Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The weight of evidence suggests that MeaTG is not genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
Histidine reversion
Species / strain:
other: Strains: TA98, TA100, TA1535 and TA1537
Metabolic activation:
with and without
Metabolic activation system:
Rat and hamster liver S9 induced with aroclor 1254
Test concentrations with justification for top dose:
0, 10, 33, 100, 333 and 1000 µg/plate
Vehicle:
no data
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without activation : sodium azide (TA1535 and TA 100),  9-aminoacridine (TA 97), 4-nitro-o-phenylenediamine (TA98).   With activation : 2-aminoanthracene (all strains).
Details on test system and conditions:
In the Salmonella assay, a test tube containing a suspension of one  strain of Salmonella typhimurium plus S9 mix or plain buffer without S9,  is incubated for 20 minutes at 37º C with the test chemical. Control  cultures, with all the same ingredients except the test chemical, are  also incubated. In addition, positive control cultures are also prepared;  these contain the particular bacterial tester strain under investigation,  the various culture ingredients, and a known potent mutagen. After 20  minutes, agar is added to the cultures and the contents of the tubes are  thoroughly mixed and poured onto the surface of Petri dishes containing  standard bacterial culture medium. The plates are incubated for 48 hours  and then counted. The substance was tested initially in a toxicity assay to determine the  appropriate dose range. 
The toxicity assay was performed by using TA 100.  Toxic concentrations were those at which a decrease in the number of his+  colonies was seen or at which there was a clearing in the density of the  background lawn.

- Test Design   . Number of replicates : 3
- Description of follow up repeat study : same conditions than the initial experiment performed 1 week later
Evaluation criteria:
The positive control plates are counted, and the number of mutant  colonies appearing on them must be significantly increased over the  spontaneous control number for the test to be considered valid.  If no increase in mutant colonies is seen after testing several strains  under several different culture conditions, the test chemical is  considered to be non mutagenic in the Salmonella test.
Statistics:
None
Key result
Species / strain:
other: Strains: TA98, TA100, TA1535 and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
> 1000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Chemical Name:

Sodium thioglycolate

CAS Number:

367-51-1

Study Type:

Salmonella

Study ID:

471613

Study Result:

Negative

Year Completed:

1979

Vehicle Control:

Water

Protocol:

Preincubation

Individual strain data is presented as mean ± standard error.

Abbreviations are noted at bottom of page.

Trial summary calls are shown in parentheses.

Strain: TA1535

Dose

No Activation

No Activation

10% HLI

10% HLI

10% RLI

(Negative)

(Negative)

(Negative)

(Negative)

(Negative)

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

7

0.7

7

0.6

6

0.9

7

2.6

7

0.7

10     

6

1

6

0.9

5

0.9

6

2

8

1.2

33     

5

1.5

6

0.9

7

1.5

9

1.2

6

1.3

100     

8

3.5

9

1.3

7

0.6

9

0.7

6

0.9

333     

6

0.6

6

0.9

5

1.2

7

1.5

8

1.2

1000     

5

1.2

8

2

5

0.3

8

1.2

8

0.9

Positive Control

288

32.3

251

40.8

106

16

43

4.1

135

9.7

Strain: TA100

Dose

No Activation

No Activation

10% HLI

10% HLI

10% RLI

10% RLI

(Negative)

(Negative)

(Negative)

(Equivocal)

(Negative)

(Negative)

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

100

5

110

5.8

185

26.5

179

11.7

175

27.8

189

8.7

10     

95

5

126

11.4

160

18

164

6.2

140

30.4

204

37.7

33     

105

31.2

129

7.5

195

30

174

7.6

170

40.9

189

3.5

100     

115

10

121

3.8

220

18

221

4.2

145

18

175

9

333     

125

13.2

122

8.5

160

5

173

10.1

135

8.7

221

8.1

1000     

120

30

124

8.8

215

13.2

202

4.3

155

35

181

18.1

Positive Control

430

27.8

599

36.4

558

5.9

836

172.5

300

11.1

430

70.3

Conclusions:
Under these experimental conditions, sodium thioglycolate is considered as non-genotoxic
Executive summary:

