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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:
weight of evidence
Study period:
11 November 1987 to 4 December 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

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
Test material form:
solid

Method

Target gene:
Histidine
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material to the tester organisms. In the preliminary toxicity study, Dispersionsblau F-60 768 exhibited no toxicity to any of the strains of Salmonella at the maximum dose of 5000 ug/well.
In the main study five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standards for mutagenicity tests.
0, 8, 40, 200, 1000, 5000 μg/plate
Vehicle / solvent:
Dispersionsblau F-60 768 was accurately weighed and dissolved in dimethyl sulphoxide and appropriate dilutions made.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
other: N-Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG); 4 NItro-O-phenyldiamine (4NOPD); 2-Aminoanthracene (2-AA)
Details on test system and experimental conditions:
Test Procedure
Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material to the tester organisms. 0.1 ml of bacterial suspension was added to 4 ml of molten, trace histidine supplemented media (histidine/biotin & top agar) and overlayed onto sterile plates of Vogel Bonner agar (minimal agar- 25 ml/plate). When the agar had set, wells were made on each plate and 0.1 ml aliquots of concentrations ranging from 80 μg/ml to 50,000 μg/ml of test material were added to each well. These plates were incubated at 37 °C for at least 24 hours after which time the toxicity of the test material was assessed by measuring the zones of inhibition around each well.
Dimethyl sulphoxide, which was used as a solvent diluent for the test material in this assay, was also added to each plate as a control.

Mutation Study
Experiment 1
Five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standards for mutagenicity tests using micro-organisms.

Test Material and Negative Controls
0.1 ml of the appropriately diluted test material or negative control solution was placed in sets of sterile test tubes containing 2.0 ml of molten, trace histidine supplemented, top agar at 45 °C. These sets comprised of two test tubes for each bacterial tester strain. A 0.1 ml aliquot of one of the bacterial suspensions was also added to each of the two test tubes. Into one of the test tubes was placed 0.5 ml of the S-9 liver microsome mix; in the other tube 0.5 ml of pH 7.4 buffer was added. This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material.

Positive Controls
Without Activation
0.1 ml of one of the appropriate positive control solutions (MNNG, 9AA, or 4NOPD) was added to a test tube containing 2.0 ml of molten, trace histidine supplemented, top agar. 0.1 ml of bacterial suspension and 0.5 ml of pH 7.4 buffer was also added to the agar. This procedure was then repeated, in triplicate, for each of the positive controls and each of the bacterial strains.
With Activation
0.1 ml of 2AA solution was added to a test tube containing 2.0 ml of molten, trace histidine supplemented, top agar. 0.1 ml of one of the test bacterial suspensions and 0.5 ml of S-9 mix were also added to the agar. The procedure was then repeated, in triplicate, for each tester strain.
The contents of each test tube were equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). These plates were incubated at 37 °C for at least 48 hours and the number of revertant colonies counted.

Experiment 2
The complete experiment was repeated using fresh bacterial cultures, test material and control solutions.

Tester Strains
The strains used in this assay were all mutants derived from Salmonella typhimurium LT2 and were those recommended for general screening.
TA1535: sensitive to agents inducing base-pair substitution
TA100: sensitive to agents inducing base-pair substitution
TA1537: sensitive to agents inducing frame-shift mutations
TA1538: sensitive to agents inducing frame-shift mutations
TA98: sensitive to agents inducing frame-shift mutations

These strains were obtained from the British Industrial Biological Research Association on 14th August 1987 and were stored at 4 °C on nutrient agar slopes. Prior to being used, characterisation checks were carried out to determine the amino-acid requirement, presence of rfa and R factors and the spontaneous reversion rate.
In this assay, overnight sub-cultures of the master slopes were prepared in nutrient broth (Oxoid Limited) and incubated at 37 °C for approximately 18 hours. These overnight cultures yielded approximately 107 - 109 bacteria per ml.

Microsomal Enzyme Fraction
Lot No. Aro. S-9/08/10, prepared on 8 October 1987 was obtained from the British Industrial Biological Research Association on 8 October 1987. For the second experiment Lot. No. Aro. S-9/17/11, prepared on 17 November 1987 was obtained from B.I.B.R.A. on 19 November 1987. They were prepared from the livers of male Sprague-Dawley rats weighing 150 - 200g. These had received a single i.p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S-9 preparation.
The S-9 was stored at -180 °C in a Statebourne liquid N2 freezer, model SXR 34.
Rationale for test conditions:
The study was conducted according to Laboratory Protocol and was designed to assess the mutagenic potential of the test material using a bacterial/microsome test system. The study was based on the in vitro technique described by Ames and his co-workers and Garner et al in which mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium to various concentrations of the test material.
Evaluation criteria:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S-9 microsomal enzymes in both experiments. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at all dose levels employed, the intervals of which should be between 2- and 5-fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5 mg/plate.
Statistics:
Statistical methods not referenced in the study report.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Study
Dispersionsblau F 60 768 exhibited no toxicity to any of the strains of Salmonella at the maximum dose of 5000 μg/well.

