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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Weight of evidence: Ames test: Test item do not induce any mutagenic change in tested bacterias (Salmonella typhimurium TA98, TA100, TA 1535 and TA 1537)  with and without metabolic activation.

In another study, test substance showed negative results with respect to genotoxicity in the DNA repair test with hepatocytes for both species (rat and mice), negative results in mutagenicity in Salmonella Typhymurrium but positive results in carcinogenicty.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
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.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
1. Four bacteria strain were used in the study. According the TG 471 - 5 strains of bacteria should be used.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine-requiring gene in Salmonella typhimurium.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 rat and hamster liver
Test concentrations with justification for top dose:
At least five doses were used in each strain in the presence of absence S9.
Test substance 33.0, 100, 333, 1000, 2167 ug/plate.
Positive control 1.5 ug/plate in case of rats S9 activation and 0.75 ug/plate in case of hamster S9 activation.

The highest dose was selected according the preliminary study,where chemicals were checked for toxicity to TAl00 up to a concentration of 10 mg/plate or the limit of solubility, both in absence and in presence of S-9mix. As the indicator of toxicity was used Viability of complete medium and reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn. If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate.


Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance is compatible with the survival of the bacteria and the S9 activity.
Untreated negative controls:
yes
Remarks:
choline chloride, glycerol, glycine, mannitol, and sodium phosphate
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2 Amino antracene tested on all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Preincubation Methodology
Briefly, 0.5 ml of S-9 mix or 0. 1 M PO4 buffer was dispensed into an appropriate number of 13 X 100 mm culture tubes maintained at 37°C in a dry-bath. Then, 0.05 ml of cells and 0.05 ml of solvent or chemical dilution were added to each tube. The mixture was vortexed and allowed to incubate with shaking in the early tests (CWR, EGG), or standing (SRl) for 20 min at 37°C. The protocol was later changed to eliminate the shaking procedure, because the commercial shakers available would not fit in the Class 11. Type B hoods and, for the purposes of laboratory safety, it was inadvisable to incubate the chemicals at 37°C in the open laboratory. Following the preincubation period, 2.5 ml (EGG) or 2.0 ml (CWR, SRI) of molten top agar (45°C) supplemented with 0.5 mM L-histidine and 0.5 mM d-biotin was pipetted into the tubes, which were immediately vortexed, and their contents poured onto 25 ml of minimal glucose bottom agar in a 15 x 100-mm plastic petri dish (Falcon Muta-Assay, 1028 [EGG, SRI] or Fisher Scientific petri dishes [CWR]). After the overlay solidified, the plates were inverted and incubated at 37°C for 48 h.

DURATION
- Preincubation period:Yes,20 minutes at 37ºC
- Exposure duration: 48 hour at 37ºC

NUMBER OF REPLICATIONS: Three plates were used, and the experiment was repeated no less than 1 week after completion of the initial test.
Statistics:
If a chemical was mutagenic or gave a questionable response, it was analyzed by Radian for identity and purity. Analyses had been performed previously by Midwest Research Institute (MRI) on selected other chemicals and on chemicals that had been tested in the National Cancer Institute’s Carcinogenesis Bioassay Program
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Test item do not induce any mutagenic change in tested bacterias (Salmonella typhimurium TA98, TA100, TA 1535 and TA 1537) with and without metabolic activation.
Executive summary:

According the present study test substance was dissolved in water and tested with the pre-incubation procedure with S.typhimurium strains TA1535, TA1537, TA98,TA100 with and without metabolic activation S9. To select the dose range, chemicals were checked for the toxicity to TA100 strain in presence and in absence of S9 obtained from rat and mouse liver.All chemcials were tested used preincubation period at the temperature 37ºC in a dry bath for 20 minutes. After the overlay solidified, the plates were inverted and incubated at 37°C for 48 h. Positive and negative solvent controls were used also. There was no evidence of any increase in the number of revertant colonies in the presence of the test item solution and dilutions without and with metabolic activation. Based on the result of this study, the test item was not found to be mutagenic under the test conditions.

Endpoint:
in vitro DNA damage and/or repair study
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
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
no
GLP compliance:
no
Type of assay:
other: Hepatocyte/DNA repair test in mammalian cells in vitro
Species / strain / cell type:
hepatocytes: Rat and mouse
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells:isolated from the liver of rats (ACI/N) weighting 200 -250 g and from liver of mice (C3H/HeN) weighing 20 g

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:William´s medium E
Additional strain / cell type characteristics:
not specified
Test concentrations with justification for top dose:
0.001,0.0001,0.00001,0.000001 and 0.0000001 M.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
other: N-2fluorenylacetoamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium-Williams Medium E.

