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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-12-19 to 2017-03-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: INTERBUSINESS S.r.l. (Via Spartaco, 2520135 Milano, Italy); batch no. 6061
- Expiration date of the lot/batch: 2018-03-01
- Purity test date: 2016-03-17

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: prepared immediately before use in culture medium. No assay of test item stability, nor its concentration and homogeneity in solvent were under-taken, as not requested by the Sponsor

OTHER SPECIFICS:
Identity of the test material: INTERCURE®1

Method

Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: For the first Main Experiment, two batches of human whole blood, provided by Biopredic International (France), were pooled. For the second Main Experiment, one batch of human whole blood, provided by Biopredic International (France) was used
- Suitability of cells: Not specified
- Sex, age and number of blood donors if applicable: First Experiment: 1) Male (n=1); Age = 23 years; 2) Female (n = 1); Age: 27 Years. Second Experiment: 1) Female (n = 1); Age: 22 Years
- Whether whole blood or separated lymphocytes were used if applicable: separated lymphocytes

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: culture medium (composition shown below) was used for lymphocytes:

RPMI 1640 1x (Dutch modification) 500 mL
Foetal Calf Serum 100 mL
L-Glutamine (200 mM) 6.25 mL
Antibiotic solution 1.25 mL

The foetal calf serum was heat-inactivated at 56°C for 20 minutes before use. For the initiationof the cultures, medium with the addition of phytohaemagglutin (PHA) was used in thefollowing proportion: 10 mL of PHA was added to 500 mL of medium.
- Properly maintained: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital – 5,6-Benzoflavone induced Rat liver
Test concentrations with justification for top dose:
General dose selection procedure:
Cytotoxicity was determined for each of the treatment levels and the highest dose level for genotoxicity assessment was selected as a dose which produces approximately 55 ± 5% cytotoxicity compared to solvent control. If the test item does not induce toxicity at any dose level, then the highest treatment level is selected as the highest dose level for scoring.

Two lower dose levels were also selected for the scoring of micronuclei. The lowest dose level for scoring should be on the borderline of cytotoxicity. The intermediate dose level are evenly spaced between the two.

The test item was found to be soluble in culture medium at the concentration of 16.0 mg/mL after few minutes of vortex stirring. This concentration was chosen since, when added to the treatment medium in the ratio 1:10, it gave a maximum dose level of 1600 µg/mL corresponding to 10.0 mMwhich is lower than 2000 µg/mL.

Two main experiments were performed. Based on the preliminary solubility assay, dose levels of 1600, 1070, 711, 474, 316, 211, 140, 93.6, 62.4 and 41.6 µg/mL were used for the first main experiment. Based on these results, the dose for the main experiment were 1600, 1070 and 711 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Test material was solubilized in the culture medium
- Justification for choice of solvent/vehicle: not specified
Controls
Untreated negative controls:
yes
Remarks:
Untreated cultures were included in the experimental scheme and acted as reference contro
Negative solvent / vehicle controls:
yes
Remarks:
culture medium (RPMI 1640, Dutch modification, ob-tained from Gibco, supplemented with fetal calf serum, antiobiotic solution and L-glutamine)
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: Colchicine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 3 hours followed by further incubation for 28 hours (recovery period)
- Expression time (cells in growth medium): 31 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin-B

STAIN (for cytogenetic assays): Acridine Orange in PBS

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The lymphocyte cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed up to approximately 5 mm from the pellet. The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension. A few drops of the cell suspension obtained in this way were dropped onto clean, wet, grease-free glass slides. Three slides were prepared for each test point and each was labelled with the identity of the culture. The slides were allowed to air dry and kept at room temperature prior to staining with a solution of Acridine Orange in PBS.

NUMBER OF CELLS EVALUATED: 1000 binucleated cells per cell culture (solvent; negative; and positive controls); For cultures treated with Colchicine 1000 mononucleated cells per cell culturewere scored

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The criteria for identifying micronuclei were as follows:

1. The micronucleus diameter was less than 1/3 of the nucleus diameter
2. The micronucleus diameter was greater than 1/16 of the nucleus diameter
3. No overlapping with the nucleus was observed
4. The aspect was the same as the chromatin

DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity of the test item treatments calculated by the cytokinesis-block proliferation index (CBPI)
Rationale for test conditions:
According to OECD Guideline 487.
Evaluation criteria:
Acceptance criteria:

The experiment is considered valid when:

- The incidence of micronucleated cells of the negative control is within the distribution range of our historical control values.
- Concurrent positive controls induce responses that are compatible with those generated in our historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
- Adequate cell proliferation is observed in solvent control cultures.
- The appropriate number of doses and cells are analysed

Criterion for outcome:

The assay is considered as clearly positive if:

- Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
- The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values.
- There is a significant dose effect relationship.

