclimation:
Preliminary dose-range finding study : 7 days after receipt
Main study I: 9 days after receipt
Main study II: (Confirmation test) : 7 days after receipt
(The only healthy animals were selected for this study by observation of general symptoms during the acclimation period.)
Age of test animal at administration: 8 weeks old
Grouping Method
Animals were weighted one day before the administration, and grouped by using the graded body weight randomly.
Identification of individual animals
Individual animals was identified by tail marking using oil marker and individual cages was identified by card labeling. The record sheet was posted at the entrance of animal room.
Weighing method of test animals
Body weight of test animals was measured at the time of animal acquisition, grouping, administration of test substance and autopsy. Body weight was measured one by one in accordance with individual animal number using electrical balance calibrated by calibration standard weight and recorded in data sheet.
Disposal of remaining animals: Animals were euthanized on the last autopsy.

Range of acceptable temperature and humidity
Preliminary dose-range finding study I
: 21.6 ± 0.9 ºC of temperature, 44.5 ± 2.6 % of relative humidity
Preliminary dose-range finding study II
: 21.4 ± 1.0 ºC of temperature, 45.0 ± 7.5 % of relative humidity
Main study I
: 21.6 ± 1.2 ºC of temperature, 54.4 ± 8.6 % of relative humidity
Main study II (Confirmation test)
: 21.7 ± 0.6 ºC of temperature, 53.1 ± 1.4 % of relative humidity

Ventilation frequency : 10~15 times/hr.

Lighting cycle : 12 hours of lighting duration (lighting up at 8 a.m.~lighting out at 8 p.m.)

Luminous intensity
Preliminary dose-range finding study I : 278 Lux
Preliminary dose-range finding study II: 278 Lux
Main study I : 275 Lux
Main study II (Confirmation test) : 275 Lux

Breeding cage (size) : During the quarantine, acclimation, administration and observation period, all animals were housed in polycarbonate cage (170W x 235L x 125H mm).

Animal number per cage
Preliminary dose-range finding study : All animals were housed 5 animals per cage during quarantine and acclimation period and 3 animals per cage during administration and observation period.

Main study and Confirmation test : All animals were housed 5 animals per cage during quarantine and acclimation period and 3 animals per cage during administration and observation period.

Feed
Type: Solid feed for laboratory animals
Source: Polas International
Produced by: Harlan
Salvage method: Free ingestion by feeding.
Identification of pollutants: Data from feed producers were used.

Water
Type : Tap water purified by reverse osmosis filtering system
Feeding method : ad libitum using polycarbonate water bottles
Contamination analysis : Water certification from nationally certified inspection organization was referred to examine contamination.
Route of administration:
intraperitoneal
Vehicle:
distilled water.
Details on exposure:
Preparation and dosage of test substance
The test substance was weighed on the day of administration in accordance with the concentration of the preparation, and heated at about 40 ºC in hot water to dissolve in distilled water. The administration level of prepared test substance was determined to 10 ml/kg based on body weight measured at the administration day. The stability test and the homogeneity test were not performed because the test solution was prepared fresh on administration day.
Duration of treatment / exposure:
single injection
Post exposure period:
24 or 48 hours
Dose / conc.:
16 mg/kg bw/day (nominal)
Remarks:
Main tests I and II
Dose / conc.:
32 mg/kg bw/day (nominal)
Remarks:
Main tests I and II
Dose / conc.:
63 mg/kg bw/day (nominal)
Remarks:
Main tests I and II
No. of animals per sex per dose:
Preliminary tests: 3 male mice per group
Main tests: 6 males mice per group
Control animals:
yes, concurrent vehicle
other: yes, concurrent positive control
Positive control(s):
Mitomycin C (MMC) 2.0 mg/kg
Positive control substances were selected according to OECD guidelines for the Testing of Chemicals No. 474.
Tissues and cell types examined:
Erythrocytes in bone marrow from thigh bone.
Details of tissue and slide preparation:
Extraction of bone marrow and preparation of slides
Three slides of bone marrow were prepared per animal. In case of preliminary dose-range finding study, the thigh bone of the test animal was collected in 24, 48 hours after administration. In case of main test and confirmation test, the thigh bone of the test animal was collected in 24 hours after administration. The thigh bone of the test animal was collected with care to avoid blood contamination. The bone marrow was collected in a centrifuge tube by flushing the thigh bone inside with in-activated FBS (Hyclone, Lot No. AWA93840). The extracted bone marrow was centrifuged at 1000 rpm for 5 minutes and then resuspended with small aliquots of FBS after discarding the supernatant. The bone marrow was smeared on a slide glass and then dried at room temperature and fixed in methanol for 5 minutes.

Staining of specimen
For the scoring of the polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) ratio, the slides were stained in 4 % Giemsa staining solution. For the scoring of the micronucleated polychromatic erythrocytes (MNPCE) from PCEs, the slides were fixed and then stained with acridine orange (40 µg/ml) and covered with cover glasses.

Slide analysis
Observation method
The observation of slides was performed with blind method and PCE to NCE ratio was examined by optical microscope at over 400× magnification. The observation of MNPCEs was performed by fluorescence microscope equipped with FITC filter.

Criteria
The PCE/NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring 200 erythrocytes per animal. The micronuclei frequency (expressed as percent micronucleated cells) was determined by analyzing the number of MNPCEs from 2000 PCEs per animal. In case of observation by fluorescence microscope, PCE was counted cells which appeared as red-fluorescence color without nucleus by acridine orange staining. NCE stained with acridine orange was appeared as only shadow entity without fluorescence. In case of observation by optical microscope, PCE stained with Giemsa staining solution was counted cells appeared as purple or blue color. NCE stained with Giemsa staining solution was counted cells appeared as pink color. According to criteria in micronucleus judgement, the largest size was defined as 1/2 size of erythrocyte diameter and the smallest size was defined as up to the limit of identification. Shape included circle, donut, semi-circle. Color was defined as same color as nucleus of cell.

