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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 05 to April 11, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
OECD Guideline For Testing Of Chemicals, 476 "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test". Adopted: July 21st, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
EEC-Direktive 2000/32/EC, L 136, Annex 4E, B.17: "Mutagenicity -In Vitro Mammalian Cell Gene Mutation Test". Adopted: May 19th, 2000
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
U.S. Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.5300, In Vitro Mammalian Cell Gene Mutation Test, August 1998
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Acid Red 299

Method

Target gene:
HPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Source of cells: cell bank of "Genetic Toxicology", Aventis Pharma Deutschland GmbH, ProTox
Test organism: cell line V79 of Chinese hamster lung fibroblasts
Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
The compound was suspended in cell culture medium and tested at the following concentrations:
without S9-mix: 160, 500, 1600 and 5000 μg/ml
with S9-mix: 160, 500, 1600 and 5000 μg/ml
In a preliminary experiment for solubility Telon Rubin A5B 01 was studied with respect to its solubility in hepatocyte attachment medium. The highest evaluable concentration was found to be 5000 ug/ml. Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 5000 ug/ml and a range of lower dose levels down to 10 ug/ml. Following treatment in the presence and in the absence of S9 metabolic activation, moderate toxicity was observed at the highest concentration of 5000 ug/ml. On the basis of these results, a concentration of 5000 ug/ml was selected for the main assays and three lower dose levels down to 160 ug/ml were included in the treatment series.
Vehicle / solvent:
Formulation of test compound: suspended in cell culture medium at appropriate concentrations immediately before use.

Formulation of reference compounds:
EMS dissolved in cell culture medium on the day of treatment, final concentration: 1.0 mg/ml = 8 mM.
DMBA dissolved in DMSO and frozen in small portions. Aliquot thawed on the day of treatment,
final concentration in cell culture medium: 7.7 μg/ml = 30 μM
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Toxicity experiments and dose range finding
A preliminary toxicity test was undertaken in order to select appropriate dose levels for the mutation assay. In this test a wide range of dose levels of test compound was used.
Cell cultures were subjected to the same treatment conditions as in mutation assays, and the survival of the cells was subsequently determined.
The test included the following treatments:
Solvent control: the maximum final concentration of organic solvents will not exceed approx. 1 % (v/v).
Test compound: the highest dose level for the preliminary toxicity test was determined by the solubility of the test compound up to the maximum of 10 mM or 5000 μg/ml.
Treatments were performed both in the presence and absence of S9 metabolic activation system using duplicate doses at each test point.

Test procedure
In preliminary toxicity experiments approximately 1500 cells were seeded in each well of a microliter plate, allowed to attach overnight and then exposed to the test and control compound for four hours.
For each concentration at least 6 wells were used. After 4 or 5 days, the cells were fixed and stained with crystal violet.
Survival was determined by measurement of the crystal violet extinction.
In the main mutation experiments the cultures for assessing toxicity were prepared and treated with the test compound in the same way as for the preliminary experiment. 24 hours after seeding of approx. 1500 cells per well in a microtiter plate, the medium was replaced with serum-reduced (5 % v/v) medium containing the test compound to which either buffer or S9-mix was added as appropriate. After 4 hours the treatment medium was removed and the cells were rinsed twice with normal medium. Thereafter normal medium was added to the wells. The cultures were stained with crystal violet and survival was determined after an incubation period of 4 or 5 days.

Mutagenicity test
Two independent mutation tests were performed.
Exponentially growing cultures which were more than 50% confluent were trypsinated by an approx. 0.25 % (v/v) trypsin ready for use (mfr. Gibco).
A single cell suspension was prepared.
Subsequently the cells were replated to determine the mutation frequency and plating efficiency. The procedures used were as described in Test Procedure
The treatment schedule of the mutagenicity test is described below:
Day 1: Subculturing of an exponentially growing culture
a) Approx. 1500 cells in each well of a micro titer plate for determination of the plating efficiency.
b) 6E+5 – 1E+6 cells in 182 cm2 flasks with 30 ml medium for the mutagenicity test, one flask per experimental point.
Day 2: Treatment of a) and b) with the test compound in the presence and absence of S9-mix (final protein concentration: approx. 0.15 mg/ml) for 4 hours.
Day 5 or 6: Fixation and staining of the cells in a) for the determination of the plating efficiency Subculturing of b) in 182 cm2 flasks
Day 9: Subculturing of b) in five 75 cm2 flasks with culture medium containing 6-thioguanine:
Mutant selection (about 300 000 cellss/flask); subculturing of b) in two 25 cm2 flasks for plating efficiency (about 400 cells per flask)
Day 16: Fixation and staining of colonies of b) - from subcultures seeded on day 9.
All incubations were carried out at approx. 37 °C and 4 % CO2.
Staining was performed with approx. 10 % (v/v) methylene blue in approx. 0.01 % (w/v) KOH solution.
Only colonies with more than 50 cells were counted.
Rationale for test conditions:
In accordance with test guidelines.
Evaluation criteria:
Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range
- the plating efficacy for the solvent control was greater than 50 %