The assay is considered as clearly negative if:

- None of the dose levels shows a statistically significant increase in the incidence of micronucleated cells.
- There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
- All the results are inside the distribution of the historical control data.
Statistics:
A modified X^2 test was used to compare the number of cells with micronuclei in control and treated cultures. Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Following treatment with the test item, a slight dose related increase of the pH was observed at the highest dose levels in the absence and presence of S9 metabolism
- Effects of osmolality: No remarkable variation of osmolality was observed at any dose level
- Precipitation: Following treatment with the test item, no precipitation or opacity of the medium was observed at beginning or end of treatment, in the absence or presence of S9 metabolism.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: See Tables 1-3.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.
- Indication whether binucleate or mononucleate where appropriate: Yes, See Tables 1-3.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Data were included in the statistical analysis
- Negative (solvent/vehicle) historical control data: Data were included in the statistical analysis

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
- Other observations when applicable: frequency of mononucleated, binucleated cells, polynucleated cells

Any other information on results incl. tables

Table 1. Main Experiment 1 – Proliferation Index

Treatment: 3 hours - without S9

Sampling Time: 32 hours

Treatment

(µg/mL)

Culture

No.

MonoN

BiN

PoliN

Total

Cells

CBPI

Mean

Value

Cytotoxicity

Untreated

1

160

260

80

500

1.840

 

 

2

166

270

64

500

1.796

1.818

0

 

Test Item

316

11

162

258

80

500

1.836

 

 

12

186

253

61

500

1.750

1.793

3

Test Item

474

9

180

223

97

500

1.834

 

 

10

168

240

92

500

1848

1.841

-3

Test Item

711

7

193

220

87

500

1.788

 

 

8

207

233

60

500

1.706

1.747

9

Test Item

1070

5

182

251

67

500

1.770

 

 

6

182

244

74

500

1.784

1.777

5

Test Item

1600

3

166

238

96

500

1.860

 

 

4

182

237

81

500

1.798

1.829

-1

MonoN: Mononucleated cells

BiN: Binucleated cells

PoliN: Polinucleated cells

CBPI: Cytokinesis block proliferation index

% Cytotoxicity: Relative to vehicle/solvent control cultures

 

Table 2. Main Experiment 1 – Proliferation Index

Treatment: 3 hours - with S9

Sampling Time: 32 hours

Treatment

(µg/mL)

Culture

No.

MonoN

BiN

PoliN

Total

Cells

CBPI

Mean

Value

Cytotoxicity

Untreated

23

160

244

96

500

1.872

 

 

24

178

233

89

500

1.822

1.847

0

 

Test Item

316

33

128

259

113

500

1.970

 

 

34

151

240

109

500

1.916

1.943

-11

Test Item

474

31

137

258

105

500

1.936

 

 

32

135

256

109

500

1.948

1.942

-11

Test Item

711

29

166

223

111

500

1.890

 

 

30

136

240

124

500

1.976

1.933

-10

Test Item

1070

27

176

236

88

500

1.824

 

 

28

146

232

122

500

1.952

1.888

-5

Test Item

1600

25

132

258

110

500

1.956

 

 

26

129

262

109

500

1.960

1.958

-13

 

Cyclophosphamide

20.0

45

350

143

7

500

1.314

 

 

46

320

167

13

500

1.386

1.350

59

Cyclophosphamide

15.0

47

338

155

7

500

1.338

 

 

48

311

168

21

500

1.420

1.379

55

MonoN: Mononucleated cells

BiN: Binucleated cells

PoliN: Polinucleated cells

CBPI: Cytokinesis block proliferation index

% Cytotoxicity: Relative to vehicle/solvent control culure

Table 3. Main Experiment 2 – Proliferation Index

Treatment: 31 hours - without S9

Sampling Time: 31 hours

Treatment

(µg/mL)

Culture

No.