Observation of general symptoms
It was conducted once at the administration date and autopsy date for observation of dead and abnormal sign of test animals.

Measurement of body weight
The body weight of the animals was measured before the administration and autopsy
date.
Evaluation criteria:
In case PCE/(PCE+NCE) ratio (Mean±SD) is more than 0.1, test is judged to be reasonable. It is determined as a positive result if there is dose-related increase or
statistically significant increase in frequency of MNPCE (MNPCE/2000PCEs) in at least single dose group.
Statistics:
Frequency of MNPCE (MNPCE/2000PCEs), PCE/(PCE+NCE) ratio
1) Comparison between negative control and treated group: ANOVA test
2) In case of P<0.05 at 1), verification of capacity correlation : Linear logistic regression
3) Comparison between negative control and positive control: ANOVA test

Weight
1) Weight of each individual at the time of autopsy: ANOVA test
2) Statistically significant case in 1) : Dunnett T3 or Duncan's multiple range test

The result of statistical evaluation is regarded as significant when the P value is less than 0.05. statistical analysis was performed with SPSS 12.0 program.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Micronuclei frequency and cytotoxicity
The frequencies of MNPCEs in 2000 PCEs per animal were 0.23 %, 0.14 %, 0.13 %, 0.20 % and 9.59 % in order of negative control group, 16, 32, 63 mg/kg administration group and positive control group. There was no statistically increase in frequency of PCEs having micronuclei in administration group compared to negative control group. There was clear statistically significant increase in positive control group compared with negative control group in frequency of MNPCE (p<0.01).
The PCE ratios of 200 erythrocytes (PCE+NCE), as an index of cytotoxicity, were 0.48, 0.52, 0.49, 0.54 and 0.36 in order of negative control, 16, 32, 63 mg/kg group and positive control. There was no significant decrease in PCE/(PCE+NCE) ratio compared to negative control group.

Clinical sign
As a result of observation, no special abnormality was observed compared to negative control group.

Body weight
There was no statistically change in body weight at any group compared to negative control group.
Conclusions:
It was concluded that, under the condition of this study, test substance ESA did not induce micronucleus in mice bone marrow.
Executive summary:

The test substance, ESA, was evaluated for its potential to induce micronucleus in bone marrow of intraperitoneal administrated mice.

Animals were about 8 weeks old at administration of test substance. The test substance was intraperitoneally administered to the mice twice with a 24-hour interval, three dose levels (16 ㎎/㎏, 32 ㎎/㎏, 63 ㎎/㎏) and then frequencies of MNPCEs and cytotoxicity (PCE ratio in the erythrocytes) were evaluated by collecting bone marrow at 18~24 hours after administration of test substance. As a results of counting the frequency of MNPCEs of 2000 PCEs, there was no statistically significant increase at any dose groups compared to negative control group. Also, there was statistically significant increase in positive control group compared to negative control group (p<0.01). The PCEs ratio of 200 erythrocytes (PCE/(PCE+NCE)), as a index of cytotoxicity, did not significantly decrease in PCE/(PCE+NCE) ratio compared to negative control group.

It was concluded that, under the condition of this study, test substance ESA did not induce micronucleus in mice bone marrow.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The substance was mutagenic in in-vitro gene mutation studies, with and without metabolic activation. The substance is also suspected of causing cancer and has been identified as a possible alkylating agent.

Additional information

ESA induced reverse mutation in Ames test conducted according to OECD TG 471. In addition, the test item induced toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells that markedly exceeded the GEF (Global Evaluation Factor) and was considered to be mutagenic under test conditions designed to be compatible with the OECD TG 490 . 

However, ESA did not induce chromosome aberration in Chinese Hamster Ovary (CHO-K1) cells under the condition of the study conducted in compliance with OECD TG 473 and also did not induce micronucleus in mice bone marrow under the condition of the study conducted in compliance with OECD TG 474.

Taking into account the positive profile of in vitro studies for gene mutation, a further in vivo study for gene mutation to confirm the findings in vitro would be Comet assay (OECD 489) or transgenic rodent somatic and germ cell gene mutation assays (OECD TG 488). A Comet assay is proposed.

The stability of the test item in the genotoxicity studies:

ESA in Ames and chromosome aberration tests was dissolved in 1,4-dioxane, therefore it may be assumed that substance was not hydrolysed. ESA was dissolved in R0 medium in the mouse lymphoma assay.

The micronucleus study in mice bone marrow was performed by intraperitoneal administration. The test substance was weighed on the day of administration in accordance with the concentration of the preparation, and heated at about 40in hot water to dissolve in distilled water. The stability test and the homogeneity test were not performed because the test solution was prepared fresh on administration day. Based on the instability of ESA in water, the validity of micronucleus study maybe questionable as to whether exposure was to ESA or its hydrolysis product.

Justification for classification or non-classification

From the conducted in vitro studies, it can be concluded that the tests for gene mutations (Ames and L5178Y mouse lymphoma cells) showed positive results, whereas the test for chromosomal abnormalities (in Chinese Hamster Ovary (CHO-K1) cells) was negative.

Only in vivo genetic toxicology test for chromosomal abnormalities was performed (mammalian erythrocyte micronucleus test) and the test was negative.

Based on the available study data it is considered that the current results are inconclusive to be able to classify the substance as a germ cell mutagen, or conclude that no classification is warranted under the CLP criteria.