Semicarbazide hydrochloride

Genetic toxicity: in vivo

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

Test material information

Constituent 1

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:

semicarbazide 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 / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Experiment 1

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Experiment 1

Doses / concentrations 3

Dose / conc.:

80 mg/kg bw/day (nominal)

Remarks:

Experiment 1

Doses / concentrations 4

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

Experiment 1

Doses / concentrations 5

Dose / conc.:

80 mg/kg bw/day (nominal)

Remarks:

Experiment 2

Doses / concentrations 6

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.