Semicarbazide hydrochloride

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

This study was published in September 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study aimed to evaluate the effects of SEM on testicular morphology of young albino rats. Reversibility of the induced changes after SEM withdrawal was also assessed.

GLP compliance:

not specified

Remarks:

study considered aceptable for use. Recent published paper that is well-documented and meets generally accepted scientific principles.

Limit test:

no

Specific details on test material used for the study:

semicarbazide hydrochloride (Sigma-Aldrich, Ref S2201)

Species:

rat

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

Thirty male albino rats aged 4 weeks weighing 40-50 gm were used in this study. All animals were kept in clean, properly ventilated cages under similar environmental conditions.

Route of administration:

oral: gavage

Details on route of administration:

orally by a gastric tube once daily

Vehicle:

not specified

Details on oral exposure:

Group I
Control -without any treatment throughout the entire experiment


Group II
Orally by a gastric tube once daily at a dose of 40 mg/kg body weight for 4 weeks

Group III
Group III (recovery) were administered SEM orally for 4 weeks as group II, then left untreated and maintained on basal diet for another 2 weeks as recovery groups.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

4 weeks

Dose / conc.:

40 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

Thirty male albino rats aged 4 weeks weighing 40-50 gm were used in this study. All animals were kept in clean, properly ventilated cages under similar environmental conditions. The animals were divided equally into three groups: Group I (control), Group II (treated) and Group III (recovery group). Animals of the control group were kept without any treatment throughout the entire experiment, whereas animals of the treated group were administered semicarbazide hydrochloride (Sigma-Aldrich, Ref S2201) orally by a gastric tube once daily at a dose of 40 mg/kg body weight for 4 weeks (Maranghi et al., 2009a). Group III (recovery) were administered SEM orally for 4 weeks as group II, then left untreated and maintained on basal diet for another 2 weeks as recovery groups. Body weight changes were recorded weekly on an electronic balance (0.lg) (Takahashi et al., 2010). At the end of the experiment, animals were weighed and anesthetized with diethyl ether inhalation. The testes were dissected out carefully from each animal without damage to tunica albugenia and were fixed and processed for light, transmission electron microscopic examinations and morphometric study.

Light microscopic study Hematoxylin and Eosin Stain
Specimens were fixed in Bouin's fixative for 24 hours, processed for paraffin sections of 5µm­ thick

Toluidine Blue Stain

Small pieces of the testis were fixed in 2.5% glutaraldehyde for 24 hours, specimens were washed in 0.1% M phosphate buffer, 7.4 at 4°C and post fixed in 1% phosphate-buffered osmium tetra-oxide for 30 min. Then, they were dehydrated and embedded in epoxy resin. Semithin sections were cut on an ultramicrotome and stained with toluidine blue stain.

Immunohistochemical Study (Fas- Ligand reaction)

Sections from the testis were fixed in acetone (4°C), dried, rehydrated in PBS, incubated with the appropriate blocking agent (Vector Laboratories) for 20 minutes. Monoclonal antibodies to Fas Ligand (Dako 9094/3610) were used, and the slides were incubated for 60 minutes. Then, the slides were washed in phosphate buffered saline (PBS) and incubated with a biotinylated antibody for 30 minutes, rinsed in PBS, incubated with ABC reagent for 45 minutes, and washed again in PBS, and the reaction product was developed with hydrogen peroxide in AEC containing acetate buffer and counterstained with Hematoxylin. Apoptotic cells are stained dark brown.

Transmission electron microscopic study

Ultra-thin sections (70-80 µm) were cut and mounted on copper grids. The grids were double stained with uranyl acetate and lead citrate (Glaurt and Lewis, 1998) for examination with a transmission electron microscope (Joel TEM), at Histology and Cell Biology Department, Faculty of Medicine, Zagazig University.

Morphometric and statistical analysis

The image analyzer computer system Leica Qwin 500 at Pathology Department, Faculty of Dentistry, Cairo University was used to measure the epithelial height of seminiferous tubules in micrometer using the interactive measuring menu. This was performed using hematoxylin and eosin-stained sections at a magnification of 400 of ten seminiferous tubules from five sections of each rat in randomly chosen five rats of each group. In addition, the mean number of positive Fas­ Ligand spermatogenic cells was counted in each high power field (HPF) in the studied groups.
Results were expressed as means± SD. The data obtained by image analyzer were analyzed statistically using one-way analysis of variance (ANOVA) for comparison between groups. ANOVA was statistically significant when P value <0.05, was considered statistically highly significant when P value <0.001 and non significant when P value >0.05

Observations and examinations performed and frequency:

At the end of the experiment, animals were weighed and anesthetized with diethyl ether inhalation. The testes were dissected out carefully from each animal without damage to tunica albugenia and were fixed and processed for light, transmission electron microscopic examinations and morphometric study.

