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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames test Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with Bayscript Gelb GGN using the Ames pre-incubation method (Prival Mitchell Modification for Azo Compounds) at up to eight dose levels, in triplicate, both with and without the addition of a hamster liver homogenate metabolizing system (30% liver S9 in modified co‑factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range for Experiment 2 was amended, following the results of Experiment 1, and was 15 to 5000 µg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item. Bayscript Gelb GGN was negative (non-mutagenic) under the conditions of this test.

Bayscript Gelb GGN did not induce gene mutations in an HPRT assay according to OECD guideline 476 in V79 cells of the Chinese hamster. Under the experimental conditions the test item did not induce gene mutations at the HPRT locus in V79 cells. Bayscript Gelb GGN was negative (non-mutagenic) in this HPRT assay.

In an OECD guideline 487 (In vitro Mammalian Cell Micronucleus Test) study, Bayscript Gelb GGN did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system.  The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Bayscript Gelb GGN is considered to be negative (non-mutagenic) in this in vitro micronucleus test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 29 March 2017 Experimental completion date: 23 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Physical state/Appearance: Amber coloured solid pieces
Expiry Date: 22 June 2018
Storage Conditions: Room temperature in the dark
Target gene:
histidine locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
hamster liver S9
Test concentrations with justification for top dose:
Experiment 1: The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item were tested: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Experiment 2: The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle / solvent:
The test item was fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed in house. Sterile distilled water was therefore selected as the vehicle. The homogeneity and stability was previously confirmed for the test item in sterile distilled water formulations (within Envigo study no. LR73TX).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Remarks:
3 µg/plate for TA100, 5 µg/plate for TA1535
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Remarks:
80 µg/plate for TA1537
Positive control substance:
9-aminoacridine
Remarks:
Absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Remarks:
0.2 µg/plate for TA98
Positive control substance:
other: 4-Nitroquinoline-1-oxide
Remarks:
Absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Remarks:
0.5 µg/plate for TA102
Positive control substance:
mitomycin C
Remarks:
Absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Remarks:
1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537
Positive control substance:
other: 2-Aminoanthracene
Remarks:
Presence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg/plate for TA98
Positive control substance:
benzo(a)pyrene
Remarks:
Presence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Remarks:
10 µg/plate for TA102
Positive control substance:
other: 1, 8-Dihydroxyanthraquinone
Remarks:
Presence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Remarks:
50 µg/plate for TA100 and TA98
Positive control substance:
congo red
Remarks:
Presence of uninduced S9-mix
Details on test system and experimental conditions:
Experimental Design and Study Conduct
Test Item Preparation and Analysis
The test item was fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed in house. Sterile distilled water was therefore selected as the vehicle. The homogeneity and stability was previously confirmed for the test item in sterile distilled water formulations (within Envigo study no. LR73TX).
The test item was accurately weighed and approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer and sonication for 20 minutes at 40 °C on the day of each experiment. Formulated concentrations were adjusted to allow for the stated impurity content (10.4%) of the test item. All formulations were used within four hours of preparation. Analysis was carried out in Experiment 1 to determine the concentration of the maximum test item formulation (50 mg/mL). Details of the analytical determinations are presented in Appendix 2 of the report.

Test for Mutagenicity: Experiment 1 – Pre-Incubation Method
The Prival-Mitchell modification to the standard Ames Test is necessary for the testing of azo dyes which can contain mutagenic aromatic amines which are not readily detected using the standard method (Prival and Mitchell (1982)). The modification differs in five key areas from the standard plate incorporation Ames Test:
• Uninduced hamster liver S9 rather than induced rat liver S9.
• 0.15 mL of S9 rather than the maximum of 0.05 mL of S9 in the standard Ames Test.
• The use of flavin mononucleotide (FMN), nicotinamide adenine dinucleotide (NADH), four times the standard amount of glucose-6-phosphate, and the inclusion of exogenous glucose 6 phosphate dehydrogenase in the co-factor mix.
• A 30 minute pre-incubation prior to the addition of the molten top agar.
• Vogel-Bonner plates containing 0.5% glucose instead of the standard 2% glucose.

Only the test item concentrations, vehicle and the positive control, Congo Red, were dosed using the Prival Mitchell modification.