DURATION
- Exposure duration:20 hours
- Selection time (if incubation with a selection agent):14 days

NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED: 50 cells/ coverslips

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth.
Evaluation criteria:
Each cell nucleus was positive when the net nuclear grain count was more than 5 grains above the cytopasmic background.
Test substance was considered positive when the mean nuclear grain count was more than 5 grains or positive cells were more tna 33%.
Statistics:
Autoradiographic grains were counted on a CRT display (Olympus type S) with microscopic attachment.
Key result
Species / strain:
hepatocytes: Rat or mice
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

Table of results:

Chemical

Dose (M)

Rat

Mouse

 

 

UDS grains/nucleus

DNA repair

UDS grains/nucleus

DNA repair

 

Semicarbazide hydrochloride

10-3

-1.2+-1.6

 

      -

-0.6+-1.3

 

-

10-4

-0.4+-1.5

-0.7+-1.2

10-5

-1.2+-1.7

-0.8+-1.2

- : negative

Positive DNA response were not present at two doses (10 -6 and 10 -7M) they are not shown in the table.

Conclusions:
Test substance was negative with respect to genotoxicity in the DNA repair test with hepatocytes for both species (rat and mice) and also it had negative results in mutagenicity in Salmonella typhimurium strains.
Executive summary:

In the present study, semicarbazide hydrochloride was tested to genotoxicity in the DNA repair test with hepatocytes from rats and mices. As medium was used Williams Medium E. After hepatocytes were isolated, they were attached to plastic coverslips in primary culture for 2 hours using Williams Medium E. Then were washed and exposed to the test compunds for 20 hours. Test item was dilluted with distilled water before addition to the cultures (10-3 and 10-7 M). Concentration tested included the highest soluble non toxic dose. At the end of incubation, the cultures were washed in medium and the coverslips were mounted on glass sides which were dipped in Sakura NR-M2 photographic emulsions and exposed for 14 days. In the concentration of 10-6 and 10 -7 DNA reponse was not presented. Based on the results obtained from that study, test substance is negative with respect to genotoxicity in the DNA repair test with hepatocytes for both species (rat and mice).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Weight of evidence: Substance tested in vivo micronucleus assay does not show any visual signs of distress (fur condition, wakefulness, etc) nor adverse effects in two different mouse strains.

In another vivo study (UDS assay), test item does not induce mutagenicity to liver UDS of female CD-1 mice in a single oral dose of 100 or 200 mg/kg bw.

Link to relevant study records

Referenceopen allclose all

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
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
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
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.
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
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.
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.

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).

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.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Remarks:
No GLP, no Guideline followed
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
GLP compliance:
no
Type of assay:
unscheduled DNA synthesis
Species:
mouse
Strain:
CD-1
Sex:
female
Route of administration:
oral: gavage
Duration of treatment / exposure:
2 and 16 hours
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Control animals:
yes
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
not specified
Conclusions:
Test item does not induce mutagenicity to liver UDS of female CD-1 mice in a single oral dose of 100 or 200 mg/kg bw.
Executive summary:

According the data from the mentioned publication, mice were administered with SEM-HCl orally at doses of 100 or 200 mg/kg bw (doses of 67 or 135 mgSEM/kg bw). Liver cells were isolated cultured at 2 and 16 h after dosing and analysed for incorporation of 3H-thymidine. Also, the positives controls were used in that study. Test substance, semicarbazide hydrochloride, showed no effect to female CD-1 mice.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

IN VITRO studies:

Ames test (Haworth et al)

Test substance was dissolved in water and tested with the plate incorporation method with S.typhimurium strains TA1535, TA1537, TA98,TA100 with and without metabolic activation S9. Firstly, chemicals were checked for the toxicity to TA100 strain in presence and in absence of S9 obtained from rat and mouse liver to check the toxicity. Prior to test, the preincubation period at the temperature 37ºC in a dry bath for 20 minutes was used. No mutagenicity was observed in any bacterial strains.

UDS assay (Mori K.):

Test substance was tested to genotoxicity in the DNA repair test with hepatocytes from rats and mices. As medium was used Williams Medium E. After hepatocytes were isolated, they were attached to plastic coverslips in primary culture for 2 hours using Williams Medium E. Then were washed and exposed to the test compunds for 20 hours. Test item was dilluted with distilled water before addition to the cultures (10-3 and 10-7 M). Concentration tested included the highest soluble non toxic dose. Test substance is negative with respect to genotoxicity in the DNA repair test with hepatocytes for both species (rat and mice), negative results in mutagenicity in Salmonella Typhymurrium but positive results in carcinogenicty.

IN VIVO studies:

Micronucleus assay (Zetterberg A.L.)

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 intraperitoneally injected (10 ul/g b.w.) with test substance, negative control and positive control. 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.

UDS assay (Nestmann E.R)

According the data from the present study, mice were administered with SEM-HCl orally at doses of 100 or 200mg/kg bw. Liver cells were isolated cultured at 2 and 16 h after dosing and analysed for incorporation of 3H-thymidine. Positives controls were used in that study. Test substance, semicarbazide hydrochloride, showed no effect to female CD-1 mice.

.

Justification for classification or non-classification

Based on the data obtained from several in vitro and in vivo studies semicarbazide hydrochloride is not classified for cell mutagenicity according to CLP Regulation no. 1272/2008.