Sacrifice and pathology:

At the end of the experiment, animals were weighed and anesthetized with diethyl ether inhalation. The testes were dissected out carefully from each animal without damage to tunica albugenia

Statistics:

Results were expressed as means ± SD. The data obtained by image analyzer were analyzed statistically using one-way analysis of variance (ANOVA) for comparison between groups. ANOVA was statistically significant when P value <0.05, was considered statistically highly significant when P value <0.001 and non significant when P value >0.05 (Dean et al., 2000).

Clinical signs:

not examined

Mortality:

not examined

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The results of this study showed a highly significant decrease in the body weight gain in the animals of treated and recovery groups when compared with control group.

Body weight (BW; g) of rats in the studied groups of rats

GROUPS/PARAMETERS Group I Group II Group III
Body weight (BW)/g 154.4 ± 13.8 79.2 ± 16.4** 94. 0 ± 14.6**#

Results were expressed as mean ±SD (n=10 rats/group)
** Significant difference from group I ** P<0.001
# Significant difference from group II# P < 0.05 and ## P<0.001

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

As regards the epithelial height of the seminiferous tubules, it was found to be highly significant decreased in group II as compared with the control group and group III. As regards the mean number of positive Fas- Ligand apoptotic cells/HPF, it was found to be highly significantly increased in group II and group III when compared with the control group (Table 1)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 1

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Table 1

Histopathological findings: neoplastic:

not examined

Dose descriptor:

dose level:

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other:

Remarks on result:

other: testicular damage and germ cell apoptosis

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

seminiferous tubules

testes

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

yes

Table 1: Body weight (BW;g )of rats, epithelial height of seminiferous tubules (µm) and mean number of positive Fas- Ligand cells in the studied groups of rats

GROUPS/PARAMETERS	Group I	Group II	Group III
Body weight (BW)/g	154.4±13.8	79.2±16.4**	94.0± 14.6**#
Epithelial height of seminiferous tubules (µm)	82.2±3.3	61.5±7.7**	77.1±10.1##
Number of positive  Fas-Ligand cells	0.4±0.04	30.2±10.2**	20.6±6.7**#

 Results were expressed as mean ±SD (n=10 rats/group)

** Significant difference from group I**P<0.001

#SignificantdifferencefromgroupII#  P<0.05and##P<0.001

Light and electron microscopic results

Group I (Control Group): Examination of H&E stained sections from control animals showed that the testicular parenchyma was formed of densely packed seminiferous tubules with rounded, regular outline and stratified germinal lining. These tubules were enclosed by a connective tissue capsule (tunica albuginea).The stratified germinal epithelium was formed of spermatogonia, primary spermatocytes, spermatids and Sertoli cells. The interstitium contained clusters of interstitial Leydig cells with acidophilic cytoplasm. Immunohistochemical stained sections of the control group revealed that few spermatogenic cells had positive Fas-Ligand reaction.The ultrastructure of control testis showed regular basement membrane surrounding the seminiferous tubules ensheathed by myoid cell. Sertoli cells with indented euchromatic nuclei and prominent nucleoli were seen. Spermatogonia appeared with ovoid nuclei and peripheral marginated heterochromatin. Their cytoplasm contained mitochondria,rough endoplasmic reticulum and ribosomes.

Group II (Semicarbazide-treated Group):Histological examination of the testis of this group showed that the testicular parenchyma was formed of markedly distorted seminiferous tubules with irregular outlines, disorganized epithelium and wide lumina. These tubules were enclosed by thick tunica albuginea with widened interstitial space in some areas.The seminiferous tubules appeared with wide spaces between the lining cells that lost the normal distribution. The epithelial lining was formed of few spermatogenic cells with vacuolar cytoplasm and darkly stained nuclei. Sloughed germ cells were present in the lumen.Spermatogonia in toluidine blue-stained sections appeared with shrunken cytoplasm, cellular processes and darkly stained nuclei.Fas- Ligand immunohistochemical stain showed many apoptotic spermatogenic cells with positive reaction.The ultrastructure of testis of this group showed that spermatogonia appeared with ill defined boundaries and had condensed heterochromatic nuclei.Primary spermatocytes with clumps of heterochromatin in their nuclei and widened perinuclear space were seen.Spermatogonia resting on irregular thickened basement membrane had indented nuclei and peripheral marginated heterochromatin. Their cytoplasm contained swollen mitochondria with disrupted cristae. Wide intercellular space was also observed.