Criteria for a positive response
The test compound is classified as mutagenic if:
- it reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
- there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants.
- survival of the responding dose group is at least 30 %.
However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.
Statistics:
The biometry of the results for the test compound is performed off-line with the MANN WHITNEY-U-TEST

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Solubility and toxicity
In a preliminary experiment for solubility Acid Red 299 was studied with respect to its solubility in hepatocyte attachment medium. The highest evaluable concentration was found to be 5000 μg/ml.
Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 5000 μg/ml and a range of lower dose levels down to 10 μg/ml.
Following treatment in the presence and in the absence of S9 metabolic activation, moderate toxicity was observed at the highest concentration of 5000 μg/ml.
On the basis of these results, a concentration of 5000 μg/ml was selected for the main assays and three lower dose levels down to 160 μg/ml were included in the treatment series.

Mutagenicity
Experimental design
Two independent mutation assays to examine resistance to 6-thioguanine were performed.
In the presence and in the absence of S9 metabolic activation dose levels of 160, 500, 1600 and 5000 μg/ml were used.
Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.

Survival after treatment
In the presence and in the absence of 89 metabolic activation survival was slightly reduced in the second mutation experiment with the concentration of 5000 μg/ml.
In the presence of S9-mix a decrease in the survival rate was also observed with the concentration of 500 μg/ml in both mutation experiments.

Mutation results
The test compound was assessed for its mutagenic potential in vitro in the HPRT-test in two independent experiments without metabolic activation and two independent experiments with metabolic activation.
In the presence of metabolic activation with the toxic concentration of 500 μg/ml a slight enhancement of the mutation rate over the range of the solvent control was induced, which did not fulfil the criteria for a positive response.
In addition, this effect was based mainly on one of the duplicate cultures. Beside this no concentration-related increases were observed and therefore the effect is considered to be without biological relevance.
No relevant reproducible increases in the mutant colonies or mutant frequency over the range of the solvent control was found with any other of the investigated concentrations, either with or without metabolic activation by S9-mix.
The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Any other information on results incl. tables

Toxicity Data (Preliminary experiment)

 

Dose μg/ml

S9-mix

Extinction in microwell plates mean less blank values

Standard deviation

Relative survival*

Solvent control

0

-

2.403

0.10

100.0

Telon Rubin

10

50

100

250

500

1000

2500

5000

-

-

-

-

-

-

-

-

2.315

2.248

2.295

2.396

2.325

2.452

2.349

1.512

0.15

0.12

0.09

0.05

0.06

0.09

0.10

0.17

96.3 §

93.5 #§

95.5 #§

99.7 #§

96.6 #§

102.0 #§

97.8 #§

62.9 #§

 

 

Dose μg/ml

S9-mix

Extinction in microwell plates mean less blank values

Standard deviation

Relative survival*

Solvent control

0

+

2.416

0.06

100.0

Telon Rubin

10

50

100

250

500

1000

2500

5000

+

+

+

+

+

+

+

+

2.415

2.360

2.364

2.076

1.973

2.355

2.357

1.777

0.04

0.06

0.07

0.20

0.33

0.06

0.06

0.10

99.9 §

97.7 #§

97.8 #§

85.9 #§

81.7 #§

97.5 #§

97.5 #§

73.5 #§

# macroscopic precipitate§ microscopic precipitate

*= relative survival (mean value/mean value corresponding control x 100)

Solvent = MEM

 

Toxicity Data (First main mutation experiment)

 