In a study performed according to the US NTP protocol, four S. typhimurium strains (TA 98, TA 100, TA 1535 and TA 1537) were exposed to sodium thioglycolate in a preincubation assay. Based on a preliminary toxicity assay, concentrations up to 1000 µg/plate were used with or without metabolic activation (rat and hamster S9) in all four strains. Sodium thioglycolate did not induce mutations in these studies. Positive and solvent controls gave the expected results.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
Histidine reversion
Species / strain:
other: Strains: TA1535, TA1537, TA98, TA100 and TA102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (from livers of male Sprague-Dawley rats  treated by Aroclor 1254)
Test concentrations with justification for top dose:
15, 50, 150, 500, 1500 and 5000 µg a.i./plate.
Two distinct experiments were performed using these doses.
Vehicle:
sterile distilled water
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and conditions:
METHOD DETAILS:
- Test concentrations:
. Preliminary study (on TA100): 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
. Main study: see the "Test concentration" field
- Number of replicates: 3
- Positive controls:
without S9-mix
. for TA1535 and TA100: N-ethyl-N'-nitro-N-nitrosoguanidine (5 and 2  µg/plate respectively)
. for TA1537: 9-aminoacridine (80 µg/plate)
. for TA98: 4-Nitroquinoline-1-oxide (0.2 µg/plate)
. for TA102: mitomycin C (0.5 µg/plate)
with S9-mix
. for TA1535, TA1357 and TA100: 2-aminoanthracene (2; 2 and 1µg/plate  respectively)
. for TA98: benzo(a)pyrene (5 µg/plate)
. for TA102: 1,8-Dihydroxyanthraquinone (10 µg/plate)
- Pre-incubation time: no
- Pre-incubation temperature: no
- Incubation time: 48h
- Incubation temperature: 37°C

EXAMINATION:
- Bacterial toxicity: determined by examination of background lawn growth
- Number of revertants / plate

ANALYTICAL DEVICE:  Colonies were counted electronically using a Domino Colony Counter.
Evaluation criteria:
The reverse mutation assay may be considered valid if the following  criteria are met: All tester strain cultures exhibit a characteristic number of spontaneous  revertants per plate in the vehicle and untreated controls. Acceptable  ranges are presented below with historical control ranges for 2001 and  2002 presented in Table 1.

Spontaneous Mutation Ranges:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
TA102 180 to 400

The test material may be considered positive in this test system if the  following criteria are met: The test material should have induced a reproducible, dose-related and  statistically significant  increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's method of linear regression
Key result
Species / strain:
other: Strains: TA1535, TA1537, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
= 5000 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
CYTOTOXICITY:
The test substance was toxic at 5000 µg/plate in the TA100 strain (without S9 mix: very weak bacterial background lawn, with S9 mix: sparse bacterial background lawn).

GENOTOXICITY:
No significant increases in the number of revertants were observed at any dose level, with or without metabolic activation.

Table 1:Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

136

9

351

15

5

139

(135)

14

(10)

350

(358)

18

(15)

5

(6)

129

7

373

13

8

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

62

18

267

12

6

97

(73)

11

(14)

318

(301)

12

(12)

5

(5)

60

13

318

12

5

Table 2: Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From : 25 March 2003

To : 28 March 2003

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

-

0

153

121

131

(135)

16.4#

9

7

8

(8)

1.0

367

310

385

(354)

39.2

13

16

24

(18)

5.7

6

7

11

(8)

2.6

-

15

128

114

130

(124)

8.7

6

9

5

(7)

2.1

353

296

434

(361)

69.3

16

17

17

(17)

0.6

5

5

3

(4)

1.2

-

50

104

129

118

(117)

12.5

12

12

5

(10)

4.0

399

360

399

(386)

22.5

16

14

12

(14)

2.0

5

6

4

(5)

1.0

-

150

144

113

118

(125)

16.6

11

8

8

(9)

1.7

406

372

360

(379)

23.9

15

7

16

(13)

4.9

4

5

6

(5)

1.0

-

500

101

106

118

(108)

8.7

11

13

11

(12)

1.2

362

359

393

(371)

18.8

15

8

12

(12)

3.5

5

3

3

(4)

1.2

-

1500

116

104

106

(109)

6.4

5

3

11

(6)

4.2

382

356

368

(369)

13.0

17

15

18

(17)

1.5

7

7

4

(6)

1.7

-

5000

0 V

0 V

0 V

(0)

0.0

3 S

9 S

5 S

(6)

3.1

204

221

153

(193)

35.4

17 S

14 S

13 S

(15)

2.1

0 S

0 S

4 S

(1)

