Semicarbazide hydrochloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

no

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

Details on test material:

White powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:4 weeks, room temperature or 8 weeks at 4ºC.
- Stability under test conditions:Yes, 89% stability under both condition.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hannover GALAS

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:CLEA Japan, Inc. (Tokyo, Japan)
- Age at study initiation: 5 weeks old
- Weight at study initiation: Females:126.3+-4.3 g, Males: 166.5+-4.5g (mean value+- standard deviations)
- Fasting period before study: One night fasting, prior to scheduled sacrifice.
- Housing: 2-4 per plastic cage with sterilized softwood chips as bedding in a barrier-maintened animal room.
- Diet (e.g. ad libitum):ad libitum,basal diet CE-2, CLEA Japan Inc.
- Water (e.g. ad libitum): ad libitum, free access
- Acclimation period:1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C):24+-1
- Humidity (%):55+-5
- Photoperiod (hrs dark / hrs light):12-h light/dark

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

SEM-HCl was well mixed at concentrations 0 (control), 250,500 or 1000 ppm into powdered basal diet (CE-2).

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): every 2 weeks, stored at 4ºC before use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of SEM-HCL was prepared by Japan Food Research Laboratories (Osaka, Japan). Concentrations after storage at room temperature for 4 weeks or at 4ºC for 8 weeks, were analyzed and more than 89 % stability of the test compound was confirmed under both conditions. SEM was no detected in basal diet (detection limit, 0.01 ppm).

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

250 ppm

Doses / concentrations 3

Dose / conc.:

500 ppm

Doses / concentrations 4

Dose / conc.:

1 000 ppm

Remarks:

The highest dose

No. of animals per sex per dose:

4 groups, each containing of 10 males and 10 females.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Preliminary examination a 2-week dose finding study (Weisburger, E.K., Ulland, B.M., Nam, J., Gart, J.J., Weisburger, J.H., 1981. Carcinogenicity tests of certain environmental and industrial chemicals. J.Natl. Cancer Inst. 67, 75–88) with 1000 ppm selected as the highest dose for subsequent study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, mortality

DETAILED CLINICAL OBSERVATIONS: Yes, daily

BODY WEIGHT: Yes: Every week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Every week
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data:Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (RBC,Hb,Ht,MCV, MCH, MCHC,Plt, WBC,band form neutrophils,segment neutrophils, eosinophils, basophils, lymphocytes, monocytes, reticulocytes.
- Anaesthetic used for blood collection: Yes (identity) - Ether

CLINICAL CHEMISTRY: Yes (TP,Alb,A/G,Total bil,glucose,TG, TC,BUN, CRB, Na, Cl,K,IP,AST,ALT,ALP,GAMA-GTP)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes,posting and gait abnormalities and deformation of four limbs, thorax and tail.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all organs (the brain, thymus, heart, lungs, liver, spleen, adrenals, kidneys and testes were removed and weighed).the pituitary, eyes, Harderian glands, salivary glands, tongue, trachea, esophagus, thyroid glands, thoracic aorta, stomach, small intestine (duodenum, jejunum, and ileum), large intestine (cecum, colon, and rectum), pancreas, mesenteric lymph nodes, thigh muscle, sciatic nerve, skin, mammary gland, urinary bladder, epididymides, seminal vesicles, prostate, ovaries, uterus and vagina were similarly resected.All organs were fixed in 10% buffered formalin, except for testes, which were fixed in Bouin’s solution overnight.For examination of osteochondral lesions, the nasal cavity, sternum, right femur, right tibia, left knee joint, right and left ankles, spine (cervical, thoracic, lumbar and caudal vertebrae with corresponding spinal cord) and macroscopic lesions (in wrist joints etc.) were fixed in 10% buffered
formalin and then decalcified in EDTA solution at room temperature for a month.

HISTOPATHOLOGY: Yes, on the bones, joints and thoracic aorta of all groups.Additionally, all organs of the control and 1000 ppm groups and the heart, lungs, liver, kidneys, thyroid, trachea, testes and prostate of the intermediate dose groups were also examined. The tissues were routinely processed for paraffin embedding, sectioned and stained with hematoxylin and eosin (HE). Victoria blue and HE staining was applied to three transverse sections of the descending thoracic aorta cut at 5 mm intervals to demonstrate elastic fibers.

Statistics:

Hematology analysis was performed using an automated hematology analyzer,K-4500 (Sysmex Corp., Hyogo, Japan). Differential leukocyte counts and reticulocyte counts were performed with a MICROX HEG-50S (Sysmex Corp.).Data for body weights, food consumption, hematology, serum biochemistry and organ weights was checked for homogeneity by Bartlett’s procedure.If the variance was homogeneous, the data were assessed by one-way analysis of variance. If not, the Kruskal–Wallis test was applied.When statistically significant differences were detected, the Dunnett’s multiple test was employed for comparison between the control and treatment groups.For histopathological findings, the incidences were compared using the Fisher’s exact probability test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Suppression was observed at 1000 ppm group from week 1 in males and from week 4 in females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In both sexes, food consumption was decreased at 1000 ppm throughout the study, and the mean values for food consumption/animal were significantly lowered compared to the control group.
No intergroup differences in the mean values for food consumption/kg body weight in males, due to suppressed body weight gain.
The mean values for food consumption/animal were significantly increased in males at 500 ppm and decreased in females at 250 and 500 ppm.
Significant decrease of the mean values for food consumption/kg body weight was found in females of the 250 and 1000 ppm groups.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Both sexes:Differential leukocyte showed significant decrease and increase in the propotions of segmented neutrophils and lymphocytes, respectively, in the 1000 ppm group. The WBC counts
were decreased in males and increased in females, although not statistically significant in the 1000 ppm group.
In males, MCH was significantly decreased at 250 and 500 ppm, but without dose-dependence.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Changes in CRN, ALT, A/G, total Bil and K were found in males at 500 and/or 1000 ppm.I n females, significant increases of BUN were observed in all treated groups, and K and IP were statistically increased at 500 and 1000 ppm. Significant alterations of TP, ALT and ALP were also detected at 1000 ppm.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Enlargement and deformation of the knee joints were apparent in males and females at 500 and 1000 ppm from week 3, and prominence of the thorax was also found from week 5.
The 1000 ppm group, enlargement and deformation of the wrist joints were observed from week 12 in both sexes, and some showed posture and gait abnormalities.
Tails of male rats in the treated groups exhibited stiff flexion from week 4.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute weights of the lungs in both sexes.
The thymus, heart and liver in males were statistically lowered at 1000 ppm.
Males - significant increase in relative kidney weights was observed at 500 and 1000 ppm, and relative weights of the brain, spleen, adrenals and testes were also increased at 1000 ppm.
Females - relative weights of the brain, heart and kidneys were significantly elevated in the 1000 ppm group.
Decreases in relative liver weight in males and relative spleen weight in females were found at 250 ppm, but such changes were not observed in the 500 and 1000 ppm groups.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Bowing of the tibia was apparent in males and females at 500 and 1000 ppm.
An increases in diameter of the femur and tibia were evident due to enlargement of the marrow cavities along with prominence of the sternum, thoracic kyphosis and deformation of the wrist joints at 1000 ppm.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The incidences of mineralization in the pulmonary arteries were significantly lowered in males of the 250 and 500 ppm group compared to the controls, but without dose-dependence.In males, significant increase of chronic inflammation of the ventral prostate was found at 1000 ppm. The incidence of cysts in the anterior lobe of the pituitary was also significantly elevated in females of the 1000 ppm group.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Histipathological findings for bones and joints:
Femur and tibia: disarrangement of epiphyseal chondrocytes was observed in both sexes at all doses tested.The epiphyseal plate at the proximal end of the tibia was thickened at 500 and 1000 ppm, and degeneration of hypertrophic chondrocytes was found in the thickened cartilage plate.There were fissures in the cartilage matrix, and these were widened and accompanied by increase of connective tissues at 500 and 1000 ppm.In the metaphysis, spongy bones were decreased and irregularly-branched.
Sternum: disarrangement of epiphyseal chondrocytes and fissures in the cartilage matrix were found at all doses tested, and severe fissures with increased connective tissues and bone deformation were more evident at 500 and 1000 ppm in a dosedependent manner.
Wrist joint :the epiphyseal plates of the radius and ulna exhibited similar histological lesions with the tibia.
Knee joints and intervertebral joints:Deformation and fissures of articular cartilage with disarrangement of chondrocytes were observed at all doses tested. Synovial inflammation
and fibrosis were also found in the 500 and 1000 ppm groups.
In the thoracic vertebrae, fissures occurred in the epiphyseal plate adjacent to the intervertebral disk, and displacement of the vertebra originating in the fissures, as well as compression of the spinal cord by displaced vertebra, were seen in males at 1000 ppm.
Thinning of bone in the vertebral body was obvious in the 1000 ppm group, suggesting loss of bone mass.the severities of the osteochondral
lesions described above were higher in males than females. There were no treatment-related changes in the nasal cavity, ankles and Achilles tendons.
Thoracic aorta: their edges became roughened in a dose-dependent manner.The interlaminar spaces in the treated groups had a rod or globular appearance, in contrast to the fibrillar appearance in the 0 ppm group.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 18.1 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Bones, cartilage and the aorta

Remarks on result:

other: < 250 ppm in both sexes.

2

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 21.1 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Bones, aorta and cartilage

Remarks on result:

other: < 250 ppm in both sexes.

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

For the test item, tested for the repeated dose toxicity, the NOEAL was evaluated as less than 250 ppm in both sexes (equivalent to 18.1 mg/kg/day in males and 21.1 mg/kg/day in females).

Executive summary:

According the study, the subchronic toxicity was examinated with the test substance in Wistar Hannover GALAS rats, obtained from CLEA Japan. Rats (males and females) were 5 weeks old and fed with diet containing the test compound at concentration 0, 250,500 and 1000 ppm.Study was carried out during 90 days at the temperature 24 -+1ºC. The diets were prepared every 2 weeks and stored at 4ºC before use. Animals were divided into 4 groups containing 10 males and ten females. During the study, body weights and food consumption were observed every weeks, observation of mortality,clinical signs, including posture, gait abnormalities, deformation of four limbs thorax and tail were conducted daily.Hematology, histopathological and gross examination were provided also. Suppression in body weight gain and food consumption was observed in both sexes at 1000 ppm. Enlargement and deformation of knee joints were obvious at 500 and 1000 ppm from week 3, together with deformation of the thorax and tail. Histopathologically, disarrangement of chondrocytes and fissures in the cartilage matrix were apparent at all doses tested in epiphyseal and articular cartilage. Compact bones at 1000 ppm became thin, suggesting loss of bone mass. In the thoracic aorta, the edges of elastic laminae became rough and the interlaminar spaces were altered from a fibrillar to a rod or globular appearance. No abnormalities were detected in any other organs. Taken into acount effects of subchronic exposure to SEM-HCl, the NOAEL was set as less than 250 ppm in both sexes (equivalent to 18.1 mg/kg/day in males and 21.1 mg/kg/day in females).