Without Metabolic Activation
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation or vehicle. Each mixture was shaken gently at 37 ± 3 ºC for 30 ± 3 minutes. Then, 2 mL of molten, trace amino-acid supplemented, top agar was added to each tube. The mixture was vortexed and poured onto Vogel-Bonner minimal agar plates containing 0.5% glucose. Each concentration of the test item, appropriate positive control, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of hamster S9 mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer. In addition, 0.1 mL of TA98 or TA100, 0.5 mL of uninduced hamster liver S9 and 0.1 mL of Congo Red at 50 µg/plate was dispensed into dosing tubes, incubated and overlaid onto 0.5% glucose Vogel Bonner plates as previously described.

The standard Ames positive controls were dosed using the pre-incubation method (previously described) where 0.1 mL of bacterial culture was mixed with 0.5 mL of rat liver S9-mix (phenobarbitone/B-naphthoflavone) or phosphate buffer and 2 mL of amino-acid supplemented top agar before overlaying onto 2% glucose Vogel-Bonner agar plates.

The negative (untreated) controls were dosed using the plate incorporation method where 0.1 mL of bacterial culture was mixed with 2 mL of amino-acid supplemented top agar before overlaying onto 2% glucose Vogel-Bonner agar plates.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.


Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

Without Metabolic Activation
Testing was performed as described above

With Metabolic Activation
Testing was performed as described above

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.


Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the induced rat liver and the uninduced hamster liver S9-mixes were validated.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. A yellow test item induced colouration was noted from 15 µg/plate, becoming orange at 5000 g/plate, this observation did not prevent the scoring of revertant colonies.
There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2.

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

93

 

24

 

305

 

11

 

11

 

90

(96)

34

(29)

247

(262)

12

(13)

15

(11)

104

 

30

 

234

 

17

 

8

 

Experiment 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

135

 

31

 

321

 

18

 

19

 

119

(126)

23

(26)

302

(321)

11

(18)

13

(15)

123

 

25

 

339

 

24

 

13

 

 

  Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 16 May 2017

To: 19 May 2017

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

TA102

TA98

TA1537

Solvent Control

(Water)

85

83

76

(81)

4.7#

23

32

33

(29)

5.5

250

264

271

(262)

10.7

14

12

16

(14)

2.0

9

7

13

(10)

3.1

1.5 µg

87

82

88

(86)

3.2

28

36

24

(29)

6.1

210

229

241

(227)

15.6

14

17

10

(14)

3.5

7

21

7

(12)

8.1

5 µg

81

94

76

(84)

9.3

12

22

28

(21)

8.1

219

177

278

(225)

50.7

10

13

18

(14)

4.0

9

14

10

(11)

2.6

15 µg

87

80

75

(81)

6.0

17

22

20

(20)

2.5

233

219

238

(230)

9.8

11

14

9

(11)

2.5

15

17

9

(14)

4.2

50 µg

88

73

80

(80)

7.5

29

18

8

(18)

10.5

238

266

277

(260)

20.1

11

17

12

(13)

3.2

15

15

5

(12)

5.8

150 µg

74

72

76

(74)

2.0

27

21

29

(26)

4.2

258

264

260

(261)

3.1

13

17

11

(14)

3.1

7

5

7

(6)

1.2

500 µg

72

75

83

(77)

5.7

18

28

16

(21)

6.4

257

291

269

(272)

17.2

11

8

14

(11)

3.0

3

6

7

(5)

2.1

1500 µg

90

77

80

(82)

6.8

24

19

18

(20)

3.2

271

236

274

(260)

21.1

14

9

9

(11)

2.9

7

5

11

(8)

3.1

5000 µg

83

80

72

(78)

5.7

21

18

20

(20)

1.5

206

250

263

(240)

29.9

10

8

10

(9)

1.2

5

6

17

(9)

6.7

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

MMC

4NQO

9AA

3 µg

5 µg

0.5 µg

0.2 µg

80 µg

1557

1719

1536

(1604)

100.1

1037

963

1198

(1066)

120.2

1919

1813

2066

(1933)

127.1

349

386

369

(368)

18.5

311

398

355

(355)

43.5

ENNG       N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO       4-Nitroquinoline-1-oxide