2.3

Positive

controls

S9-Mix

-

Name

Concentration

(µg/plate)

No. colonies

per plate

ENNG

ENNG

MMC

4NQO

9AA

3

5

0.5

0.2

80

553

474

505

(511)

39.8

287

284

256

(276)

17.1

862

859

903

(875)

24.6

75

81

80

(79)

3.2

2605

2689

2688

(2661)

48.2

Table 3: Test Results: Experiment 1 – With Metabolic Activation

Test Period

From : 25 March 2003

To : 28 March 2003

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

+

0

173

145

180

(166)

18.5#

16

11

15

(14)

2.6

380

373

370

(374)

5.1

21

26

28

(25)

3.6

5

6

7

(6)

1.0

+

15

150

125

163

(146)

19.3

17

14

12

(14)

2.5

361

352

376

(363)

12.1

24

21

25

(23)

2.1

5

7

12

(8)

3.6

+

50

156

158

149

(154)

4.7

9

6

12

(9)

3.0

332

362

403

(366)

35.6

22

22

19

(21)

1.7

5

6

5

(5)

0.6

+

150

127

140

150

(139)

11.5

13

19

17

(16)

3.1

419

369

363

(384)

30.7

33

20

18

(24)

8.1

9

3

6

(6)

3.0

+

500

119

136

164

(140)

22.7

17

15

18

(17)

1.5

389

337

345

(357)

28.0

19

24

20

(21)

2.6

12

3

6

(7)

4.6

+

1500

133

130

121

(128)

6.2

8

6

9

(8)

1.5

404

412

304

(373)

60.2

23

25

19

(22)

3.1

3

14

2

(6)

6.7

+

5000

98

82

77

(86)

11.0

6

6

7

(6)

0.6

75

88

94

(86)

9.7

7

25

16

(16)

9.0

0

0

0

(0)

0.0

Positive

controls

S9-Mix

+

Name

Concentration

(µg/plate)

No. colonies

per plate

2AA

2AA

DAN

BP

2AA

1

2

10

5

2

1982

2219

1854

(2018)

185.2

378

376

401

(385)

13.9

789

787

779

(785)

5.3

220

262

259

(247)

23.4

284

336

315

(312)

26.2

Table 4: Test Results: Experiment 2 – Without Metabolic Activation

Test Period

From : 11 April 2003

To : 14 April 2003

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

-

0

95

102

128

(108)

17.4#

17

11

16

(15)

3.2

345

350

358

(351)

6.6

19

16

14

(16)

2.5

14

6

C

(10)

5.7

-

15

66

50

63

(60)

8.5

4

19

11

(11)

7.5

364

322

393

(360)

35.7

16

C

16

(16)

0.0

4

7

6

(6)

1.5

-

50

63

66

55

(61)

5.7

4

11

7

(7)

3.5

391

383

362

(379)

15.0

16

18

17

(17)

1.0

5

9

1

(5)

4.0

-

150

84

82

66

(77)

9.9

13

8

11

(11)

2.5

380

345

398

(374)

27.0

13

14

9

(12)

2.6

9

7

4

(7)

2.5

-

500

82

92

95

(90)

6.8

8

17

14

(13)

4.6

376

326

397

(366)

36.5

15

12

15

(14)

1.7

7

6

5

(6)

1.0

-

1500

86

103

85

(91)

10.1

11

8

6

(8)

2.5

326

390

329

(348)

36.1

11

13

22

(15)

5.9

9

6

3

(6)

3.0

-

5000

0 S

0 S

0 S

(0)

0.0

7 S

3 S

0 S

(3)

3.5

22

23

25

(23)

1.5

0 S

0 S

0 S

(0)

0.0

0 S

0 S

XS

(0)

0.0

Positive

controls

S9-Mix

-

Name

Concentration

(µg/plate)

No. colonies

per plate

ENNG

ENNG

MMC

4NQO

9AA

3

5

0.5

0.2

80

419

381

274

(358)

75.2

305

227

222

(251)

46.5

1080

1040

C

(1060)

28.3

160

170

151

(160)

9.5

1379

1196

1552

(1376)

178.0

Table 5: Test Results: Experiment 2 – With Metabolic Activation

Test Period

From : 11 April 2003

To : 14 April 2003

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

+

0

94

113

115

(107)

11.6#

11

8

11

(10)

1.7

397

326

384

(369)

37.8

22

26

24

(24)

