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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
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

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Ammonium Thioglycolate 71%
Source: Bruno Bock Chemische Fabrik GmbH & Co KG
Description: colourless liquid
Batch number: 4804
Content of active ingredient: 71.2%

Method

Target gene:
Histidine reversion
Species / strain
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
Controls
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

Results and discussion

Test results
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.