9AA       9-Aminoacridine

MMC       Mitomycin C

#       Standard deviation

Test Results: Experiment 1 – With Metabolic Activation

Test Period

From: 16 May 2017

To: 19 May 2017

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

TA102

TA98

TA1537

Solvent Control

(Water)

118

117

104

(113)

7.8#

29

30

21

(27)

4.9

359

257

320

(312)

51.5

40

28

32

(33)

6.1

21

6

5

(11)

9.0

1.5 µg

112

95

125

(111)

15.0

25

19

21

(22)

3.1

247

277

319

(281)

36.2

21

32

22

(25)

6.1

10

5

7

(7)

2.5

5 µg

111

100

94

(102)

8.6

21

21

20

(21)

0.6

291

263

262

(272)

16.5

24

24

13

(20)

6.4

5

3

7

(5)

2.0

15 µg

99

93

115

(102)

11.4

17

27

12

(19)

7.6

349

218

249

(272)

68.5

12

31

43

(29)

15.6

11

12

9

(11)

1.5

50 µg

102

103

111

(105)

4.9

16

22

14

(17)

4.2

320

305

328

(318)

11.7

27

40

9

(25)

15.6

6

11

13

(10)

3.6

150 µg

84

103

92

(93)

9.5

26

22

23

(24)

2.1

299

299

357

(318)

33.5

27

43

26

(32)

9.5

14

12

6

(11)

4.2

500 µg

91

92

111

(98)

11.3

24

30

26

(27)

3.1

283

318

309

(303)

18.2

20

33

26

(26)

6.5

6

4

16

(9)

6.4

1500 µg

100

102

112

(105)

6.4

27

20

27

(25)

4.0

303

314

308

(308)

5.5

30

20

22

(24)

5.3

16

8

15

(13)

4.4

5000 µg

80

99

85

(88)

9.8

24

23

18

(22)

3.2

254

283

302

(280)

24.2

14

27

28

(23)

7.8

12

11

11

(11)

0.6

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

DAN

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

301

285

302

(296)

9.5

374

451

478

(434)

54.0

850

782

759

(797)

47.3

122

161

153

(145)

20.6

329

275

320

(308)

28.9

Positive controls

Hamster S9-Mix

(+)

Name

CR

 

CR

 

Dose Level

50 µg

50 µg

No. of Revertants

484

533

358

(458)

90.3

341

317

300

(319)

20.6

BP       Benzo(a)pyrene

2AA       2-Aminoanthracene

DAN       1,8-Dihydroxyanthraquinone

CR              Congo Red

P       Test item precipitate

S       Sparse bacterial background lawn

T       Toxic, no bacterial background lawn

V       Very weak bacterial background lawn

#       Standard deviation

 

Test Results: Experiment 2 – Without Metabolic Activation

Test Period

From: 20 June 2017

To: 23 June 2017

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

TA102

TA98

TA1537

Solvent Control

(Water)

104

89

98

(97)

7.5#

24

23

21

(23)

1.5

289

303

285

(292)

9.5

11

16

11

(13)

2.9

19

10

9

(13)

5.5

15 µg

115

91

110

(105)

12.7

18

20

30

(23)

6.4

292

258

310

(287)

26.4

11

10

13

(11)

1.5

10

15

7

(11)

4.0

50 µg

117

93

90

(100)

14.8

24

19

25

(23)

3.2

284

302

309

(298)

12.9

12

9

16

(12)

3.5

5

12

8

(8)

3.5

150 µg

109

84

101

(98)

12.8

24

22

27

(24)

2.5

305

272

268

(282)

20.3

10

14

11

(12)

2.1

9

5

14

(9)

4.5

500 µg

98

107

95

(100)

6.2

22

20

19

(20)

1.5

264

226

268

(253)

23.2

11

12

12

(12)

0.6

11

6

4

(7)

3.6

1500 µg

96

113

111

(107)

9.3

14

20

20

(18)

3.5

251

237

315

(268)

41.6

9

11

10

(10)

1.0

7

5

7

(6)

1.2

5000 µg

93

87

105

(95)

9.2

26

23

23

(24)

1.7

248

236

300

(261)

34.0

13

10

13

(12)

1.7

2

7

5

(5)

2.5

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

MMC

4NQO

9AA

3 µg

5 µg

0.5 µg

0.2 µg

80 µg

761

663

680

(701)

52.4

799

755

744

(766)