2.0

5

7

7

(6)

1.2

+

15

135

92

107

(111)

21.8

5

11

14

(10)

4.6

380

399

396

(392)

10.2

23

23

29

(25)

3.5

12

7

5

(8)

3.6

+

50

128

107

129

(121)

12.4

13

8

14

(12)

3.2

345

381

394

(373)

25.4

29

29

28

(29)

0.6

7

16

7

(10)

5.2

+

150

110

137

114

(120)

14.6

8

11

11

(10)

1.7

422

424

425

(424)

1.5

21

29

33

(28)

6.1

6

11

4

(7)

3.6

+

500

116

119

134

(123)

9.6

5

12

13

(10)

4.4

399

405

361

(388)

23.9

31

C

29

(30)

1.4

6

3

5

(5)

1.5

+

1500

76

85

92

(84)

8.0

11

7

9

(9)

2.0

434

380

376

(397)

32.4

36

22

25

(28)

7.4

7

5

6

(6)

1.0

+

5000

46

46

37

(43)

5.2

5

5

7

(6)

1.2

39

74

44

(52)

18.9

15

12

21

(16)

4.6

3

7

1

(4)

3.1

Positive

controls

S9-Mix

+

Name

Concentration

(µg/plate)

No. colonies

per plate

2AA

2AA

DAN

BP

2AA

1

2

10

5

2

1099

1410

1350

(1286)

165.0

236

292

243

(257)

30.5

1135

1103

1090

(1109)

23.2

383

477

536

(465)

77.2

876

723

678

(759)

103.8

S            Sparse bacterial background lawn

V           Very weak bacterial background lawn

#            Standard deviation

C               Contaminated

Conclusions:
Interpretation of results (migrated information):
negative

Ammonium thioglycolate was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Ammonium thioglycolate was tested in a bacterial gene mutations assay performed following a protocol compliant with the OECD guideline # 471. The direct plate incorporation procedure was performed with Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 at concentrations up to 5,000 µg/plate. Cytotoxic effects were observed in the absence and in the presence of a metabolic activator (Aroclor 1254-induced rat liver S9) at the concentration of 5,000 µg/plate. Ammonium thioglycolate did not induce mutations in the bacterial mutation test in either the absence or presence of metabolic activator in any strain tested. The positive and negative controls included in the experiment showed the expected results.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Cited as Directive 2000/32/EC, B.17
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Test material information:
Composition 1
Target gene:
Locus Examined: thymidine kynase, the selection agent used was 5 µg/ml trifluorothymidine
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
- Type and identity of media: the medium used was RPMI 1640 (complete medium) with 3 or 15 % horse serum.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 derived from male Sprague Dawley (phenobarbital/B-naphtoflavone induced rat liver)
Test concentrations with justification for top dose:
Non-activated conditions: Initial trial: 13, 50, 100, 200, 400, 800 and 1600 µg/mL; Confirmatory trial: 13, 25, 50, 100, 200, 400 and 600 µg/mL
S9-activated conditions: 50, 100, 200, 400, 800 and 1600 µg/mL
Duplicate cultures were processed for all trials.
Vehicle:
- Solvent used: deionised water
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and conditions:
Experimental Performance:
In the mutation experiment 1x10e7 cells/flask suspended in 10 ml RPMI medium with 3 % horse serum (15 % during 24 h treatment) were exposed to various concentrations of the test item either in the presence or absence of metabolic activation.

After 4 h (after 24 h in the second experiment) the test item was removed by centrifugation and washing twice in "saline G". Subsequently the cells were resuspended in 30 ml complete culture medium and incubated for an expression and growth period of totally 72 h. In the second experiment the expression time without metabolic activation was 48 hours (RPMI medium with 15 % horse serum).

The cell density was determined each day and adjusted to 3x10e5 cells/ml, if necessary. Relative suspension and total growth (RSG and RTG) of the treated tell cultures were calculated after 48 h (72 h following continuous treatment) according to the method of Clive and Spector. One sample of the cells was taken at the end of the treatment (4 h and 24 h, respectively), diluted and seeded into microtiter plates (about 2.5 cells/well), to determine the survival of the cells after treatment (cloning efficiency 1).

After the expression period the tells were seeded into microtiter plates. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4x10e3 tells in selective medium with TFT. The viability (cloning efficiency 2) was determined by seeding about 2.5 cells per well into 2 microtiter plates (same medium without TFT). The plates are incubated at 37 °C in 4.5 %C02 and 95.5 % humidified air for 10 - 15 days to determine the cloning efficiency and to evaluate mutagenicity. Then the plates were evaluated manually.