MonoN

BiN

PoliN

Total

Cells

CBPI

Mean

Value

Cytotoxicity

Untreated

49

188

229

83

500

1.790

 

 

50

137

285

78

500

1.882

1.836

0

 

Test Item

316

59

136

278

86

500

1.900

 

 

60

153

265

82

500

1.858

1.879

-5

Test Item

474

57

138

265

97

500

1.918

 

 

58

147

277

76

500

1.858

1.888

-6

Test Item

711

55

146

270

84

500

1.876

 

 

56

157

263

80

500

1.846

1.861

-3

Test Item

1070

53

203

248

49

500

1.692

 

 

54

178

258

64

500

1.772

1.732

12

Test Item

1600

51

261

202

37

500

1.552

 

 

52

263

220

17

500

1.508

1.530

37

 

Colchicine

0.080

71

449

41

10

500

1.122

 

 

72

468

27

5

500

1.074

1.098

88

Colchicine

0.040

73

477

20

3

500

1.052

 

 

74

490

10

0

500

1.020

1.036

96

Colchicine

0.020

75

355

122

23

500

1.336

 

 

76

387

93

20

500

1.266

1.301

64

MonoN: Mononucleated cells

BiN: Binucleated cells

PoliN: Polinucleated cells

CBPI: Cytokinesis block proliferation index

% Cytotoxicity: Relative to vehicle/solvent control cultures

 

Table 4. Main Experiment 1 – Scoring of Micronuclei

Treatment: 3 hours - without S9

Sampling Time: 32 hours

Treatment

(µg/mL)

Culture

No.

Cells scored (BiN cells)

BiN cells with 1 Mn

BiN cells with 2 Mn

BiN cells with >2 Mn

BiN cells with Mn

Untreated

1

1000

2

0

0

2

2

1000

2

1

0

3

 

Test Item

711

7

1000

3

0

0

3

8

1000

1

1

0

2

Test Item

1070

5

1000

5

0

0

5

6

1000

2

0

0

2

Test Item

1600

3

1000

0

0

0

0

4

1000

4

0

0

4

BiN: Binucleated cells

Mn: Micronucleus/micronuclei

 

Table 5. Main Experiment 1 – Scoring of Micronuclei

Treatment: 3 hours - with S9

Sampling Time: 32 hours

Treatment

(µg/mL)

Culture

No.

Cells scored (BiN cells)

BiN cells with 1 Mn

BiN cells with 2 Mn

BiN cells with >2 Mn

BiN cells with Mn

Untreated

23

1000

3

0

0

3

24

1000

4

0

0

4

 

Test Item

711

29

1000

2

0

0

2

30

1000

5

0

0

5

Test Item

1070

27

1000

2

0

0

2

28

1000

2

0

0

2

Test Item

1600

25

1000

1

0

0

1

26

1000

3

0

0

3

 

Cyclophosphamide

20.0

45

1000

22

2

0

24

46

1000

24

1

1

26

BiN: Binucleated cells

Mn: Micronucleus/micronuclei

 

Table 6. Main Experiment 2 – Scoring of Micronuclei

Treatment: 31 hours - without S9

Sampling Time: 31 hours

Treatment

(µg/mL)

Culture

No.

Cells scored (BiN cells)

BiN cells with 1 Mn

BiN cells with 2 Mn

BiN cells with

>2 Mn

BiN cells with Mn

Untreated

49

1000

3

0

0

3

50

1000

2

0

0

2

 

Test Item

711

55

1000

2

0

0

2

56

1000

3

0

0

3

Test Item

1070

53

1000

3

0

0

3

54

1000

3

0

0

3

Test Item

1600

51

1000

5

0

0

5

52

1000

4

0

0

4

 

Treatment

(µg/mL)

Culture

No.

Cells scored (MonoN cells)

MonoN cells with 1 Mn

MonoN cells with 2 Mn

MonoN cells with

>2 Mn

MonoN cells with Mn

Colchicine

0.040

73

1000

17

4

0

21

74

1000

22

2

0

24

BiN: Binucleated cells

Mn: Micronucleus/micronuclei

 

Table 7. Main Experiment 1 – Statistical Analysis

Treatment: 3 hours - without S9

Sampling Time: 32 hours

Treatment

(µg/mL)

Cells scored (BiN cells)

BiN cells with

Micronuclei

χ2

Significance

Untreated

2000

5

 

 

711

2000

5

0.100

N.S.

