Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In reverse gene mutations assays with multiple strains of Salmonella typhimurium performed with methods compliant or comparable to the OECD guideline # 471, mercaptoacetic acid and its ammonium and sodium salts were not mutagenic in the presence and absence of metabolic activation. In a gene TK+/- mutation assay in mouse lymphoma L5178Y cells, performed following the OECD guideline # 476, ammonium mercaptoacetate was also not mutagenic in the presence and absence of metabolic activation. As well, mercaptoacetic acid was not clastogenic, with or without metabolic activation, in an in vitro chromosomal aberration assay in human lymphocytes performed following the OECD guideline # 473.

 

Gene Mutations Assays

Several bacterial and mammalian mutation assays have been conducted and demonstrated no mutagenic activity.

The most recent bacterial gene mutations assay was conducted with ammonium mercaptoacetate 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 mercaptoacetate 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 (Thompson, 2003).

 

In former studies performed according to the US NTP protocol, four S. typhimurium strains were exposed to mercaptoacetic acid (TA97, TA 98, TA 100 and TA 1535) or sodium mercaptoacetate (TA 98, TA 100, TA 1535 and TA 1537) in a preincubation assay. Based on a preliminary toxicity assay, concentrations up to 3,333 (TGA) or 1000 (NaTG) µg/plate were used with or without metabolic activation (rat and hamster S9) in all four strains. Mercaptoacetic acid and its sodium salt did not induce mutations in these studies. Positive and solvent controls gave the expected results (Zeiger et al., 1987).

 

Ammonium mercaptoacetate was tested in a Mouse Lymphoma Forward Mutation Assay performed according to the OECD guideline # 476. At concentrations up to 1,600 µg/ml (10 mM), ammonium mercaptoacetate did not induce gene mutations in the mouse lymphoma L5178Y heterozygous TK+/-cells, with or without metabolic activation. The spontaneous mutation frequencies and the levels of activity of the positive controls confirmed the sensitivity of the test system (Wollny, 2004).

 

Chromosomal aberrations Assays

Mercaptoacetic acid was tested at concentrations of 0, 30, 100 and 300 µg/ml, without metabolic activation and 0, 100, 300 and 1000 µg/ml with metabolic activation (rat S9), in an in vitro chromosome aberration test in human lymphocytes performed according to the OECD guideline # 473. Exposures were for 24 and 48 hours in absence of S9 and 2 hours in presence of S9. Two hundreds cells per dose level were evaluated for chromosomal aberrations. Cytotoxicity was observed at concentration of 300 µg/plate without S9 and>1000 µg/ml with S9. The positive controls (mitomycin-C, without activation and cyclophosphamide with activation) gave the expected values. Mercaptoacetic acid did not induce structural chromosome aberrations in this test (Molinier, 1994).

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine reversion
Species / strain / cell type:
other: 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 / solvent:
sterile distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene, 1,8-Dihydroxyanthraquinone
Details on test system and experimental 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
Species / strain:
other: TA1535, TA1537, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
= 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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 bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
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
Target gene:
Histidine reversion
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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 / solvent:
no data
Untreated negative controls:
no
Negative solvent / vehicle 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 experimental 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
Species / strain:
other: TA98, TA100, TA1535 and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Mutagenicity of Sodium Thioglycolate in Salmonella typhimuriuma

Strain

Dose (µg/plate)