Size Distribution of the Colonies:
The numbers of colonies were counted manually. The colony size distribution was determined in the controls and at all concentrations of the test item


Data Recording:
All plates were evaluated manually.
The mutation frequency was derived from the cloning efficiency under selective conditions compared to the corresponding viability under non-selective conditions
Evaluation criteria:
Acceptability of the Assay:
A mutation assay is considered acceptable if it meets the following criteria:
a) Both plates, from either the survival or the TFT resistance-testing portion of the experiment are analysable.
b) The absolute cloning efficiency 2 of the negative and/or solvent controls is > 0.5 (50 %).
c) The spontaneous mutant frequency in the negative and/or solvent controls are in the range of the historical control data .
d) The positive controls (MMS and CPA) induce significant (at least 2-fold) increases in the mutant frequencies. The values of the cloning efficiencies and the relative total growth are greater than 10 % of the concurrent vehicle control group.

Evolution of results:
A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points
Statistics:
The survival rate and viability was determined based on the Poisson distribution method. The zero term of the Poisson distribution, [P(0)] method, was used.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: 4-h experiment (+/-S9): > 1600 µg/ml / 24-h experiment (-S9): >= 800 µg/ml
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.
Conclusions:
Interpretation of results (migrated information):
negative

Ammonium Thioglycolate did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Executive summary:

Ammonium thioglycolate was tested in an in vitro gene mutation assay using mammalian cells cultures both in the absence and presence of metabolic activation (S9 mix), according to the Directive 2000/32/EC, B17 and in compliance with the Principles of Good Laboratory Practice.

In a mammalian cell gene mutation assay (TK+/-), mouse lymphoma L5178Y cells cultured in vitro were exposed to ammonium thioglycolate (purity 71.1%) in deionised water. Two parallels culture were used. The first main experiment was performed without microsomal activation at concentrations of 0, 13, 50, 100, 200, 400, 800 and 1600 µg/ml, and with activation at concentrations of 0, 50, 100, 200, 400,800 and 1600 µg/ml

with a treatment period of 4 h. The second experiment was solely performed in the absence of metabolic activation at concentrations of 0, 13, 25, 50, 100, 200, 400 and 600 µg/ml, with a treatment period of 24 h. Appropriate positive controls were used and showed a statistical increase in mutant colonies, indicating that the tests were sensitive and valid.

After a 48 rest period, cells were incubated mutagenicity evaluation with trifluorothymidine (TFT).

No substantial and reproductible dose dependant increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Under these experimental conditions, ammonium thioglycolate did not induce any increase in mutant colonies and is not considered as mutagenic in this mouse lymphoma mouse.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Species / strain:
other: Human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
10% S9 mix, prepared from a liver microsomal fraction (S9) of  rats induced with Aroclor 1254. 
Test concentrations with justification for top dose:
Without S9: 0, 30, 100 and 300 µg/ml
With S9: 0, 100, 300 and 1000 µg/ml
Vehicle:
distilled water
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and conditions:
For each culture, heparinised whole blood were added to culture medium  containing a mitogen (phytohaemogglutinin) and incubated at 37°C. After  48 hours, the conditions of treatment were as follows, using 2  cultures/experimental point:  . without S9 mix, the test or control substances remained in the culture  medium either for 24 hours or for 48 hours, until harvest, . with S9 mix, the test or control substances remained in the culture  medium for 2 hours. The cells were then rinsed and fresh culture medium  was added. The cultures were then incubated until the appropriate harvest  time, 24 and 48 hours. Each culture was then treated for 2 hours with a colcemid solution to  block them at the metaphase-stage of mitosis and harvested. The  chromosomal preparations were stained and screened microscopically for  mitotic index and for aberrations: 200 well-spread metaphases per dose  were read, whenever possible.
Evaluation criteria:
A reproducible and statistically significant increase in the aberrant  cells frequency for at least one of the doses is considered as a positive  response.
Statistics:
 X² test
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
other: With S9 : 1000 µg/ml. Without S9 : 300 µg/ml
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: Human lymphocytes
Remarks:
Migrated from field 'Test system'.