29.1

2009

1633

1594

(1745)

229.2

263

253

299

(272)

24.2

381

267

303

(317)

58.3

ENNG       N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO       4-Nitroquinoline-1-oxide

9AA       9-Aminoacridine

MMC       Mitomycin C

#       Standard deviation

 

Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 16 May 2017

To: 19 May 2017

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

TA102

TA98

TA1537

Solvent Control

(Water)

124

112

117

(118)

6.0#

29

21

23

(24)

4.2

349

308

314

(324)

22.1

18

21

22

(20)

2.1

20

18

20

(19)

1.2

15 µg

113

127

109

(116)

9.5

26

23

17

(22)

4.6

332

316

340

(329)

12.2

23

26

17

(22)

4.6

27

18

17

(21)

5.5

50 µg

110

114

102

(109)

6.1

27

25

22

(25)

2.5

294

343

348

(328)

29.8

24

14

16

(18)

5.3

16

32

23

(24)

8.0

150 µg

129

117

123

(123)

6.0

27

23

25

(25)

2.0

340

334

353

(342)

9.7

17

14

22

(18)

4.0

24

18

18

(20)

3.5

500 µg

107

120

112

(113)

6.6

20

28

21

(23)

4.4

328

304

305

(312)

13.6

18

21

16

(18)

2.5

26

10

15

(17)

8.2

1500 µg

116

109

110

(112)

3.8

24

28

28

(27)

2.3

281

328

340

(316)

31.2

12

18

17

(16)

3.2

13

16

14

(14)

1.5

5000 µg

115

110

116

(114)

3.2

25

29

18

(24)

5.6

344

236

270

(283)

55.2

16

18

18

(17)

1.2

19

13

9

(14)

5.0

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

DAN

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1035

908

927

(957)

68.5

207

213

241

(220)

18.1

718

627

693

(679)

47.0

133

124

109

(122)

12.1

119

173

322

(205)

105.1

Positive controls

Hamster S9-Mix

(+)

Name

CR

 

CR

 

Dose Level

50 µg

50 µg

No. of Revertants

816

778

792

(795)

19.2

220

223

232

(225)

6.2

BP       Benzo(a)pyrene

2AA       2-Aminoanthracene

DAN       1,8-Dihydroxyanthraquinone

CR              Congo Red

#       Standard deviation

Conclusions:
Bayscript Gelb GGN was considered to be non-mutagenic under the conditions of this test.
Executive summary:

 Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimuriumstrains TA1535, TA1537, TA102, TA98 and TA100 were treated withBayscript Gelb GGNusing the Ames pre-incubation method (Prival Mitchell Modification for Azo Compounds)at up to eight dose levels, in triplicate, both with and without the addition of a hamster liver homogenate metabolizing system (30% liver S9 in modified co‑factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range for Experiment 2 was amended, following the results of Experiment 1, and was 15 to 5000 µg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the potential toxic limit of the test item.

Formulation analysis was carried out in Experiment 1 to determine the concentration of the test item concentration (maximum dose). 

Results

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the induced rat liver and the uninduced hamster liver S9-mixes were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. A yellow test item induced colouration was noted from 15 µg/plate, becoming orange at 5000mg/plate, this observation did not prevent the scoring of revertant colonies.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. 

Conclusion

Bayscript Gelb GGNwas considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 16 November 2017. Experimental completion date: 18 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian forward mutation assay
Specific details on test material used for the study:
Appearance Solid, crystalline, red-brown
Storage Conditions: At room temperature
Stability in Solvent: Stable in water at concentrations of 1 to 200 mg/mL for one day at 15 to 25 °C, and for 15 days at 2 to 8 °C: based on Envigo Study no. LR73TX.

Dose calculation was adjusted to purity.
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase) gene locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Cell Cultures
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants may be reduced by treatment with HAT-medium. Each master cell stock is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3 x 10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.
All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

Culture Medium
For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-Test on Toxicity
Test item concentrations between 17.4 µg/mL and 2232 µg/mL were used. The highest concentration was chosen with respect to the OECD guideline 476 regarding the purity of the test item (89.6%).
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. No precipitation or phase separation was observed up to the highest concentration with and without metabolic activation.
In the pre-experiment no relevant toxic effects were observed after 4 hours in the presence and absence of metabolic activation.