Without S9

Without S9

With 10%

hamster S9

With 10%

hamster S9

With 10%

rat S9

With 10%

rat S9

TA100

0

100 ± 5

110 ± 6

185 ± 26

179 ± 12

175 ± 28

189 ± 9

10

95 ± 5

126 ± 11

160 ± 18

164 ± 6

140 ± 30

204 ± 38

33

105 ± 31

129 ± 8

195 ± 30

174 ± 8

170 ± 41

189 ± 4

100

115 ± 10

121 ± 4

220 ± 18

221 ± 4

145 ± 18

175 ± 9

333

125 ± 13

122 ± 8

160 ± 5

173 ± 10

135 ± 9

221 ± 8

1,000

120 ± 30

124 ± 9

215 ± 13

202 ± 4

155 ± 35

181 ± 18

Trial summary

Negative

Negative

Negative

Equivocal

Negative

Negative

Positive controlb

430 ± 28

599 ± 36

558 ± 6

836 ± 173

300 ± 11

430 ± 70

TA1535

0

7 ± 1

7 ± 1

6 ± 1

7 ± 3

7 ± 1

10

6 ± 1

6 ± 1

5 ± 1

6 ± 2

8 ± 1

33

5 ± 1

6 ± 1

7 ± 2

9 ± 1

6 ± 1

100

8 ± 4

9 ± 1

7 ± 1

9 ± 1

6 ± 1

333

6 ± 1

6 ± 1

5 ± 1

7 ± 2

8 ± 1

1,000

5 ± 1

8 ± 2

5 ± 0

8 ± 1

8 ± 1

Trial summary

Negative

Negative

Negative

Negative

Negative

Positive control

288 ± 32

251 ± 41

106 ± 16

43 ± 4

135 ± 10

TA1537

0

6 ± 3

5 ± 1

8 ± 1

12 ± 2

5 ± 1

7 ± 2

10

7 ± 1

6 ± 1

4 ± 1

9 ± 3

4 ± 1

8 ± 2

33

6 ± 1

6 ± 1

6 ± 0

11 ± 2

8 ± 2

12 ± 2

100

5 ± 1

3 ± 2

5 ± 1

6 ± 1

6 ± 2

8 ± 2

333

6 ± 1

8 ± 2

6 ± 1

9 ± 3

5 ± 1

9 ± 1

1,000

7 ± 2

6 ± 0

6 ± 0

9 ± 0

6 ± 1

10 ± 2

Trial summary

Negative

Negative

Negative

Negative

Negative

Negative

Positive control

133 ± 12

248 ± 79

92 ± 9

48 ± 7

48 ± 8

60 ± 29

TA98

0

17 ± 2

14 ± 2

22 ± 2

21 ± 1

18 ± 2

16 ± 1

10

17 ± 2

11 ± 2

25 ± 2

19 ± 4

27 ± 5

20 ± 2

33

21 ± 2

14 ± 2

23 ± 2

16 ± 3

15 ± 3

18 ± 3

100

21 ± 3

13 ± 2

18 ± 2

17 ± 1

18 ± 3

15 ± 1

333

20 ± 2

13 ± 2

23 ± 2

19 ± 2

19 ± 3

15 ± 1

1,000

22 ± 3

14 ± 3

18 ± 3

18 ± 1

16 ± 1

16 ± 3

Trial summary

Negative

Negative

Negative

Negative

Negative

Negative

Positive control

246 ± 23

256 ± 44

468 ± 16

444 ± 51

142 ± 23

134 ± 15

a Data are presented as revertants/plate (mean ± standard error) from three plates. Study was performed at Case Western Reserve University. The detailed protocol and these data are presented by Zeiger et al. (1987). 0 µg/plate was the solvent control.

b The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA1537), and 4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine reversion
Species / strain / cell type:
other: Strains: TA97, TA98, TA100 & TA1535
Metabolic activation:
with and without
Metabolic activation system:
10% and 30 of Rat and Hamster liver S9 induced with Aroclor 1234
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 2000, 3333 µg/plate
Vehicle / solvent:
water
Untreated negative controls:
no
Negative solvent / vehicle 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 experimental 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.
- 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
Species / strain:
other: TA97, TA98, TA100 & TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
With S9: >= 2000 µg/plate. Without S9: >= 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Chemical Name:

Thioglycolic acid

CAS Number:

68-11-1

Study Type:

Salmonella

Study ID:

301735

Study Result:

Negative

Year Completed:

1983

Vehicle Control:

Water

Protocol:

Preincubation

Table Instructions and Notes: You are currently viewing the results for this Salmonella study's detailed data.