1070

2000

7

0.084

N.S.

1600

2000

4

0.000

N.S.

BiN: Binucleated cells

N.S. Not significant

 

Table 8. Main Experiment 1 – Statistical Analysis

Treatment: 3 hours - with S9

Sampling Time: 32 hours

Treatment

(µg/mL)

Cells scored (BiN cells)

BiN cells with

Micronuclei

χ2

Significance

Untreated

2000

7

 

 

711

2000

7

0.072

N.S.

1070

2000

4

0.365

N.S.

1600

2000

4

0.365

N.S.

Cyclophosphamide

20.0

2000

50

31.395

***

BiN: Binucleated cells

N.S. Not significant

*** Significant atp<0.001

 

Table 9. Main Experiment 2 – Statistical Analysis

Treatment: 31 hours - without S9

Sampling Time: 31 hours

Treatment

(µg/mL)

Cells scored (BiN cells)

BiN cells with

Micronuclei

χ2

Significance

Solvent 1%

2000

5

 

 

711

2000

5

0.100

N.S.

1070

2000

6

0.000

N.S.

1600

2000

9

0.645

N.S.

Treatment

(µg/mL)

Cells scored (MonoN cells)

MonoN cells with

Micronuclei

χ2

Significance

Colchicine

0.040

2000

45

30.805

***

BiN: Binucleated cells

N.S. Not significant

*** Significant atp<0.001

 

Table 10. Main Experiment 1 – Summary Table

Treatment: 3 hours

Sampling Time: 32 hours

Treatment

 

Presence of S9 Metabolism

Absence of S9 Metabolism

Dose Level (µg/mL)

% Mn cells

Sig.

%

Cytotoxicity

Dose Level (µg/mL)

% Mn cells

Sig.

%

Cytotoxicity

Untreated

0.00

0.35

 

0

0.00

0.25

 

0

Test Item

711

0.35

N.S.

-10

711

0.25

N.S.

9

Test Item

1070

0.20

N.S.

-5

1070

0.35

N.S.

5

Test Item

1600

0.20

N.S.

-13

1600

0.20

N.S.

-1

Cyclophosphamide

20.0

2.50

***

59

 

-

 

-

%Mn cells Percentage of cells bearing micronuclei

N.S. Not significant

*** Significant atp<0.001

 

Table 11. Main Experiment 2 – Summary Table

Treatment: 31 hours

Sampling Time: 31 hours

Treatment

 

Absence of S9 Metabolism

Dose Level (µg/mL)

% Mn cells

Sig.

% Cytotoxicity

Untreated

0.00

0.25

 

0

Test Item

711

0.25

N.S.

-3

Test Item

1070

0.30

N.S.

12

Test Item

1600

0.45

N.S.

37

Colchicine

0.040

2.25

***

96

%Mn cells Percentage of cells bearing micronuclei

N.S. Not significant

*** Significant atp<0.001

Applicant's summary and conclusion

Conclusions:
Based on the results observed, the test material did not induce micronuclei in human lymphocytes after in vitro treatment.
Executive summary:

In a key Guideline (OECD 487) in vitro micronucleus test, the test material was assayed to evaluate its ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation.

Two main experiments were performed in this study. In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 32 hours corresponding to approximately 2.0 cell cycles was used. As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using a continuous treatment until harvest at 31 hours. Solutions of the test material were prepared in culture medium. For both experiments, the highest dose level of 1600 μg/mL was used (corresponding to10 mM), the upper limit to testing indicated in the Study Protocol. The following lower concentrations were assayed: 1070, 711, 474, 316, 211, 140, 93.6, 62.4 and 41.6 μg/mL. Each experiment included appropriate negative and positive controls and two cell cultures were prepared at each test point. The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. Dose levels were selected for the scoring of micronuclei on the basis of the cytotoxicity of the test material treatments calculated by the cytokinesis-block proliferation index (CBPI). For both experiments, the following dose levels were selected for scoring: 1600, 1070 and711 μg/mL. One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.

 

Following treatment with the test material, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicin and all results were within the distribution of historical negative control data.

 

Based on the results observed, the test material did not induce micronuclei in human lymphocytes after in vitro treatment.