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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:
03.05.-01.07.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
yes
Remarks:
See any other information...
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Direct Blue 78- Physical state: solid, powder- Analytical purity: 95% (w/w)- Impurities (identity and concentrations): NaCl (CAS: 7647-14-5) 10% (w/w)- Lot/batch No.: 7013/207- Expiration date of the lot/batch: unlisted- Storage condition of test material: The test substance should be stored in dry room in dark in closed container at the room temperature.

Method

Species / strain
Species / strain / cell type:
mammalian cell line, other:
Remarks:
peripheral blood lymphocytes mammalian cell line
Details on mammalian cell type (if applicable):
The human peripheral blood lymphocytes used for testing were obtained from healthy non smokingdonors (up to 35 years of age). Peripheral blood (heparinized) is taken from donors in certified medicallaboratory (MeDiLa) in the morning and as soon as possible transported into the test facility.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rat liver homogenate and mixture of cofactors
Controls
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Colchicine
Details on test system and experimental conditions:
Principle of test is the detection of binucleated cells with micronuclei, which are induced by the test substance in human peripheral blood lymphocytes. Lymphocytes are cultured in growth medium and test substance is added to them. Cell cycle is then stopped by cytochalasin B, cultures are sampled and microscopic preparations are prepared. Preparations are then analysed by microscope. Genotoxicity is indicated by increased incidence of binucleated cells with micronuclei.Experiments with and without metabolic activation with short treatment (3 hours) are done at first. If both experiments with the short treatments are negative or equivocal, subsequently, extended exposure treatment without metabolic activation is performed.

Results and discussion

Test results
Key result
Species / strain:
mammalian cell line, other: peripheral blood lymphocytes
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:
Results overviewResults of experiments are summarized in the tables in the Annexes 1 (cytotoxicity) and 2 (genotoxicity). The tables contain the dose applied per culture in µg/mL (concentrations of test substance were applied to cultures at a volume of 50 µl), amount of S9 per culture in µl, number of mononucleated, binucleated and multinucleated cells, CBPI index and % cytotoxicity, numbers of binucleated cells with micronuclei and average numbers of binucleated cells with micronuclei in 1000 cells, number of micronuclei and average number of micronuclei in 1000 cells, parameter Mt / Mc, i.e. ratio of number of binucleated cells with micronuclei at tested dose (Mt) to number of binucleated cells with micronuclei at negative control (Mc, UTC or S9-mix). Numbers of binucleated cells with micronuclei in negative and positive controls were compared with historical controls in our laboratory. The current ranges are given in Annex 3 (Table 7 and Table 8). Values of negative and positive controls in this study are within the ranges of historical data, so that test system responds adequately and the experiment is acceptable.The cytotoxic effect was characterized as % of cytotoxicity. Results of the cytotoxic effect are given in the Table 1 - 3 (Annex 1). All of test concentrations did not show the cytotoxicity higher than 55±5 % in the time of exposure 3 hours. The second experiment with the prolonged exposition without activation (23 hours) gave in three tested concentrations (2000, 1000 and 500 µg/mL) high cytotoxicity (small and dark cells) therefore microscopic slides could not be analyzed for cytotoxicity and genotoxicity. The concentrations 250 µg/mL was evaluated only for cytotoxicity, but could not be used for genotoxicity evaluation because of poor appearance of microscopic slides (small and dark cells). On the basis of these results, the third experiment without metabolic activation with extended exposure and lower concentrations 125, 62.5 and 31.25 µg/mL had to be done. In the third experiment with the prolonged exposition without activation (23 hours), all of tested concentrations did not show the cytotoxicity higher than 55±5 %. Therefore the concentration of 125 µg/mL was selected as the highest one for the analysis of genotoxic effect.

Any other information on results incl. tables

Cytotoxic effect

Table No. 1: Evaluation of cytotoxic effect without metabolic activation-3h exposure

- MA I

Culture No.

Treatment/Test substance concentration

Number of MNC

Number of BNC

Number of MTNC

CBPI

Cytotoxicity (%)

1

UTC

756

282

39

1.334

0.0

2

2000 μg/mL

928

182

9

1.179

46.5

3

1000 μg/mL

691

386

26

1.397

-18.8

4

500 μg/mL

811

348

55

1.377

-12.9

5

250 μg/mL

607

422

77

1.521

-55.8

6

125 μg/mL

707

290

56

1.382

-14.2

13

Colchicine

857

176

76

1.296

11.5

 

Table No. 2: Evaluation of cytotoxic effect with metabolic activation (S9-mix) -3h exposure

+MA I

Culture No.