Group III (Recovery Group):Histological examination of testicular tissue of this group showed still distortion of the seminiferous tubules with widened interstitial space in some areas and acidophilic hyaline material.The seminiferous tubules appeared with stratification in their epithelial lining. Several layers of spermatogenic cells appeared with darkly stained nuclei and vacuolar cytoplasm. Sloughed germ cells were observed in the lumen. Multiple vacuoles appeared within the acidophilic hyaline material in the interstitium.Spaces between the cells were still present. Some scattered apoptotic spermatogenic cells were observed with positive Fas- Ligand reaction.The ultrastructure of the testis of the same group showed spermatogonia with condensed heterochromatin in the nuclei resting on the basement membrane. Their cytoplasm contained mitochondria with disrupted cristae. Primary spermatocyte had nuclei with clumps of heterochromatin,mitochondriawithdisruptedcristaeanddilatedendoplasmicreticulumwhile Sertoli cells appeared with indented nuclei and prominent nucleoli.

Morphometric and statistical results

The results of this study showed a highly significant decrease in the body weight gain in the animals of treated and recovery groups when compared with control group. As regards the epithelial height of theseminiferous tubules,it was found to be highly significant decreased in group II as compared with the control group and group III. As regards the mean number of positive Fas- Ligand apoptotic cells/HPF, it was found to be highly significantly increased in group II and group III when compared with the control group(Table1).

Conclusions:

The present results showed that oral administration of semicarbazide induced important changes during juvenile period in rat testicular morphology in the form of testicular damage and germ cell apoptosis which still present after withdrawal and probably may affect reproductive functions. This can be considered relevant for food safety in particular for children who represent a group of major exposure and susceptibility to semicarbazide

Executive summary:

Semicarbazide (SEM) is an azodicarbonamide by-product present in glass jar packaged foods including babyfoods,inbleachingstepsandflourtreatment. Arelativelyhighconsumptionof these products by infants can result in higher exposure compared with other consumers.This study aimed to evaluate effects of SEM on the testis of young albino rat and possibility of recovery after its withdrawal. This study was carried out on 30 male albino rats (4 weeks age) that were divided into three equal groups. Group I (control) and group II (SEM- treated) that were administered 40 mg of SEM orally once daily for 4 weeks. Group III (recovery) were administered SEM orally for 4 weeks as group II, then left untreated for further 2 weeks. At the end of the experiment, the testes of all groups were dissected out and prepared for light and electron microscopic examination. Mean of body weight of control and experimental groups was measured. Morphometric study was performed to measure the epithelial height of seminiferous tubules and the mean number of Fas-ligand positive apoptotic cells.SEM-treated rats showed a significant decrease of body weight gain. SEM induced variable degrees of tubular affection in the form of distorted seminiferous tubules, cellular disorganization, sloughing and cytoplasmic vacuolation. The tubules were enclosed by thick tunica albuginea. Immunohistochemically, SEM treatment induced a significant increase in the mean number of Fas-ligand positive apoptotic cells. Ultrastructural alterations of spermatogenic cells with wide intercellular spaces were observed. The testis of recovery group still contained distorted seminiferous tubules and did not return to its normal histological structure. Acidophilic hyaline material and vacuolations were present in the interstitial spaces. In conclusions, the present study indicated that SEM administration during the growing period induced important changes in rat testicular morphology in the form of testicular damage and germ cell apoptosis which probably may affect reproductive functions; thus, it is recommended to avoid food products sold in glass jars, especially during juvenile period.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

other: Publication

Remarks:

Food and Chemical Toxicology 47 (2009) 472—479

Adequacy of study:

weight of evidence

Study period:

This study was accepted for publication on 02 December 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study aimed to evaluate effects of Semicarbazide hydrochloride oral administration for 28 days at 0. 40. 75. 140 mg/kg bw day during the juvenile period in Sprague—Hawley rats. Histopatological examinations of: epiphyseal cartilage — potential target of lathyrogen action — testes, ovary, uterus, thyroid. ihymus. spleen. adrenals, representative of the main developing organs relevant to juvenile toxicity, and neurobehavioural tests in males, were performed.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

semicarbazide hydrochloride / SEM(CAS No 563-41-7, purity >98—99%) was purchased from Sigma—Aldrich (Milan, Italy)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Forty dams (10/group) o Sprague—Dawiey rats (Harlan, Italy) with offspring were kept under standard laboratory conditions ( 22±0.5 ”C room temperature, 50-60% relative humidity, 12h light-dark alternation with 12-14 air changesper hour) with water and foor (4RF25 GLP "Top Certificate" diet purchased from Mucedola, Milan, Italy) available ad libitum.
Alter six days of acclimatising.wearing rats (23-day old. 50—60g males and 40— 50g females) were housed in singie cages in order to obtain a tsacrihce a least 10 aniinals/sex/group.

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The test animals were treated for 28 days per os by gavage from postnatal day (PND) 23 with 0 (control.vehicle only),40,75 and140 mg/kg bodyweiglt / day of SEM dissolved indistilled water,

To ensure the stability of the test compound.5EM solutions were prepared every moming. Dose levels were selected on the basis of the available literature data on animal experiments (Nesimann et al.,2005), The concentration of the SEM solutions were calculatedt in order to supply volumes of I ml/200g of body weight. Animals were checked daily for their health status. Every two days, individual body weights and food consumption were recorded and dose levels of SEM were ad justed according to the weight gain for each animal. After 28 days. at PNO50, the treatment ceased.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

140 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

The test animals were treated for 28 days per os by gavage form postnatal day (PND) 23 with 0 (control.vehicle only),40,75 and140 mg/kg bodyweiglt / day of SEM dissolved indistilled water,

To ensure the stability of the test compound.5EM solutions were prepared every moming. Dose levels were selected on the basis of the available literature data on animal experiments (Nesimann et al.,2005), The concentration of the SEM solutions were calculatedt in order to supply volumes of I ml/200g of body weight. Animals were checked daily for their health status. Every two days, individual body weiglst and food cunsumption were recorded and dose levels of SEM weue ad justed according to the weight gain for each animal. After 28 days. at PNO50, the treatment ceased. To screen for the possible effects of SEM on the development and maturation of central nervous system (CNS), a basic set of behavioural tests were per formed on adult male rats only at 40 and 75 mg/kg bw/day atter the end of treatment (PND 51-60):males at the highest dose level were not included due to the strong general toxicity which could have altered the test results. Female rats of the same age have not been submitted to the neurobehavioural tests since they were sactificed befo reaching there regular estrous cyclicity (Schoenfelder et al., 2004) which in turn highly influences the test performances in female rat (Liang et al, 2OO8). At full sexual maturity (PND 60) all the animals were sacrificed byasphixiaiton withCO2. The fo!lowing target organs were excised, weighed and stored in Bouin’s solution for histologic andt histomorphological examinaiton: thyroid, adrenals. thymus, spleen, uterus, ovary and testis. Coxal-femoral joints werefixed in 10% buffered formalin.

Observations and examinations performed and frequency:

Selected organs of all groups were fixed in Bouin•s solution and stored in 80X ethyl alcohol They were embedded in paraffin, cut intou 5µm sections and stained with haematoxylin and eosin for the examination under a light microscopy (Nikon Microphot FX) with different lenses.
Coxal-femoral joints were fixed in buffered formalin followed by the decalcification processes and stained with Mallory Trichrome, the standard procedure for connective tissue (collagen. rclicutum, cartilage. bone, amyloid).
A preliminary semi.quantitative evaluation was performed, by scoring thc alterations observed as • (slight). ++ {moderate). or +++ {marked): comparison to controls provided guidance. The findings of the histological evatuation were also utilised to target a detailed quantitative assessment performed by means of histomorphometry,

HIstomorphometry

Mortality:

mortality observed, treatment-related

Description (incidence):