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

30% HLI

10% RLI

30% RLI

(Negative)

(Negative)

(Negative)

(Negative)

(Negative)

(Negative)

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

30

4.9

43

3.4

11

1.2

15

3.2

13

1.5

17

2.6

33     

26

3.2

38

7.8

7

1.5

15

1.5

7

0.3

14

0

100     

28

4.2

41

1.2

9

1.5

16

1.7

11

1.5

14

3.1

333     

23

1.8

41

4.3

9

2.3

12

2.3

9

0.3

14

2.5

1000     

9

4.9

28

5

7

1.2

11

2

9

0.9

11

0.9

2000     

 

 

 

 

10

1.2

10s

1.2

6

2

10s

2

3333     

1s

0.3

13s

1.5

 

 

 

 

 

 

 

 

Positive Control

1065

17.4

531

10.7

122

11.5

154

5.9

89

2

121

8.4

Strain: TA100

Dose

No Activation

No Activation

10% HLI

30% HLI

10% RLI

30% RLI

(Negative)

(Negative)

(Negative)

(Negative)

(Negative)

(Negative)

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

91

3.2

129

5

83

9

109

4.7

100

3.7

133

8.7

33     

91

10.1

108

9.9

85

1.5

111

7.9

90

7.2

103

2.2

100     

92

6.7

113

9.2

77

2.5

100

11.9

94

7.2

127

6.4

333     

81

1.3

113

6.9

101

3.3

89

4.4

107

9.3

101

8.1

1000     

78

2.3

88

4.7

87

7.5

104

1.7

89

3

123

7.5

2000     

 

 

 

 

63

9.6

88s

8.3

63

5.8

90s

3

3333     

2s

0.9

39s

6.3

 

 

 

 

 

 

 

 

Positive Control

1407

48.6

1122

27.6

1373

25.2

400

39.4

1218

11.7

416

10.4

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
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
Target gene:
Locus Examined: thymidine kynase, the selection agent used was 5 µg/ml trifluorothymidine
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (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 / cell type 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:
- Solvent used: deionised water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methyl methanesulfonate (13 µg/mL) without activation; cyclophosphamide with activation (3 µg/mL)
Details on test system and experimental 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.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 4-h experiment (+/-S9): > 1600 µg/ml / 24-h experiment (-S9): >= 800 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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:
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
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
Species / strain / cell type:
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 / solvent:
distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:  mitomycin C 0.2 µG/ml (without S9) and  cyclophosphamide 50 µg/ml (with S9)
Details on test system and experimental 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
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: With S9 : 1000 µg/ml. Without S9 : 300 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

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

In a micronucleus assay on the peripheral blood of mice treated dermally for 13 weeks with sodium mercaptoacetate, a slight but statistically significant increase of the frequency of the micronucleated normochromatic erythrocytes was only observed in female mice at the top dose level of 360 mg/kg bw/d. The Scientific Committee on Consumer Safety (SCCS/1520/13) agrees with the conclusion that this result is of doubtful significance because mercaptoacetic acid did not induce structural chromosomal aberrations in vitro, and mercaptoacetic acid and its sodium salt failed to show any evidence of clastogenic potential when administered acutely by the dermal and oral routes up to the maximum tolerated dose in the two mouse bone marrow micronucleus assays performed following the OECD guideline 474. In the sex-linked recessive lethal mutations test, sodium mercaptoacetate was not mutagenic. The weight of evidence suggests that mercaptoacetic acid and its salts are not genotoxic.

 

The clastogenic potential of mercaptoacetic acid and sodium mercaptoacetate was evaluated in 2 micronucleus assays on mouse bone marrow performed according to the OECD guideline # 474 and in a micronucleus assay on mouse peripheral blood performed according to the US NTP protocol.

 

Mercaptoacetic acid, partially neutralized to pH4, was administered dermally over a 2-day period to three groups of five male and five female Swiss mice at dose-levels of 0, 250, 500 and 1000 mg/kg bw/day for males or 0, 125, 250 and 500 mg/kg/day for females (Haddouk, 2006) and sodium mercaptoacetate 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 (Honarvar, 2005). For each study, the positive control was the cyclophosphamide administered orally.