Treatment/Test substance concentration

Number of MNC

Number of BNC

Number of MTNC

CBPI

Cytotoxicity (%)

7

S9-mix

925

298

45

1.306

0.0

8

2000mg/mL +

S9-mix

923

204

16

1.206

32.5

9

1000mg/mL +

S9-mix

939

183

20

1.195

36.2

10

500mg/mL +

S9-mix

828

187

28

1.233

23.9

11

250mg/mL +

S9-mix

764

267

69

1.368

-20.3

12

125mg/mL +

S9-mix

724

406

67

1.451

-47.4

14

Cyclophosphamide + S9-mix

630

453

35

1.468

-52.9

1

UTC

756

282

39

1.334

-9.2

 

Table No. 3: Evaluation of cytotoxic effect without metabolic activation -23h exposure

- MA III

Culture No.

Treatment/Test substance concentration

Number of MNC

Number of BNC

Number of MTNC

CBPI

Cytotoxicity (%)

15

UTC

528

426

61

1.54

0.0

17

125 μg/mL

642

338

54

1.43

20.1

18

62.5 μg/mL

607

382

50

1.46

14.1

19

31.25 μg/mL

603

497

47

1.52

4.6

16

Colchicine

823

256

67

1.34

37.0

Genotoxic effect

Table No. 4: Evaluation of genotoxicity without metabolic activation-3h exposure

Culture No.

Treatment/Test substance concentration

Number of binucleated cells with MN

Number of MN

Average number of BN cells with MN per 1000 binucleated cells

Average number of MN per 1000 binucleated cells

Mt/Mc

1

UTC

22

24

11

12

1.00

2

2000mg/mL

42

45

21

22.5

1.91

3

1000mg/mL

15

17

7.5

8.5

0.68

4

500mg/mL

35

40

17.5

20

1.59

13

Colchicine

273

323

136.5

161.5

12.41

 

Table No. 5:Evaluation ofgenotoxicitywith metabolic activation (S9-mix) -3h exposure

Culture No.

Treatment/Test substance concentration

Number of binucleated cells with MN

Number of MN

Average number of BN cells with MN per 1000 binucleated cells

Average number of MN per 1000 binucleated cells

Mt/Mc

7

S9-mix

23

24

11.5

12

1.00

8

2000 mg/mL +

S9-mix

20

22

10

11

0.87

9

1000mg/mL +

S9-mix

20

21

10

10.5

0.87

10

500mg/mL +

S9-mix

24

25

12

12.5

1.04

1

UTC

22

24

11

12

0.96

14

Cyclophosphamide + S9-mix

58

64

29

32

2.52


 

Table No. 6: Evaluation of genotoxicity without metabolic activation- 23h exposure

Culture No.

Treatment/Test substance concentration

Number of binucleated cells with MN

Number of MN

Average number of binucleated cells with MN per 1000 binucleated cells

Average number of MN per 1000 binucleated cells

Mt/Mc

15

UTC

17

18

8.5

9

1.00

17

125mg/mL

20

21

10

10.5

1.18

18

62.5mg/mL

22

23

11

11.5

1.29

19

31.25mg/mL

26

29

13

14.5

1.53

16

Colchicine

50

55

25

27.5

2.94

Applicant's summary and conclusion

Conclusions:
Under the experimental design described above, the test substance, Direct Blue 78, had no genotoxic effects in the micronucleus test performed in human peripheral blood lymphocytes in experiments both without and with metabolic activation.The result of micronucleus test was negative, test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.
Executive summary:

In Vitro Mammalian Cell Micronucleus Test assayed genotoxicity of the test substance, Direct Blue 78. The test was performed according to OECD Test Guideline No. 487 -In Vitro Mammalian Cell MicronucleusTest, Adopted 26thSeptember, 2014.

The human peripheral blood lymphocytes from healthy donors were used for testing. The test substance was suspended in RPMI medium and assayed in five concentrations 31.25-2000  µg/mL, which were applied to cultures in volume of 50 mL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

Under the experimental design described above, the test substance, Direct Blue 78,had no genotoxic effects in the micronucleus test performed in human peripheral blood lymphocytesin experiments both without and with metabolic activation.

The result of micronucleus test was negative, test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.