Significant mortality was observed in SEM-treated rats at 75 and 140 mg/kg bw per day of both sexes (respectively, +19X and +20% vs. in controls).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant dose-dependent decreased body weight gain was observed in male rats at all dose levels during treatment (PND 2350) (F- 176.1825: 0.00011, After the end of treatment (PND 51-60). male rats at all dose levels showed a daily weight gain comparable to control group. In female rats, body weight gain was significantly decreased only at the higher dose level during treatment [F" 28.6114: P < 0.0001 1. After the end of treatment (PND 51-60) female rats showed a daily weight gain significantly increased at 40 and 140 mg/kg bw per day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was significant decreased at the higher dose level both in male (F- 93.1900; P < 0.0001) and female rats IF- 52.9149: P $ 0.0001}, whereas was slightly but significantly increased at 75 mg/kg bw per day in females only.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Epiphyseal cartilage showed an absence of mineralization in males and females rats at all dose levels tested.
In the thymus, a dose-related trend toward increased number of adipocytes in capsular and septal areas as well as fibrotic zones was observed in females (statistically significant a top dose). Thyroid showed a dose-dependent increase of necrotic follicular epithelial cells exfoliated into the lumen and colloidal fluid in males of all SEM—treated groups; in females, an increased number of follicles with necrotizing cells in all SEM-treated groups were observed. Uterus showed no major modifications in the microscopic structures; the endometrium/myometrium area ratio was de creased at mid and high dose levels.
In the ovaries of the females treated with SEM a dose-related in crease of primary and secondary oocytes with condensed chromatin (statistically significant at mid and high dose levels) was observed. The number of corpora lutea as well as the atretic follicles were significantly reduced at highest dose level.
Testes did not show any qualitative alterations in the structure or in the spermatogenesis; a trend toward reduced testicular tubular diameter in all SEM-treated groups (-5%, -4% and -8% at 40, 75 and 140 mg/kg. respectively) was present with statistical significance at top dose.
Adrenal alterationswere present in females only: an unclear differentiation of the three main layers in the cortex (glomerulosa, fasciculata and X-zones)was observed at top dose and increased cellular debris in the lumen of vessels in medulla were shown at mid and high dose levels.
In the spleen red pulp, an increased haematopoiesis and presence of megakaryocytes in both sexes at high dose level were evident.

Key result

Dose descriptor:

NOAEL

Effect level:

< 40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

mortality

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day (actual dose received)

System:

other: behaviours were evident even at 40 mg/ kg. Histological alterations were observed in all tissues; thyroid and ovary effects ware present also at 40mg/kg

Organ:

other: behaviours were evident even at 40 mg/ kg. Histological alterations were observed in all tissues; thyroid and ovary effects ware present also at 40mg/kg

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

The present study indicate that the NOAEL in juvenile rats is lower than 40mg/kg for Semicarbazide oral administration.

Executive summary:

Semicarbazide (SEM) is an azodicarbonamide by-product present in glass jar packaged foods including babyfoods. in bleaching steps and flour treatment. Experimental data showed SEM acting as osteolathyr ngen agent, but few toxicological data are available in susceptible life-stages. This study aimed to eval uate effects of SEM oral administration for 28 days at 0. 40. 75. 140 mg/kg bw day during the juvenile period in Sprague—Hawley rats. Histopatological examinations of: epiphyseal cartilage — potential target of SEM lathyrogen action — testes, ovary, uterus, thyroid. ihymus. spleen. adrenals, representative of the main developing organs relevant to juvenile toxicity, and neurobehavioural tests in males, were per formed. Mortality at high and mid dose levels and significantly decreased body weight gain were observed in males even at the lowest dose. Lack of mineralization in cartilage at all dose levels was present. Marked alterations of spontaneous motor and exploratory behaviours were evident even at 40 mg/ kg. Histological alterations were observed in all tissues; thyroid and ovary effects ware present also at 40mg/kg. The present study indicate that the NOAEL in juvenile rats is lower than 40mg/kg for SEM oral administration. SEM administration during juvenile period exerted pleiotropic effects and further studies are suggested to elucidate mechanisms.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

other: Publication

Adequacy of study:

weight of evidence

Study period:

This study was accepted for publication on 13 September 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study aimed to evaluate the effects of semicarbazide on testicular morphology of juvenile Wistar rats. Thirty weaning male Wistar rats, four-week-old, were individually weighed, identified, divided into three groups (ten animals/group). The animals of control group received standard diet; the animals of the twom test groups received semicarbazide hydrochloride in the diet at a concentration of 3g/kg and 6g/kg, respectively.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Sigma-Aldrich Ref S2201

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Dose / conc.:

3 000 mg/kg diet

Dose / conc.:

6 000 mg/kg diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

The aim of this study was to evaluate the effects of semicarbazide on the testicular morphology of juvenile Wistar rats.
After ten days of acclimatisation, thirty weaning male Wistar rats, four-week-o;d, were individually weighed, identified, divided into three groups (tena nimals/group) and kept in polupropylene cages with wood ship bedding junder a 12h ;ight/dark cycle and room temperature of 22-24”C, with water and food ad libitum.The animals of the control group (G0) recieved the standard diet, the animals of the G3 and G6 groups recieved the semicarbazide hydrochloride in the diet at a concentration of3g/kg and 6g/kg, respectively. Body weight changes were recorded weekly on an electronic balance (0.1g). At the end of the thirty days of experimental period, the animals were weighed and sacrificed be anesthesia overdose. After sacrifice, the testes were excised and fixed in 10% neutral phosphate-buffered (pH 7.4) formalin, dehydrated, embedded in paraffin, cut into 5µm secrions and stained with hematoxylin and eosin, for the exminaiton under light microscopy (Nikon Eclipse 600 microscope). All ten animals from each group were used and for each animal, 15 pictures from random semineferous tubules were collected with a digital camera at 200x magnification. For each picture, the area and the perimeter of the semineferous tubules, the area and the perimeter of the lumenof the semiineferous tubules and the height of the germinative epithelium were measured. For each semineferous tubules, the height of the germinative epithelium was the mean of ten measurements. From the results obtained, the average values for each animal and group were evaluated.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The animals in the semicarbazide treatment groups showed significant reductions in bodywheight in a dose-dependent manner

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Untreated rats showed mostly normal testicular architechture with an orderly arrangement of germinal cells and Sertoli cells and normal successive stages of sprematogenesis. Semicarbazisde treatment induced testicular atrophy accompanied by degeneration of germ cells withinn the semineferous tubules, and the tubules were shrunken and greatly depleted of germ cells.

Dose descriptor:

dose level:

Effect level:

>= 3 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other:

Remarks on result:

other: Semicarbazisde treatment induced testicular atrophy accompanied by degeneration of germ cells within the semineferous tubules, and the tubules were shrunken and greatly depleted of germ cells.

Critical effects observed:

yes

Lowest effective dose / conc.:

3 000 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

yes

Conclusions:

Semicarbazide treatment induced testicular atrophy accompanied by degeneration of germ cells within the seminiferous tubules, and the tubules were shrunken and greatly depleted of germ cells.

Executive summary:

The aim of this study was to evaluate the effects of semicarbazide on the testicular morphology of juvenile Wistar rats.

After ten days of acclimatisation, thirty weaning male Wistar rats, four-week-o;d, were individually weighed, identified, divided into three groups (tena nimals/group) and kept in polupropylene cages with wood ship bedding junder a 12h ;ight/dark cycle and room temperature of 22-24”C, with water and food ad libitum.The animals of the control group (G0) recieved the standard diet, the animals of the G3 and G6 groups recieved the semicarbazide hydrochloride in the diet at a concentration of3g/kg and 6g/kg, respectively.  Body weight changes were recorded weekly on an electronic balance (0.1g).  At the end of the thirty days of experimental period, the animals were weighed and sacrificed be anesthesia overdose. After sacrifice, the testes were excised and fixed in 10% neutral phosphate-buffered (pH 7.4) formalin, dehydrated, embedded in paraffin, cut into 5µm secrions and stained with hematoxylin and eosin, for the exminaiton under light microscopy (Nikon Eclipse 600 microscope).  All ten animals from each group were used and for each animal, 15 pictures from random semineferous tubules were collected with a digital camera at 200x magnification.  For each picture, the area and the perimeter of the semineferous tubules, the area and the perimeter of the lumenof the semiineferous tubules and the height of the germinative epithelium were measured. For each semineferous tubules, the height of the germinative epithelium was the mean of ten measurements.  From the results obtained, the average values for each animal and group were evaluated.

Untreated rats showed mostly normal testicular architechture with an orderly arrangement of germinal cells and Sertoli cells and normal successive stages of sprematogenesis.  Semicarbazisde treatment induced testicular atrophy accompanied by degeneration of germ cells within the semineferous tubules, and the tubules were shrunken and greatly depleted of germ cells.

Reference 4

Endpoint:

chronic toxicity: oral

Type of information:

other: Publication

Adequacy of study:

supporting study

Study period:

This publication was accepted on 08 July 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Semicarbazide hydrochloride (SEM-HCl, CAS No. 563–41-7) was purchased from Hayashi Pure Chemical Ind., Ltd. (Osaka, Japan), as a white powder with a purity of 99.3%.