In both studies, the polychromatic erythrocytes/normochromatic erythrocytes ratios (PE/NE) in the treated groups were equivalent to those of the control groups. However, systemic exposure was confirmed by the mortality observed in males given 1000 mg/kg/day mercaptoacetic acid, as well as by the clinical signs observed in males treated with 1000 and 500 mg/kg/day mercaptoacetic acid and in males and females receiving 250 mg/kg bw of sodium mercaptoacetate. No increase of the frequency of the micronucleated polychromatic erythrocytes was observed in the bone marrow harvested 24 hours after the treatment(s). Positive and vehicle controls gave the expected results.

 

At the end of the 3-month dermal toxicity study with sodium mercaptoacetate (0, 22.5, 45.0, 90.0, 180.0 and 360.0 mg/kg bw /d), peripheral blood samples were obtained from male and female B6C3F1 mice (NTP). Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) in each of five animals per dose group. In addition, the percentage of polychromatic erythrocytes in a population of 1,000 erythrocytes was determined as a measure of bone marrow toxicity. A slight but statistically significant increase (p = 0.0021) of the frequency of the micronucleated normochromatic erythrocytes was observed in female mice at the top dose level of 360 mg/kg bw/day (4.4‰ vs 2.1‰ in control females). The NIEHS regards this result as negative in males and positive in females.

 

In a former study, the mutagenic potential of sodium mercaptoacetate was evaluated using the sex-linked recessive lethal mutations test (Gocke et al., 1981). One dose (close to the LD50) of 25 mM sodium mercaptoacetate in 5.0% saccharose was fed to Berlin K (wild type) and Basc strains of Drosophila melanogaster. Approximately 1200 X chromosomes were tested per experiment in each of three successive broods. F2 progeny cultures with two or fewer wild-type males were routinely retested in the F3 generation to confirm X-linked recessive lethal mutations. Sodium mercaptoacetate was not mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
- Animals
. Preliminary toxicity test: 12 male and 15 female mice were used,
. Main cytogenetic test: 55 mice, 27 males and 28 females were used,
. Blood sampling: six mice, three males and three females were used.
. Strain: Swiss Ico: OF1 (IOPS Caw).
. Breeder: Charles River Laboratories, l'Arbresle, France.
. Age: on the day of treatment, the animals were approximately 6 weeks  old.
. Acclimation: at least 5 days before the day of treatment.

- Environmental conditions
· Temperature: 22 ± 2°C,
· Relative humidity: 30 to 70%,
· Light/dark cycle: 12 h/12 h (07:00 - 19:00),
· Ventilation: at least 12 cycles/hour of filtered non-recycled fresh air.
. Housing: individually in polycarbonate cages (24.0 x 13.5 x 13.0 cm)  containing autoclaved sawdust (SICSA, Alfortville, France).

- Food and water
. Food: A04 C pelleted maintenance diet (SAFE, Villemoisson-sur-Orge,  France), ad libitum.
. Water: drinking water filtered by a FG Millipore membrane (0.22  micron), ad libitum.
Route of administration:
dermal
Vehicle:
- Formulation of the test substance:
. Vehicle: 
Purified water (CIT, Millipore) degassed by sonication for at  least 15 minutes and saturation with nitrogen gas for at least 15 min . 
. Treatment volume: 
3 mL/kg.
. Formulation procedure: 
All the dose-levels and concentrations were  expressed as active item, taking into account the active material content  of 80.22% (w/w).
.. For the preliminary tests:
Treatment with neutralized test item: the maximum achieved  concentration of the preparation neutralized  using NaOH (10 M) was 497  mg/mL (obtained pH ranging from 6.92 to 7.24). Using a treatment volume  of 3 mL/kg, the target dose-level was 1491 mg/kg/day.
.. Remaining treatments: 
The test item preparations were brought to pH  ranging from 3.75 to 4.21, by addition of NaOH (10 M). The preparations  were used at the concentrations of 666.6, 500, 333.3, 250 and 166.67  mg/mL, in order to reach the target dose-levels of 2000, 1500, 1000, 750  and 500 mg/kg/day, respectively, using a treatment volume of 3 mL/kg.  All the test item dosage forms were prepared extemporaneously. In  addition, except for the preparation at 666.6 mg/mL (target dose-level of  2000 mg/kg/day), the preparations were made under nitrogen atmosphere and  were stored at room temperature and under nitrogen atmosphere until  treatment.
.. For the main cytogenetic test: 
The test item dosage forms were  prepared extemporaneously under nitrogen atmosphere and were stored at  room temperature and under nitrogen atmosphere until treatment. The pH of the preparations was adjusted to a value of approximately 4  (ranging from 4.0 to 4.10) by addition of NaOH (10 M).  The preparations were used at the concentrations of 41.67, 83.33, 166.67  and 333.3 mg/mL, in order to reach the target dose-levels of 125, 250,  500 and 1000 mg/kg/day, respectively, using a treatment volume of 3  mL/kg. 
Details on exposure:
- Preparation of the animals:
On the day before the beginning of the treatment period, an area of  approximately 1,5 x 2 cm or 2,5 x 2 cm (3 to 5 cm2), estimated to be up  to 10% of the total body surface (1) was clipped free from hair, as close  to the skin as possible on the dorsum of the animals, with an electric  clipper. Before the first dosage form application, the treatment area was examined  and any animals showing skin abnormality and/or irritation was replaced  from the spare animals ordered.  During the treatment period, the animals were clipped whenever necessary,  at least 4 hours before dosing.