Mutation Study
The overnight culture of each strain was found to be in the required range of 107 to 109 bacteria per ml and the spontaneous reversion rate for each was found to be within the expected range.
A marked precipitate of Dispersionsblau F-60 768 was seen at 5000 μg/plate in both experiments and intermittently at 2500 ug/plate in the second experiment. In some cases the precipitate prevented accurate recording of the numbers of revertant colonies, especially where the revertant colonies were small.
Dispersionsblau F-60 768 induced a highly significant dose-related increase in the number of bacterial revertant colonies in all the Salmonella strains both with and without metabolic activation. An increase was noted between a range of 8 - 5000 μg/plate with the maximum response recorded at a dose level of 2500 μg/plate. In all cases the response was greater in the presence of metabolic activation than in its absence. Also, the response seen in the bacterial strains that detect frameshift mutations (TA1537, TA1538 and TA98) was moderately stronger than that seen in the base-substitution strains (TA1535 and TA100).

The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S-9 fraction was found to be satisfactory.

Any other information on results incl. tables

VIABILITY AND SPONTANEOUS REVERSION TESTS

EXPERIMENT 1

Strain of

Salmonella typhimurium

Bacteria per 1 ml of overnight nutrient broth

Total counts on trace histidine supplemented top agar

TA1535

 

TA 1537

 

TA 1538

 

TA98

 

TA100

15 x 107

 

1 x 107

 

8 x 107

 

11 x 107

 

12 x 107

22

 

8

 

20

 

25

 

150

36

 

9

 

28

 

20

 

138

31

 

11

 

30

 

18

 

153

EXPERIMENT 2

Strain of

Salmonella typhimurium

Bacteria per 1 ml of overnight nutrient broth

Total counts on trace histidine supplemented top agar

TA1535

 

TA 1537

 

TA 1538

 

TA98

 

TA100

15 x 107

 

34 x 107

 

5 x 107

 

127 x 107

 

52 x 107

19

 

4

 

15

 

22

 

144

12

 

8

 

18

 

28

 

129

15

 

9

 

14

 

17

 

97

 

 

EXPERIMENT 1 – MUTATION STUDY INDIVIDUAL PLATE COUNTS

FOR TEST MATERIAL AND NEGATIVE CONTROLS

Strain of Salmonella typhimurium

Test Material

Conc.

μg/plate

Metabolic activation

Mean No. of revertants

Individual plate count

TA1535

DISPERSIONSBLAU F-60 768

5000

1000

200

40

8

0

-

-

-

-

-

-

36

29

23

20

18

12

40P

30

26

20

17

12

35P

31

21

22

19

12

32P

27

23

19

°

11

5000

1000

200

40

8

0

+

+

+

+

+

+

135

62

35

15

13

15

144P

61

27

16

17

13

124P

66

47

11

9

15

137P

58

32

17

3

16

TA1537

DISPERSIONSBLAU F-60 768

5000

1000

200

40

8

0

-

-

-

-

-

-

34

27

26

20

11

10

37P

22

18

13

15

13

30P

31

33

24

11

9

35P

29

27

23

8

8

5000

1000

200

4

8

0

+

+

+

+

+

+

471

410

141

31

15

15

430P

360

136

29

11

15

492P

416

126

29

18

12

492P

454

160

35

16

198

TA1538

DISPERSIONSBLAU F-60 768

5000

1000

200

40

8

0

-

-

-

-

-

-

591

496

272

167

76

9

578P

500

295

162

80

10

636P

500

262

170

67

7

558P

488

260

168

82

11

5000

1000

200

40

8

0

+

+

+

+

+

+

1084

1868

1485

191

45

15

1169P

1967

1376

188

52

15

1412P

1880

1640

201

39

9

671P

1758

1440

184

43

22

TA98

DISPERSIONSBLAU F-60 768

5000

1000

200

40

8

0

-

-

-

-

-

-

863

847

447

240

113

19

890P

864

432

246

113

22

940P

798

476

274

110

16

760P

878

432

200

115

19

5000

1000

200

40

8

0

+

+

+

+

+

+

1567

1931

1387

189

58

22

1680P

1888

1520

156

56

22

1620P

1930

1200

182

66

23

1400P

1974

1440

230

52

20

TA100

DISPERSIONSBLAU F-60 768

5000

1000

200

40

8

0

-

-

-

-

-

-

567

394

319

195

156

93

660P

381

306

175

161

77

493P

391

336

205

146

92

548P

411

315

205

161

109

5000

1000

200

40

8

0

+

+

+

+

+

+

1451

1601

761

236

141

133

1578P

1622

751

236

148

152

1369P

1666

764

243

126

109

1405P

1516

768

230

148

138

° = plate unscorable           P = precipitate

 

EXPERIMENT 2 – MUTATION STUDY INDIVIDUAL PLATE COUNTS

FOR TEST MATERIAL AND NEGATIVE CONTROLS

Strain of Salmonella typhimurium

Test Material

Conc.