Frequency of aberrant cells (%)

-S9

+S9

Dose

24 hours

48 hours

24 hours

48 hours

(µg/ml)

+Gap -Gap

+Gap  -Gap

+Gap -Gap

+Gap   -Gap

0

0

0

1.5

0

0

0

0.5

0

30

0

0

1.0

0.5

100

0.5

0.5

0.5

0

0

0

0

0

300

2.0

0

4.5

1.5

2.0

0.5

0.5

0

1000

1.0

0.5

0

0

+ve control

23.8

21.3

13.5

13.5

Mitotic index (% of control)

Dose

-S9

+ S9

µg/ml

24 hours

48 hours

24 hours

48 hours

30

136

78

82

81

100

100

95

124

81

300

39

89

93

100

1000

0

0

91

73

3000

0

0

0

12

5000

0

+ve control

67

33

Executive summary:

The potential of 2-thioglycolic acid to induce structural chromosome aberrations in human lymphocytes was evaluated according to OECD 473 and EC 92/69/EEC B.10 guidelines in compliance with the Principles of Good Laboratory Practice. The 2-thioglycolic acid was tested with or without a metabolic activation system (rat liver S9 mix) (2 cultures/experimental point) at the dose levels of 30, 100 and 300 µg/ml without S9 mix, and 100, 300 and 1000 µg/ml with S9 mix. Without S9 mix: the cultures were incubated with the test or control substances which remained in the culture medium, until the appropriate harvest time : 24 and 48 hours, with S9 mix: the test or control substances remained in a culture medium containing 15% S9 mix (10% S9/S9 mix) for 2 hours. The cells were then centrifuged, the treatment medium removed, the cells resuspended in fresh culture medium. The cultures were then incubated until the appropriate harvest time: 24 and 48 hours. Two hours before harvesting, the cells were treated with a colcemid solution to block them at the metaphase-stage of mitosis. The chromosomal preparations were stained and screened microscopically for mitotic index and for aberrations: 200 well-spread metaphases per dose were read, whenever possible. 2-thioglycolic acid did not induce any noteworthy increase in the number of cells with structural chromosome aberration, both with and without S9 mix, in either harvest time.

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

Genetic toxicity in vivo

Description of key information

The weight of evidence suggests that MeaTG is not genotoxic.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Test material information:
Composition 1
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
- Animals . 
. Source: Harlan Winkelmann GmbH D-33178 Borchen
. Number of Animals: 72 (36 males/36 females), 6 males and 6 females per  group
. Initial Age at Start of Acclimatisation: 8-10 weeks
. Acclimatisation: minimum 5 days
. Initial Body Weight at Start of Treatment: males mean value 37.1 g (SD  ± 2.9 g) females mean value 31.5 g (SD ± 1.9 g)

- Environmental conditions
. Housing: single Cage Type: Makrolon Type I, with wire mesh top (EHRET  GmbH, D-79302 Emmendingen)
. Bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178  Borchen)
. Temperature 22 ± 3 °C
. Relative humidity 30 - 70 %
. Artificial light 6.00 a.m. - 6.00 p.m.

- Food and water
. Feed: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH,  D-33178 Borchen)
. Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
Route of administration:
oral: gavage
Vehicle:
Name: deionised water         
Route and Frequency of Administration: orally, once         
Volume Administered: 10 mL/kg b.w.
Duration of treatment / exposure:
single administration
Frequency of treatment:
single
Post exposure period:
24 and 48 hours
Dose / conc.:
62.5 mg/kg bw/day
Dose / conc.:
125 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Name: CPA; Cyclophosphamide         
Dissolved in: deionised water         
Dosing: 40 mg/kg b.w.         
Route and frequency of administration: orally, once         
Volume administered: 10 mL/kg b.w.
Tissues and cell types examined:
Bonne marrow
Details of tissue and slide preparation:
- Preparation of the bone marrow smears
Ten animals (5 males, 5 females) per test group (all groups after 24  hours and only the high dose group after 48 hours) were killed by CO2  inhalation, following by bleeding. The femurs of the animals were removed  and the bone marrow was flushed out using foetal calf serum. After  centrifugation, the supernatant was removed and the cells in the sediment  were resuspended by shaking. A drop of this cell suspension was placed  and spread on a slide. The slides were air dried and stained with  May-Grünwald. The slides were coded so that the scorer is unaware of the  treatment group of the slide under evaluation ("blind" scoring).