Dose μg/ml

S9-mix

Extinction in microwell plates mean less blank values

Standard deviation

Relative survival*

Solvent control

0

-

2.143

0.07

100.0

Positive control

1000

-

1.784

0.06

83.2

Telon Rubin

160

500

1600

5000

-

-

-

-

2.158

2.135

2.002

1.717

0.12

0.12

0.08

0.13

100.7 #§

99.7 #§

93.4 #§

80.1 #§

 

 

Dose μg/ml

S9-mix

Extinction in microwell plates mean less blank values

Standard deviation

Relative survival*

Solvent control

0

+

2.081

0.19

100.0

Positive control

7.7

+

1.882

0.07

90.4

Telon Rubin

160

500

1600

5000

+

+

+

+

2.146

1.67

1.731

1.688

0.08

0.19

0.07

0.09

103.1 #§

56.1 #§

83.2 #§

81.1 #§

# macroscopic precipitate§ microscopic precipitate

*= relative survival (mean value/mean value corresponding control x 100)

Solvent = MEM

Positive control without S9-mix = EMS

Positive control with S9-mix = DMBA

 

Toxicity Data (Second main mutation experiment)

 

Dose μg/ml

S9-mix

Extinction in microwell plates mean less blank values

Standard deviation

Relative survival*

Solvent control

0

-

2.210

0.10

100.0

Positive control

1000

-

1.694

0.147

76.6

Telon Rubin

A5B 01

160

500

1600

5000

-

-

-

-

2.235

2.197

2.091

1.625

0.15

0.07

0.15

0.15

101.1 #§

99.4 #§

94.6 #§

73.6 #§

 

 

Dose μg/ml

S9-mix

Extinction in microwell plates mean less blank values

Standard deviation

Relative survival*

Solvent control

0

+

2.030

0.34

100.0

Positive control

7.7

+

2.000

0.12

98.5

Telon Rubin

A5B 01

160

500

1600

5000

+

+

+

+

0.1940

0.246

2.149

1.295

0.12

0.04

0.12

0.27

95.6 #§

12.1 #§

105.8 #§

63.8 #§

# macroscopic precipitate§ microscopic precipitate

*= relative survival (mean value/mean value corresponding control x 100)

Solvent = MEM

Positive control without S9-mix = EMS

Positive control with S9-mix = DMBA

 

Mutagenicity Data – Part 1 (First main mutation experiment)

 

Dose μg/ml

S9-mix

Number of cells per flask

Factor* calculated

Cells** seeded

Cells*** survived

Seeded

Found

Mean

I / II

I

II

Solvent control 1 (MEM)

0.0

-

396

254.0

257.5

255.8

0.65

302250

195203

Solvent control 2 (MEM)

0.0

-

399

254.0

259.5

256.8

0.64

330150

212446

Positive control (EMS)

1000.0

-

404

239.0

247.0

243.0

0.60

315000

189468

Telon Rubin A5B 01

160.0

-

403

328.3

316.5

322.4

0.80

280800

224640

160.0

-

402

217.5

218.0

217.8

0.54

327150

177206

500.0

-

399

279.0

280.5

279.8

0.70

318900

223590

500.0

-

398

278.5

267.0

272.8

0.69

290400

199012

1600.0

-

400

263.0

271.7

267.4

0.67

299700

200312

1660.0

-

399

293.0

283.0

288.0

0.72

321150

231808

5000.0

-

395

253.5

248.0

250.8

0.63

278100

176541

5000.0

-

393

251.5

259.5

255.5

0.65

351900

228780

 

Solvent control 1 (MEM)

0.0

+

400

246.7

245.7

246.2

0.62

328350

202099

Solvent control 2 (MEM)

0.0

+

407

223.0

222.0

222.5

0.55

345900

189098

Positive control (DMBA)

7.7

+

401

226.0

213.5

219.8

0.55

311400

170649

Telon Rubin A5B 01

160.0

+

404

298.0

296.8

297.4

0.74

342600

252201

160.0

+

399

273.0

275.5

274.3

0.69

252500

242289

500.0

+

399

258.0

252.5

255.3

0.64

336600

215331

500.0

+

397

280.3

282.0

281.2

0.71

353250

250167

1600.0

+

397

271.5

265.0

268.3

0.68

318150

214972

1600.0

+

402

269.5

285.7

277.6

0.69

327300

226016

5000.0

+

400

283.0

294.0

288.5

0.72

351750

253700

5000.0

+

402

291.0

293.0

292.0

0.73

342900

249072

* factor calculated: mean value / number of cells per flask seeded

** cells seeded in 6-thioguanine (TG) containing medium

*** cells survived after plating in (TG) containing medium (cells seeded x factor calculated)

 