Main Experiment
without S9 mix:69.8; 139.5; 279.0; and 558.0 µg/mL
with S9 mix: 69.8; 139.5; 279.0; and 558.0 µg/mL
The concentrations used in the main experiment were selected based on precipitation observed in the pre-experiment. The individual concentrations were spaced by a factor of 2.
Vehicle / solvent:
The test item was dissolved in deionised water. The final concentration of deionised water in the culture medium was 10 %. The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Remarks:
300 µg/mL = 2.4 mM
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Deionised water
True negative controls:
no
Positive controls:
yes
Remarks:
2.3 µg/mL = 8.9 µM
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Pre-Test on Toxicity
A pre-test was performed in order to determine the toxicity of the test item. In addition the pH and osmolarity were measured. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
In this pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeds for 4 hours (duplicate cultures per concentration level). Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.

Experimental Performance
Seeding
Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline.
Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2 x 10^7 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.
Treatment
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µL/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0 x10^6 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below.
Three or four days after first sub-cultivation approximately 2.0 x10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5 x 10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
Evaluation criteria:
Evaluation of Results
A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:

a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.
Statistics:
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test item Bayscript Gelb GGN was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation.

The main experiment was evaluated at the following concentrations:
without S9 mix: 69.8; 139.5; 279.0; and 558.0 µg/mL
with S9 mix: 69.8; 139.5; 279.0; and 558.0 µg/mL

Precipitation visible at the end of treatment was noted at 558.0 µg/mL with and without metabolic activation.
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% was observed neither in absence nor presence of metabolic activation.
No biologically relevant increase in mutant colony numbers was observed in the main experiment up to the maximum concentrations scored for gene mutations.
The mean mutant frequency obtained in the solvent controls was 21.5 and 22.0 mutants per 10^6 cells. The values were well within the 95% confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mean mutant frequencies of the groups treated with the test item was from 10.1 up to 25.5 mutants per 10^6 cells.
The linear regression analysis showed no significant dose dependend trend of the mutation frequency regarding the mean p-values with and without metabolic actvation. The significant trend calculated in the second culture with metabolic activation is judged as irrelevant, since it actually is reciprocal, going down versus increasing concentrations.
EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.
Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Bayscript Gelb GGN is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The study was performed to investigate the potential of Bayscript Gelb GGN to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The main experiment was performed with a treatment period of 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (2232 µg/mL) was chosen with respect to the OECD guideline 476 regarding the purity of the test item.

No relevant increase in mutant colony numbers/106cells was observed in the main experiments up to the maximum concentration.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusion

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, Bayscript Gelb GGN is considered to be non-mutagenic in this HPRT assay.


 

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 09 May 2017 Experimental completion date: 12 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Identification: Bayscript Gelb GGN
Alternative Names: Bayscript Yellow GGN
Benzenesulfonic acid, 3,3’-(carbonylbis(imino(3-methoxy-4,1-phenylene)-2,1-diazenediyl))bis-, compd. with 2,2’-iminobis(ethanol) (1:2)
Physical state/Appearance: Amber coloured solid pieces
Total Correction Factor: 1.12
Expiry Date: 22 June 2018
Storage Conditions: Room temperature in the dark
Formulated concentrations were adjusted to allow for the stated water/impurity content (10.4%) of the test item.

Target gene:
not applicable
Species / strain / cell type:
primary culture, other: whole blood
Details on mammalian cell type (if applicable):
Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 25 years
Main Experiment: male, aged 29 years


Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Cytokinesis block (if used):
cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/B-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test:
All exposure groups: 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL.

Main experiment:
4-hour exposure group (-S9): 0, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
4-hour exposure group (+S9): 0, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
24-hour exposure group (-S9): 0, 62.5, 125, 250, 500, 1000 and 2000 µg/mL




Vehicle / solvent:
The test item was soluble in water at 20 mg/mL in solubility checks performed in house, therefore water was used as the vehicle
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile water
True negative controls:
no
Positive controls:
yes
Remarks:
0.2 µg/mL for 4-hour exposure
Positive control substance:
mitomycin C
Remarks:
Absence of S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile water
True negative controls:
no
Positive controls:
yes
Remarks:
0.075 µg/mL for 24-hour continuous exposure
Positive control substance:
other: Demecolcine
Remarks:
Absence of S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile water
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg/mL for 4-hour exposure
Positive control substance:
cyclophosphamide
Remarks:
Presence of S9-mix
Details on test system and experimental conditions:
Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.