- Administration
The test item formulated in a vehicle was applied in a film as thin and  uniform as possible, to the clipped area of the skin. The dosage forms  was applied to the dorsum using a micropipette.  No dressing or protective plastic collar was used. Each animal was given the dosage forms once a day, on two consecutive  days, at approximately the same time.

- Preliminary toxicity test
In order to determine the highest dose-level, several preliminary tests  were performed on groups of six animals (three males and/or three  females). Clinical signs, any local reaction at the treatment site  (following the below-mentioned scoring scale) and any mortality was  recorded over a period of 48 hours. At the end of this period, the  animals were killed by CO2 inhalation in excess.
Duration of treatment / exposure:
2 administrations at 24-h interval
Frequency of treatment:
daily
Post exposure period:
none
Remarks:
Doses / Concentrations:
Males: 1000, 500 and 250 mg/kg. Females: 500, 250 and 125 mg/kg
Basis:

No. of animals per sex per dose:
5 in the control groups and in the low and intermediate dose groups. 7 or 8 in the high dose group
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPA), batch No. 4A121 (Endoxan, Baxter, Maurepas,  France) dissolved in distilled water at a concentration of 5 mg/mL.
. route: oral
. treatment volume: 10 mL/kg
. number of administrations: one
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
- Preparation of the bone marrow smears:
At the time of sacrifice, all the animals were killed by CO2 inhalation  in excess. The femurs of the animals were removed and the bone marrow was  flushed out using fetal 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 Giemsa. 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:
For a result to be considered positive, a statistically significant  increase in the frequency of MPE must be demonstrated when compared to  the concurrent vehicle control group. Reference to historical data, or  other considerations of biological relevance was also taken into account  in the evaluation of data obtained.
Statistics:
Normality and homogeneity of variances was tested using a Kolmogorov  Smirnov test and a Bartlett test. If normality and homogeneity of variances are demonstrated, the  statistical comparisons were performed using a Student t-test (2 groups)  or a one-way analysis of variance (>= 3 groups) followed by a Dunnett  test (if necessary). If normality or homogeneity of variances are not demonstrated, a  Mann/Withney test (2 groups) or a Kruskall Wallis test (>= 3 groups) was  performed followed by a Dunn test (if necessary). All these analyses were performed using the software SAS Enterprise Guide  V2 (2.0.0.417, SAS Institute Inc), with a level of significance of 0.05  for all tests.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

