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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No GLP

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2005

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
Five animals per each group should be used. In the study, 3 and 4 animals were used per each group (Balb/C mice)
GLP compliance:
no
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Semicarbazide hydrochloride
EC Number:
209-247-0
EC Name:
Semicarbazide hydrochloride
Cas Number:
563-41-7
Molecular formula:
CH5N3O.ClH
IUPAC Name:
hydrazinecarboxamide hydrochloride
Test material form:
solid: crystalline

Test animals

Species:
mouse
Strain:
other: CBA and Balb/C
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Scanbur BK, Sollentuna, Sweden
- Age at study initiation: CBA - 6-8 weeks old, Balb/C - 6-7 weeks ols
- Weight at study initiation: CBA - 20-25 g, Balb/C - 15-25 g
- Assigned to test groups randomly: Yes,randomly divided into the different dose groups.
- Diet (e.g. ad libitum): Yes,standard diet , free access
- Water (e.g. ad libitum):Yes, tap water, free access

Administration / exposure

Route of administration:
intraperitoneal
Details on exposure:
Semicarbazide and positive control (Colchicine) were disolved in PBS to give solutions with different cocnentrations of the tested compound. All mice were given an acute single i.p. (10 ul/g b.w.) injection with test substance, negative control and positive control.
Post exposure period:
Blood samples were collected at 42 hours after injections.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Experiment 1
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
Experiment 1
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
Experiment 1
Dose / conc.:
120 mg/kg bw/day (nominal)
Remarks:
Experiment 1
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
Experiment 2
Dose / conc.:
120 mg/kg bw/day (nominal)
Remarks:
Experiment 2
No. of animals per sex per dose:
Experiment 1 - each dose group - Three of four animals.
Experiment 2 - 5 per group.
Control animals:
yes, concurrent no treatment
Positive control(s):
Colchicine
- Route of administration: INTRAPERITONEAL
- Doses / concentrations: 1.0 mg/kg b.w

Examinations

Tissues and cell types examined:
Peripheral blood of mice.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Limited pre-study was carried out to set the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Blood samples were collected, under light anaesthesia with Fluothane (Zeneca, Goteborg), from the orbital plexus of each animal, both CBA and Balb/C mice, at 42 h after injections. The choice of the sampling time is based on the knowledge that the time between the appearance of polychromatic erythrocytes (PCE) in bone marrow and peripheral blood is about 20 h. From each animal, about 50 ul of blood was drawn. Three parallel aliquots of 5ul of blood were layered for purification on a 65% Percoll (Pharmacia Biosystems, Uppsala, Sweden) gradient.

METHOD OF ANALYSIS:
Flow cytometry: Samples were analyzed on FACSVantage SE flow cytometer (BD Immunocytometry systems, Sunnyvale, CA) equipped with an argon ion laser (Enterprise II, Coherent, Santa Clara, CA) operating at both multiline UV and 488 nm. UV output power was 50 mW. Cells, one by one, pass the two beams. Here, the analysis rate was about 1000 cells per second. From each cell different electric signals are sent out, e.g. from forward scatter (FSC) and side scatter (SSC) information’s about structure and size are given. In this analysis a threshold was set in FSC to include all intact cells. Using CellQuest software, peak values for FSC, SSC, Thiazole orange fluorescence and Hoechst 33342 (HO342) fluorescence signals were collected. The FSC signals were acquired using a linear scale and the SSC, TO and HO342 signals were acquired using a log scale.
From each sample between 20 000 and 100 000 events (cells) were collected. CellQuest software (BD) was used for data acquisition and analysis.

For the calculation of MPCE frequencies, dot plots of FSC versus SSC, and DNA content (HO342), indicating the presence of MN, versus RNA (TO) content, indicating the age of the erythrocytes were displayed for each analysed sample.

The cells in the pellet were fixed in a solution of glutaraldehyde. The fixed cells were then stored during 2–5 days at 4 ◦C and the following staining was made in a buffer prepared by adding the fluorescent dyes Hoechst 33342 (HO 342) (Sigma–Aldrich, Sweden) and Thiazole orange (TO) (Molecular probes, Eugene, OR, USA) to PBS. HO
342 is a DNA dye and TO is a RNA dye.
Statistics:
The two-tailed, Student t-test was used for a statistical evaluation of the results.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 200 (four mice), 80 (three mice), and 50 mg/kg b.w. (three mice).
- Clinical signs of toxicity in test animals: The highest dose (200 mg/kg b.w.) of semicarbazide hydrochloride - all four tested mice died within 1 hour. At the doses of 80 mg/kg or 50 mg/kg b.w. - no sign of adverse effect was noticed.
On the bases of these results and LD50 from another study, the selected dose in the micronucleus test were 40,80 and 120 mg/kg bw.

Any other information on results incl. tables

Results from the micronucleus assay with semicarbazide in Experiment 1

Strain

Animal code

Dose

Mg/kg bw

%PCE

S.D.

PCE

Number of MPCE

fMPCE

per mille

S.D.