Mutagenicity Data – Part 1 (Second main mutation experiment)

 

Dose μg/ml

S9-mix

Number of cells per flask

Factor* calculated

Cells** seeded

Cells*** survived

Seeded

Found

Mean

I / II

I

II

Solvent control 1 (MEM)

0.0

-

400

308.8

308.0

308.4

0.77

356850

275131

Solvent control 2 (MEM)

0.0

-

397

310.8

292.0

301.4

0.76

289650

219901

Positive control (EMS)

1000.0

-

400

280.0

282.0

281.0

0.70

315150

221393

Telon Rubin A5B 01

160.0

-

400

266.5

285.0

275.8

0.69

330750

228011

160.0

-

399

307.3

308.0

307.7

0.77

351600

271102

500.0

-

401

411.0

397.5

404.3

1.01

320850

323450

500.0

-

402

314.8

306.0

310.4

0.77

326850

252374

1600.0

-

398

271.5

268.0

269.8

0.68

340200

230575

1660.0

-

395

230.0

227.0

228.5

0.58

296100

171288

5000.0

-

407

285.0

273.0

279.0

0.69

368500

184058

5000.0

-

399

286.5

288.0

287.3

0.72

340200

244918

 

Solvent control 1 (MEM)

0.0

+

397

225.5

213.0

219.3

0.55

326850

180508

Solvent control 2 (MEM)

0.0

+

404

325.0

314.0

319.5

0.79

332250

262757

Positive control (DMBA)

7.7

+

399

270.0

274.0

272.0

0.68

350700

239074

Telon Rubin A5B 01

160.0

+

402

287.0

287.0

287.0

0.71

325200

232170

160.0

+

395

267.0

265.0

266.0

0.67

309750

208591

500.0

+

402

292.3

304.0

298.2

0.74

337350

250201

500.0

+

402

268.0

271.0

269.5

0.67

344850

231197

1600.0

+

402

317.5

315.5

316.5

0.79

328650

258751

1600.0

+

400

294.5

274.7

284.6

0.71

325350

231487

5000.0

+

403

264.5

254.0

259.3

0.64

332100

213640

5000.0

+

407

299.9

295.0

297.5

0.73

297750

217606

* factor calculated: mean value / number of cells per flask seeded

** cells seeded in 6-thioguanine (TG) containing medium

*** cells survived after plating in (TG) containing medium (cells seeded x factor calculated)

 

Mutagenicity Data – Part 2 (First main mutation experiment)

 

Dose μg/ml

S9-mix

Number of mutant colonies

Standard deviation

Mutation frequency

Stat. sig.

I

II

III

IV

V

Mean

 

Solvent control 1 (MEM)

0

-

4

3

3

3

5

3.6

0.89

18.4

21.5

 

Solvent control 2 (MEM)

0

-

6

3

4

4

9

5.2

2.39

24.5

 

Positive control (EMS)

1000

-

213

195

209

203

215

207.0

8.12

1092.5

 

*

Telon Rubin A5B 01

160

-

2

7

5

5

1

4.0

2.45

17.8

28.7

 

160

-

7

4

8

3

13

7.0

3.94

39.5

 

500

-

3

3

3

1

5

3.0

1.41

13.4

11.7

 

500

-

1

4

2

1

2

2.0

1.22

10.0

 

1600

-

0

3

3

7

2

3.0

2.55

15.0

22.2

 

1600

-

6

10

7

4

7

6.8

2.17

29.3

 

5000

-

3

5

4

6

7

5.0

1.58

28.3

23.4

 

5000

-

4

4

4

7

2

4.2

1.79

18.4

 

 

Solvent control 1 (MEM)

0

+

7

4

6

4

6

5.4

1.34

26.7

24.5

 

Solvent control 2 (MEM)

0

+

5

3

4

4

5

4.2

0.84

22.2

 