After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.

4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.

24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, at approximately 37 ºC with 5% CO2 in humidified air, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 µg/mL, and then the cells were incubated for a further 24 hours.
The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.

The preliminary toxicity test was performed using the exposure conditions as described for the Main Experiment but using single cultures only, whereas the Main Experiment used replicate cultures.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used was 7.81 to 2000 µg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the experiments of the main test.

Main Experiment
Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used for all three exposure conditions was 62.5, 125, 250, 500, 1000 and 2000 µg/mL.
A sample was taken from the maximum dose formulation (20 mg/ml) for dose formulation analysis.

Cell Harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

Preparation of Microscope Slides
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations. The Study Director may make a judgment based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions (i.e. S9 concentration or S9 origin)) could be useful. Any additional work to verify an equivocal response will incur extra charges.

Statistics:
The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.
Species / strain:
primary culture, other: whole blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000, and 2000 µg/mL. The maximum dose was the maximum recommended dose level.
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, in any of the three exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 2000 µg/mL in the 4-hour exposure, both in the presence and absence of metabolic activation (S9) and in the 24-hour exposure group. The test item induced no evidence of toxicity in either of the 4(20)-hour exposure groups and demonstrated very modest toxicity in the 24-hour exposure group.
The maximum dose level selected for the Main Experiment was the maximum recommended dose level, 2000 µg/mL for all three exposure groups.

Micronucleus Test – Main Experiment
The qualitative assessment of the slides determined that the toxicity was similar to that seen in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the maximum dose level of test item, 2000 µg/mL in all three exposure groups.
The CBPI data confirm the qualitative observations in that no marked dose-related inhibition of CBPI was observed in either of the 4(20)-hour exposure groups. In the 24-hour exposure group, dose related toxicity was observed with 23%, 40% and 55% cytostasis at 500, 1000 and 2000 µg/mL, respectively.

The maximum dose level selected for analysis of binucleate cells was the maximum recommended dose level (2000 µg/mL).
The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.
The formulation analysis demonstrated accurate formulation of the test item.

The dose levels of the controls and the test item are given in the table below:

Exposure Group

Final concentration of test itemBayscript Gelb GGN(µg/mL)

4-hour without S9

0*, 62.5, 125, 250, 500*, 1000*, 2000*, MMC0.2*

4-hour with S9 (2%)

0*, 62.5, 125, 250, 500*, 1000*, 2000*, CP5*

24-hour without S9

0*, 62.5, 125, 250, 500*, 1000*, 2000*,DC0.075*


*  = Dose levels selected for analysis of micronucleus frequency in binucleate cells

MMC = Mitomycin C

CP = Cyclophosphamide

DC = Demecolcine

Conclusions:
The test item, Bayscript Gelb GGN, did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
Executive summary:

Introduction

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells three dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be the maximum recommended dose level. The dose levels selected for the Main Test were as follows:

Exposure Group

Final concentration of test itemBayscript Gelb GGN(µg/mL)

4-hour without S9

0, 62.5, 125, 250, 500, 1000, 2000

4-hour with S9 (2%)

0, 62.5, 125, 250, 500, 1000, 2000

24-hour without S9

0, 62.5, 125, 250, 500, 1000, 2000

Dose formulation analysis was performed on the top dose formulation (20 mg/mL) of the main test.

Results

All vehicle (sterile water) controls had frequencies of cells with micronuclei in the binucleate cells within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the lowest precipitating dose level. The test item did not demonstrate any toxicity in the 4-hour exposure groups and only modest toxicity at the upper end of the dose range in the 24-hour exposure groups.

The dose formulation analysis demonstrated accurate formulation of the test item at 20 mg/mL.

Conclusion

The test item, Bayscript Gelb GGN was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Bayscript Gelb GGN was non-mutagenic (negative) in all in vitro tests. In a valid Ames test, in an HPRT assay according to OECD guideline 476 in V79 cells and an in vitro Mammalian Cell Micronucleus Test according to OECD guideline 487 Bayscript Gelb GGN was negative. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is therefore not justified.