Balb/C

101

0

20

 

204 589

400

1.95

 

Balb/C

102

0

2.3

 

220 700

345

1.56

 

Balb/C

103

0

3.6

 

224 589

434

1.93

 

Balb/C

104

0

2.3

 

221 667

392

1.77

 

Balb/C

101-104

0

2.5

0.72

871 545

1571

1.8

0.18

Balb/C

105

40

1.5

 

96 745

166

1.71

 

Balb/C

106

40

1.7

 

209 819

355

1.69

 

Balb/C

107

40

2.3

 

216 084

399

1.84

 

Balb/C

105-107

40

1.8

0.40

522 648

920

1.76

0.08

Balb/C

112

80

2.3

 

220 071

380

1.72

 

Balb/C

113

80

1.7

 

201 970

381

1.88

 

Balb/C

114

80

2.1

 

202 810

434

2.14

 

Balb/C

112-114

180

2.1

0.33

624 851

1195

1.91

0.34

Balb/C

115

120

2.2

 

222 684

413

1.85

 

Balb/C

116

120

2.2

 

208 059

422

2.02

 

Balb/C

117

120

2.4

 

226 696

502

2.21

 

Balb/C

118

120

2.1

 

216 086

435

2.01

 

Balb/C

115-118

120

2.2

0.17

873 525

1772

2.02

0.12

Balb/C

118

Colch.

0.8

 

30 217

216

7.10

 

Balb/C

109

Colch.

1.0

 

58 302

376

6.41

 

Balb/C

110

Colch.

0.9

 

44 331

401

8.96

 

Balb/C

108-110

Colch.

0.8*

0.14

132 850

993

7.4*

1.32

∗ p < 0.001 (Student t-test, two-tailed).

Results from the micronucleus assay with semicarbazide in Experiment 2

Strain

Animal code

Dose

Mg/kg bw

%PCE

S.D.

PCE

Number of MPCE

fMPCE

per mille

S.D.

Number of MPCE (II)

fMPCE (II)

per mille

S.D.

CBA

1

0

2.3

 

65 645

68

1.03

 

38

0.58

 

CBA

2

0

1.6

 

170 903

237

1.38

 

112

0.65

 

CBA

3

0

2.2

 

201 091

201

1.00

 

96

0.48

 

CBA

4

0

2.4

 

213 483

217

1.02

 

103

0.48

 

CBA

5

0

2.0

 

191 284

166

0.87

 

76

0.40

 

CBA

1-5

0

2.0

0.31

842 406

889

1.05

0.9

425

0.50

0.010

CBA

6

80

1.7

 

176 295

168

0.95

 

88

0.50

 

CBA

7

80

2.2

 

230 744

222

0.96

 

107

0.46

 

CBA

8

80

2.1

 

245 383

242

0.99

 

119

0.48

 

CBA

9

80

1.9

 

224 674

197

0.88

 

87

0.39

 

CBA

10

80

1.8

 

193 270

165

0.85

 

79

0.41

 

CBA

6-10

80

1.9

0.21

1 070 366

994

0.93

0.06

480

0.45

0.05

CBA

11

120

2.0

 

219 505

199

0.91

 

98

0.45

 

CBA

12

120

1.6

 

206 114

171

0.83

 

77

0.37

 

CBA

13

120

2.0

 

204 669

178

0.87

 

88

0.45

 

CBA

14

120

1.9

 

196 744

181

0.92

 

92

0.47

 

CBA

15

120

2.3

 

237 726

186

0.78

 

78

0.33

 

CBA

11-15

120

2.0

0.23

1 064 758

915

0.86

0.06

433

0.41

0.06

CBA

16

Colch.

0.46

 

22 381

88

3.92

 

62

2.76

 

CBA

17

Colch.

0.74

 

34 740

137

3.93

 

98

2.81

 

CBA

18

Colch.

0.55

 

29 939

112

3.73

 

81

2.70

 

CBA

1-18

Colch.

0.6*

0.14

87 060

337

3.9*

0.11

241

7.86*

0.06

∗ p < 0.001 (Student t-test, two-tailed).

Applicant's summary and conclusion

Conclusions:
The test substance does not show any visual signs of distress (fur condition, wakefulness, etc) nor adverse effects in two different mouse strains.
Executive summary:

According the present study, test substance was tested for genotoxicity in the flow cytometry-based micronucleus assay in vivo. Two strains of male mice were used (CBA and Balb/C). They were injected intraperitonealy with test substance. Before the main test, range finding study was performed to set the maximum tolerated dose. Positive (Colchicine) and negative control (PBS) groups were used too. Test substance and positive control (Colchicine) were disolved in PBS to give solutions with different cocnentrations of the tested compound. All mice were given an acute single i.p. (10 ul/g b.w.) injection with test substance, negative control and positive control.The mice were randomly divided into groups. Two experiments were carried out. Experiment 1 with doses 0, 40,80 and 120 mg/kg/bw and experiment 2, only with the highest doses (80 and 120 mg/kg bw). Blood samples were collected and the frequency of micronucleated polychromatic erythrocytes (MPCE), fMPCE and the cell proliferation, % PCE was determinated. All animals were observed during the experiment for side effects of the treatment. Based on the results from different analyses, the test substance has negative effect on tested animals.