- Microscopic examination of the slides
For each animal, the number of the micronucleated polychromatic  erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the  polychromatic (PE) and normochromatic (NE) erythrocyte ratio was  established by scoring a total of 1000 erythrocytes (PE + NE). 
Evaluation criteria:
The study was considered valid as the following criteria are met:
- the negative controls are in the range of our historical control data.
- the positive controls are in the range of our historical control data.
- at least 4 animals per group and sex can be evaluated
- PCE to erythrocyte ratio should not be less than 20 % of the negative  control.

A test item is classified as mutagenic if it induces either a  dose-related increase or a clear increase in the number of micronucleated  polychromatic erythrocytes in a single dose group. 
Statistics:
Statistical methods (nonparametric Mann-Whitney test (8)) will be used  as an aid in evaluating the results. However, the primary point of  consideration is the biological relevance of the results. A test item that fails to produce a biological relevant increase in the  number of micronucleated polychromatic erythrocytes is considered  non-mutagenic in this system.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs
Vehicle controls valid:
yes
Negative controls valid:
not examined
Positive controls valid:
yes
Additional information on results:
As estimated by a pre-experiment 250 mg Sodium Thioglycolate 98%, Pure  per kg b.w. was suitable.
The mean number of polychromatic erythrocytes was not decreased after  treatment with the test item as compared to the mean value of PCEs of the  vehicle control indicating that Sodium Thioglycolate 98%, Pure had no  cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no  statistically significant or biologically relevant enhancement in the  frequency of the detected micronuclei at any preparation interval and  dose level after administration of the test item. 
The mean values of micronuclei observed after treatment with Sodium Thioglycolate 98%,  Pure were below or near to the value of the vehicle control group.
40 mg/kg b.w. cyclophosphamide administered orally was used as positive  control which showed a statistically significant increase of induced  micronucleus frequency.

Summary of Micronucleus Test Results

test group

dose (mg/kg b.w)

sampling time (h)

PCEs with micronuclei (%)

range

PCE per 2000 erythocytes

vehicle

0

24

0.050

0 - 2

1098

test item

62.5

24

0.060

0 - 2

1124

test item

125

24

0.025

0 - 1

1123

test item

250

24

0.055

0 - 3

1100

Positive control

40

24

1.500

10 -43

1099

test item

250

48

0.095

0 - 6

1123

Historical Controls (1999 – 2004)

Vehicle Controls

Positive Controls (CPA)

Males

Females

Total

Males

Females

Total

Mean*±SD

0.078±0.04

0.058±0.033

0.069±0.028

1.867±0.57

1.368±0.497

1.632±0.468

Range**

0.01 - 0.23

0.0 - 0.19

0.01 - 0.15

0.70 -3.46

0.49 -3.55

0.77 - 3.48

No. of Experiments

229

217

230

228

217

229

*: mean value (percent micronucleated cells)

**: range of the mean group values (percent micronucleated cells)

Conclusions:
Interpretation of results (migrated information): negative
Sodium Thioglycolate 98%, Pure is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

The clastogenic potential of sodium thioglycolate was evaluated in a micronucleus assay on mouse bone marrow performed according to the OECD guideline # 474. Sodium thioglycolate was administered by single gavage to three groups of five male and five female NMRI mice at dose-levels of 0, 62.5, 125 and 250 mg/kg bw. The positive control was the cyclophosphamide administered orally. The polychromatic erythrocytes/normochromatic erythrocytes ratio (PE/NE) in the treated groups were equivalent to those of the control groups. However, systemic exposure (and thus bone marrow exposure) was confirmed by the clinical signs observed in males and females receiving 250 mg/kg bw of sodium thioglycolate. No increase of the frequency of the micronucleated polychromatic erythrocytes was observed in the bone marrow harvested 24 or 48 hours after the treatment. Positive and vehicle controls gave the expected results.

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

Additional information

Additional information from genetic toxicity in vivo:

Several in vitro and in vivo genotoxicity studies were performed with thioglycolic acid and its salts. The conducted genotoxicity studies on thioglycolic acid or its salts described in this chapter can be bridged to each other, because in aquous solutions only the organic thioglycolate anion may have the potential to cause genotoxic effects in vitro or in vivo. All genotoxicity studies conducted to date have either negative results or are of douptful significance. Therefore, the weight of evidence suggests that thioglycolic acid and its salts are non-genotoxic.

Justification for classification or non-classification

Conclusive, but not sufficient for classification.