- PRELIMINARY TOXICITY TEST
. Treatment with neutralized test item (target pH of 7):
At 1491 mg/kg/day, no mortality and no clinical signs were noted in the  three males and three females treated with the test item. In one male  (1/3), a very slight erythema was noted since 24h after the first  treatment until sacrifice.
. Treatment with partially neutralized test item (target pH of 4):
For all dose-levels tested no cutaneous reactions were noted in any of  the treated animals.
At 2000 mg/kg/day, 2/3 males and 2/3 females were found dead 16 hours  following the first treatment. The two surviving animals (one male and  one female) were sacrificed and no second treatment was performed.
At 1500 mg/kg/day (males only), 2/3 males were found dead 17 hours  following the first treatment. The surviving animal was sacrificed and no  second treatment was performed.
At 1000 mg/kg/day, no deaths occurred in males, either hypoactivity,  half-closed eyes and piloerection or only piloerection were observed  following the second treatment. In females, one animal (1/3) was found  dead more than 16 hours following the second treatment. The two surviving  females showed hypoactivity, lateral recumbency, tremors, piloerection  and/or half-closed eyes.
At 750 mg/kg/day (females only), one animal (1/3) was found dead 21 hours  following the first treatment. The two surviving females showed poor  clinical conditions and were therefore sacrificed immediately.
At 500 mg/kg/day (females only), no deaths occurred. Dyspnea and  piloerection were noted in 3/3 females following the second treatment. 

- CYTOGENETIC TEST
In males treated at 1000 mg/kg/day, mortality was observed (3/7).  Piloerection was noted in the surviving animals.
In males treated at 500 mg/kg/day, piloerection was noted (3/5).
No clinical signs and deaths were observed in males treated at 250  mg/kg/day as well as in females during the study.
For all dose-levels tested no cutaneous reactions were noted in any of  the treated animals.
For both males and females, the mean values of MPE as well as the PE/NE  ratio in the groups treated with the test item, were equivalent to those  of the vehicle group.
Cyclophosphamide induced a significant increase in the frequency of MPE,  indicating the sensitivity of the test system under our experimental  conditions. The study was therefore considered valid.

Results of the cytogenetic test: data summary

Group

Doses(1) (mg/kg/day)

MPE/1000PE

PE/NE ratio

Time of sacrifice after the last administration

mean

(sd)

mean

(sd)

Males

Vehicle

 -

2.1

(0.5)

0.5

(0.2)

24 h

Test item

250

2.3

(1.7)

0.6

(0.1)

500

1.6

(1.1)

0.6

(0.1)

1000

1.1

(0.5)

0.6

(0.2)

Cyclophosphamide

50

19.6

(4.2)*

0.5

(0.0)

Females

Vehicle

-

0.9

(0.5)

0.6

(0.1)

24 h

Test item

125

1.7

(0.8)

0.8

(0.2)

250

0.9

(0.7)

0.9

(0.4)

500

1.5

(0.7)

0.8

(0.3)

Cyclophosphamide

50

15.1

(6.2)**

0.7

(0.1)

(1)expressed as active item

Five animals per group (except for the high-dose treated group of males: four animals)

MPE: Micronucleated Polychromatic Erythrocytes

PE: Polychromatic Erythrocytes

NE: Normochromatic Erythrocytes

sd: standard deviation

Statistical significance:* p < 0.05 ** p < 0.01

Conclusions:
Interpretation of results (migrated information): negative
THIOGLYCOLIC ACID did not induce any damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two cutaneous administrations, at a 24 hour interval, at the dose-levels of 250, 500 and 1000 mg/kg/day for males or 125, 250 and 500 mg/kg/day for females.
Executive summary:

The clastogenic potential of thioglycolic acid was evaluated in a micronucleus assay on mouse bone marrow performed according to the OECD guideline # 474. Thioglycolic acid, partially neutralized to pH 4, was administered dermally over a 2-day period to three groups of five male and five female Swiss mice at dose-levels of 0, 250, 500 and 1000 mg/kg bw/day for males or 0, 125, 250 and 500 mg/kg/day for females. The positive control was the cyclophosphamide administered orally. The polychromatic erythrocytes/normochromatic erythrocytes ratios (PE/NE) in the treated groups were equivalent to those of the control groups. However, systemic exposure was confirmed by the mortality observed in males given 1000 mg/kg/day thioglycolic acid, as well as by the clinical signs observed in males treated with 1000 and 500 mg/kg/day thioglycolic acid. No increase of the frequency of the micronucleated polychromatic erythrocytes was observed in the bone marrow harvested 24 hours after the last treatment. Positive and vehicle controls gave the expected results.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
other: Micronucleus assay on mouse bone marrow
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
Remarks:
Doses / Concentrations:
0, 62.5, 125 and 250 mg/kg bw
Basis:
actual ingested
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.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
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 erythrocytesratios (PE/NE) in the treated groups were equivalent to those of the control groups. However, systemic 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:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
For the time being, the draft report (TOX-80) is still undergoing a multi-tiered review that includes NTP staff review and external peer review. The publication of the final TOX-80 is expected in 2009. However, the data available on the NTP website are sufficiently detailed for a preliminary assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
other: NTP standard protocol
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Route of administration:
dermal
Vehicle:
water/ethanol
Details on exposure:
In a subchronic dermal toxicity study in mice, a blood sample was obtained at the end of the 13-week exposure period from male and female mice in each dose group (5 animals per treatment  group per sex).
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
5 days/week
Post exposure period:
None
Dose / conc.:
22.5 mg/kg bw/day (actual dose received)
Dose / conc.:
45 mg/kg bw/day (actual dose received)
Dose / conc.:
90 mg/kg bw/day (actual dose received)
Dose / conc.:
180 mg/kg bw/day (actual dose received)
Dose / conc.:
360 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
None
Tissues and cell types examined:
Peripheral blood
Details of tissue and slide preparation:
Slides are prepared, air-dried, fixed, and stained  with a fluorescent DNA-specific stain that easily illuminates any  micronuclei that may be present. 2,000 mature erythrocytes  (normochromatic erythrocytes or NCEs) are scored per animal for presence  of micronuclei. These mature erythrocytes represent about 95% or more of  the circulating erythrocytes. The percent PCE is determined in the blood  as a measure of chemical-induced toxicity to the bone marrow. All data  are analyzed separately for male and female mice.
Evaluation criteria:
Factors which must be considered in analyzing micronucleus test data  include number of animals per dose group (a minimum of 3 is required),  dose levels and number of doses administered, route of administration,  tissue and cell type analyzed, sample time (interval between last dosing  and harvesting of cells for analysis), frequencies of micronucleated  cells in the negative and positive controls, and the results of the  statistical analyses.
Statistics:
A formal statistical analysis of the data is performed, including a trend  test, to determine if there is an overall increase across all doses in  the frequency of cells containing micronuclei, and a pairwise comparison  of each dose group to the corresponding control, to see if any one dose  group is statistically different from the control group in frequency of  micronucleated cells. Data are typically presented as the mean number of  micronucleated cells per 1,000 cells for each treatment group. A positive  trend test is one in which the P value is equal to or less than 0.025.  For the micronucleus frequency in any dose group to be considered  significantly elevated over the control group, the P value must be equal  to or less than 0.025 divided by the number of chemical-treatment groups.  Thus, if the number of treated groups is 3, then the required pairwise P  value is 0.008.
Sex:
male/female
Genotoxicity:
ambiguous
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not examined
Additional information on results:
A statistically significant increase (p=0.0021) of the frequency of the  micronucleated normochromatic erythrocytes was observed in female mice at  the top dose level of 360 mg/kg (4.4 o/oo vs 2.1 o/oo in control  females).  However, the same values were observed in the treated male mice (4.0 to  4.6 o/oo) but without statistical significance due to a higher control  value in male mice (3.4 o/oo).

Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Dermal Application of Sodium Thioglycolate for 3 Monthsa

Dose (mg/kg)

Number of Mice with Erythrocytes Scored

Micronucleated NCEs/1,000 NCEsb

P Valuec

PCEsb

(%)

Male

95% Ethanol:deionized waterd

5

3.4 ± 0.29

3.20 ± 0.18

Sodium thioglycolate

22.5

5

4.1 ± 0.51

0.2090

3.28 ± 0.11

 

45

5

4.6 ± 0.73

0.0894

4.08 ± 0.23

 

90

5

4.3 ± 0.56

0.1521

4.04 ± 0.35

 