Positive control (DMBA)

7.7

+

28

24

20

36

28

27.2

5.93

159.4

 

*

Telon Rubin A5B 01

160

+

4

5

1

6

4

4.0

1.87

15.9

22.0

 

160

+

6

7

6

9

6

6.8

1.30

28.1

 

500

+

10

15

10

16

15

13.2

2.95

61.3

41.5

 

500

+

5

7

6

6

3

5.4

1.52

21.6

 

1600

+

3

8

3

4

2

4.0

2.35

18.6

17.3

 

1600

+

5

4

5

3

1

3.6

1.67

15.9

 

5000

+

4

6

5

5

5

5.0

0.71

19.7

17.5

 

5000

+

5

6

3

3

2

3.8

1.64

15.3

 

Mutation frequency (mutant colonies per 1 million cells): mean value / cells surviving

*Statistical significant (p<0.05) Mann-Whitney-U-Test

 

Mutagenicity Data – Part 2 (Second main mutation experiment)

 

Dose μg/ml

S9-mix

Number of mutant colonies

Standard deviation

Mutation frequency

Stat. sig.

I

II

III

IV

V

Mean

 

Solvent control 1 (MEM)

0

-

6

4

7

8

1

5.2

2.77

18.9

21.3

 

Solvent control 2 (MEM)

0

-

6

6

3

5

6

5.2

1.30

23.6

 

Positive control (EMS)

1000

-

110

97

118

131

114

114.0

12.35

514.9

 

*

Telon Rubin A5B 01

160

-

7

2

5

4

5

4.6

1.82

20.2

13.4

 

160

-

0

2

1

3

3

1.8

1.30

6.6

 

500

-

1

1

6

4

1

2.6

2.30

8.0

10.4

 

500

-

1

3

5

4

3

3.2

1.48

12.7

 

1600

-

8

4

4

9

5

6.0

2.35

26.0

18.3

 

1600

-

2

1

1

2

3

1.8

0.84

10.5

 

5000

-

1

3

3

6

1

2.8

2.05

15.2

13.7

 

5000

-

3

2

4

3

3

3.0

0.71

12.2

 

 

Solvent control 1 (MEM)

0

+

5

4

3

8

5

5.0

1.87

27.7

20.0

 

Solvent control 2 (MEM)

0

+

4

4

3

1

4

3.2

1.30

12.2

 

Positive control (DMBA)

7.7

+

17

25

28

25

20

23.0

4.42

96.2

 

*

Telon Rubin A5B 01

160

+

5

6

4

0

9

4.8

3.27

20.7

16.1

 

160

+

3

1

1

3

4

2.4

1.34

11.5

 

500

+

13

23

23

26

16

20.2

5.45

80.7

46.4

 

500

+

1

0

6

5

2

2.8

2.59

12.1

 

1600

+

3

3

1

2

3

2.4

0.89

9.3

15.9

 

1600

+

9

5

3

5

4

5.2

2.28

22.5

 

5000

+

5

2

2

2

2

2.6

1.34

12.2

15.8

 

5000

+

2

5

8

0

6

4.2

3.19

19.3

 

Mutation frequency (mutant colonies per 1 million cells): mean value / cells surviving

*Statistical significant (p<0.05) Mann-Whitney-U-Test

Applicant's summary and conclusion

Conclusions:
Acid Red 299 did not induce gene mutation, i.e. was not mutagenic, in this HPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of metabolic activation.
The substance is not classified in accordance with CLP criteria.
Executive summary:

The study was performed to investigate the potential of Acid Red 299 (Batch No. 0481 09300) to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster in vitro.

 

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix).

 

The compound was suspended in cell culture medium and tested at the following concentrations:

without S9-mix: 160, 500, 1600 and 5000 μg/ml

with S9-mix: 160, 500, 1600 and 5000 μg/ml

 

With all investigated concentrations microscopically and macroscopically precipitation was observed.

 

The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The highest concentration showed slight toxic effects with and without metabolic activation.

 

Up to the highest investigated dose no statistically significant and reproducible increase in mutant colony numbers was obtained in two independent experiments.

 

Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix.

 

In conclusion, Acid Red 299 does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described.

 

Acid Red 299 (Batch No. 048109300) is therefore considered to be non-mutagenic in this HPRT assay.