μg/plate

Metabolic activation

Mean No. of revertants

Individual plate count

TA1535

DISPERSIONSBLAU F-60 768

5000

2500

1250

625

312.5

0

-

-

-

-

-

-

38

27

30

32

26

20

31P

37

30

36

30

27

37P

21

41

31

16

22

46P

24

18

28

33

12

5000

2500

1250

625

312.5

0

+

+

+

+

+

+

113

88

65

54

40

21

132P

84

56

49

43

21

91P

100

69

57

34

21

117P

80

70

57

44

20

TA1537

DISPERSIONSBLAU F-60 768

500

2500

1250

625

312.5

0

-

-

-

-

-

-

65

41

21

27

21

8

82P

35P

28

22

17

9

66P

33P

17

32

25

9

48P

55P

17

26

20

7

5000

2500

1250

625

312.5

0

+

+

+

+

+

+

609

641

458

266

135

11

686P

611

441

248

150

10

563P

624

495

278

126

13

577P

687

438

271

129

11

TA1538

DISPERSIONSBLAU F-60 768

5000

2500

1250

625

312.5

0

-

-

-

-

-

-

-

638

341

290

268

17

°P

640P

358

272

286

21

°P

635P

309

301

239

16

°P

638P

355

298

278

13

5000

2500

1250

625

312.5

0

+

+

+

+

+

+

1202

1624

1177

851

751

24

1100P

1706

1125

841

699

28

1011P

1577

1225

910

803

19

1495P

1588

1181

803

752

25

TA98

DISPERSIONSBLAU F-60 768

5000

2500

1250

625

312.5

0

-

-

-

-

-

-

866

1009

700

541

431

16

842P

1069P

706

544

460

19

907P

939P

709

544

401

13

849P

1018P

684

535

431

15

5000

2500

1250

625

312.5

0

+

+

+

+

+

+

2415

2637

2167

1400

1106

29

2775P

2640

2078

1576

1173

20

2465P

2652

2064

1481

1124

38

2004P

2620

2360

1144

1020

30

TA100

DISPERSIONSBLAU F-60 768

5000

2500

1250

625

312.5

0

-

-

-

-

-

-

819

638

299

268

222

78

694P

518P

292

264

232

83

993P

819P

304

274

201

81

770P

578P

300

263

233

70

5000

2500

1250

625

312.5

0

+

+

+

+

+

+

1362

1592

1083

835

566

126

1706P

1331

958

828

638

127

1298P

1984

1184

840

488

128

1082P

1461

1106

837

572

124

° = plate unscorable           P = precipitate

 

MUTABILITY TESTS WITH POSITIVE CONTROLS

Strain of Salmonella typhimurium

Test Material

Conc.

μg/plate

Metabolic activation

Mean No. of revertants

Individual plate count

EXPERIMENT 1

TA1535

TA1537

TA1538

TA98

TA100

TA1535

TA1537

TA1538

TA98

TA100

MNNG

9AA

4NOPD

4NOPD

MNNG

2AA

2AA

2AA

2AA

2AA

2

100

10

10

2

3.3

3.3

3.3

3.3

3.3

-

-

-

-

-

+

+

+

+

+

737

1283

867

1223

1489

589

458

344

310

522

1009

1369

826

1200

1454

737

630

495

379

540

224

1190

781

1306

1458

532

383

388

277

506

979

1291

993

1162

1556

499

362

148

273

520

EXPERIMENT 2

TA1535

TA1537

TA1538

TA98

TA100

TA1535

TA1537

TA1538

TA98

TA100

MNNG

9AA

4NOPD

4NOPD

MNNG

2AA

2AA

2AA

2AA

2AA

2

100

10

10

2

3.3

3.3

3.3

3.3

3.3

-

-

-

-

-

+

+

+

+

+

774

681

759

715

887

137

188

268

1044

1666

781

626

693

758

888

125

187

267

1070

1712

758

705

746

693

923

142

194

285

1005

1629

782

711

839

694

849

144

182

252

1057

1657

 

Applicant's summary and conclusion

Conclusions:
The test material, Dispersionsblau F 60 768, was found to exhibit evidence of mutagenic activity under the conditions of this test. The substance is mutagenic in bacteria with and without a metabolic activation system.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 amd TA100 were treated with Dispersionsblau F-60 768 by the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system at 10% in standard co-factors. The dose range was determined in a preliminary toxicity assay and was 8 to 5000 μg/plate in the first experiment. The experiment was repeated on a separate day using different cultures of the bacterial strains and fresh chemical solutions. In this case the dose range of Dispersionsblau F-60 768 was 312.5 to 5000 μg/pJate.

 

All solvent (DMSO) control plates gave counts of revertant colonies within the normal range.

 

All positive control chemicals gave increases in revertants, both with and without the metabolising system, within expected ranges.

 

Dispersionsblau F-60 768 did not cause a reduction in the growth of the bacterial lawn at any dose level in any of the strains of Salmonella.

Dispersionsblau F-60 768 was, therefore, tested up to the maximum recommended dose of 5000 μg/plate.

 

A significant dose-related increase in the numbers of revertant colonies was recorded for all of the bacterial strains at doses ranging from 8 - 5000 μg/plate both with and without metabolic activation in both experiments. Dispersionsblau F-60 768 was found to exhibit evidence of marked mutagenic activity under the conditions of this test.