180

5

4.0 ± 0.16

0.2423

3.80 ± 0.43

 

360

5

4.4 ± 0.37

0.1283

3.58 ± 0.29

                                                                                                      P=0.290e

Female

95% Ethanol:deionized water

5

2.1 ± 0.10

3.88 ± 0.17

Sodium thioglycolate

22.5

5

3.0 ± 0.32

0.1035

3.22 ± 0.26

 

45

5

2.6 ± 0.24

0.2326

3.48 ± 0.44

 

90

5

3.1 ± 0.48

0.0825

2.68 ± 0.15

 

180

5

3.3 ± 0.20

0.0510

3.30 ± 0.44

 

360

5

4.4 ± 0.29

0.0021

3.32 ± 0.18

                                                                                                      P=0.002

a Study was performed at ILS, Inc. The detailed protocol is presented by MacGregor et al. (1990). NCE=normochromatic erythrocyte; PCE=polychromatic erythrocyte

b Mean ± standard error

c Pairwise comparison with the vehicle control group; dosed group values are significant at P=0.005

d Vehicle control at a 1:1 ratio

e Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed trend test; significant at P=0.025

Conclusions:
The Scientific Committee on Consumer Safety (SCCS/1520/13) agrees with the conclusion that this result is of doubtful significance because thioglycolic acid did not induce structural chromosomal aberrations in vitro, and thioglycolic acid and its sodium salt failed to show any evidence of clastogenic potential when administered acutely by the dermal and oral routes up to the maximum tolerated dose in the two mouse bone marrow micronucleus assays performed following the OECD guideline 474.
Executive summary:

At the end of the 3-month dermal toxicity study with sodium thioglycolate (0, 22.5, 45.0, 90.0, 180.0 and 360.0 mg/kg bw /d), peripheral blood samples were obtained from male and female B6C3F1 mice. Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with acridine orange and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) in each of five animals per dose group. In addition, the percentage of polychromatic erythrocytes in a population of 1,000 erythrocytes was determined as a measure of bone marrow toxicity.

A slight but statistically significant increase (p = 0.0021) of the frequency of the micronucleated normochromatic erythrocytes was observed in female mice at the top dose level of 360 mg/kg bw/day (4.4‰ vs 2.1‰ in control females). The NIEHS regards this result as negative in males and positive in females.

Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
no guideline followed
Principles of method if other than guideline:
Won (1977) Abstr Ann Meet Am Soc Microbiol, 77, 276.
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay
Species:
Drosophila melanogaster
Strain:
other: Berlin K and Basc
Sex:
not specified
Route of administration:
oral: feed
Details on exposure:
In Drosophila one dose close to the LD50 was applied by the adult feeding  method in 5% saccharose. About 1200 X-chromosomes were tested per  experiment in each of 3 successive broods (3-3-4 days). In repeat  experiments, sometimes only single broods were tested. F2 progeny  cultures with 2 or fewer wild-type males were routinely retested in the  F3 generation to confirm X-linked recessive lethal mutations (RLs).  Mosaics were not counted. "Clusters" of 2 were included because their  occurrence was compatible with statistical expectation of independent  origin. No additional details available.
Duration of treatment / exposure:
3 broods
Dose / conc.:
25 other: mM
Control animals:
yes, historical
Positive control(s):
Positive control:  treminon
Sex:
not specified
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Negative
Executive summary:

The mutagenic potential of sodium thioglycolate was evaluated using the sex-linked recessive lethal mutations test. One dose (close to the LD50) of 25 mM sodium thioglycolate in 5.0% saccharose was fed to Berlin K (wild type) and Basc strains of Drosophila melanogaster. Approximately 1200 X chromosomes were tested per experiment in each of three successive broods. F2 progeny cultures with two or fewer wild-type males were routinely retested in the F3 generation to confirm X-linked recessive lethal mutations. Sodium thioglycolate was not mutagenic.

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

Additional information

Justification for classification or non-classification

According to REGULATION (EC) No 1272/2008 of 16 December 2008 and the available genotoxicity data, no classification is warranted for mercaptoacetic acid.