Species:

rat

Strain:

Wistar

Remarks:

Wistar Hannover GALAS

Details on species / strain selection:

Wistar Hannover GALAS rats at 5 weeks of age were obtained from CLEA Japan, Inc. (Tokyo, Japan). Wistar Hannover GALAS rat has been accepted as a suitable strain of toxicology studies (http://www.galas.org/index.html).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals
Male and female Wistar Hannover GALAS rats at 5 weeks of age were obtained from CLEA Japan, Inc. (Tokyo, Japan). Wistar Hannover GALAS rat has been accepted as a suitable strain of toxicology studies (http://www.galas.org/index.html). They received powdered basal diet (CE-2, CLEA Japan, Inc.) and tap water ad libitum, housed 2–4 per plastic cage with sterilized softwood chips as bedding in a barrier-maintained animal room conditioned at 24 ± 1 C and 55 ± 5% humidity with a 12-h light/dark cycle. After a 1-week acclimatization period, animals showing no abnormalities were used at 6 weeks of age. The animal protocol was reviewed and approved by the Animal Care and Use Committee of the National Institute of Health Sciences, Japan

Route of administration:

oral: feed

Details on route of administration:

Semicarbazide hydrochloride (SEM-HCl, CAS No. 563–41-7) was purchased from Hayashi Pure Chemical Ind., Ltd. (Osaka, Japan), as a white powder with a purity of 99.3%. SEM-HCl was well mixed at concentrations of 0, 250, 500 and 1000 ppm into powdered basal diet (CE-2). Concentrations after storage at room temperature for 4 weeks or at 4 C for 8 weeks were analyzed at Japan Food Research Laboratories (Osaka, Japan), and more than 89% stability of the test compound was confirmed under both conditions. SEM was not detected in the basal diet (detection limit, 0.01 ppm). Test diets were prepared every 2 weeks, and stored at 4 C before use.

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations after storage at room temperature for 4 weeks or at 4 C for 8 weeks were analyzed at Japan Food Research Laboratories (Osaka, Japan), and more than 89% stability of the test compound was confirmed under both conditions. SEM was not detected in the basal diet (detection limit, 0.01 ppm).

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily; ad libitum

Dose / conc.:

250 ppm

Remarks:

in powdered basal diet (CE-2)

Dose / conc.:

500 ppm

Remarks:

in powdered basal diet (CE-2)

Dose / conc.:

1 000 ppm

Remarks:

in powdered basal diet (CE-2)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

Experimental design
As a preliminary examination, a 2-week dose finding study was conducted based on a previous report (Weisburger et al., 1981), with 1000 ppm selected as the highest dose for subsequent study. Animals, weighing 166.5 ± 4.5 g for males and 126.3 ± 4.3 g for females (mean ± SD), were randomly allocated to 4 groups, each consisting of 10 males and 10 females, and given diet containing 0 (control), 250, 500 or 1000 ppm SEM-HCl for 90 days. The test diets were available ad libitum, except for one-night fasting prior to the scheduled sacrifice, and rats had free access to tap water throughout the study. Observations for mortality and clinical signs, including posture and gait abnormalities and deformation of four limbs, thorax and tail, were conducted daily. Body weight and food consumption were recorded every week. At necropsy, all animals were anesthetized with ether, weighed, and blood samples were collected from the abdominal aorta for hematology and serum biochemistry. Relative organ weights were calculated as the values relative to body weights.

Observations and examinations performed and frequency:

Hematology and serum biochemistry
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.). Parameters for Hematology and serum biochemistry, shown in Tables 1 and 2, were analyzed at SRL, Inc. (Tokyo, Japan) using sera frozen after centrifugation of whole blood.

Sacrifice and pathology:

Histopathological examination
After macroscopic examination, the brain, thymus, heart, lungs, liver, spleen, adrenals, kidneys and testes were removed and weighed. In addition, 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. Histopathological assessment was performed 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:

Variance in 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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The findings of clinical observation at week 13 are summarized in Table 3.
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. In 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.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant suppression of body weight gain was observed at 1000 ppm 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. However, there were 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:

not examined

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):

Hematology data are summarized in Table 1. In both sexes, differential leukocyte counts showed significant decrease and increase in the proportions of segmented neutrophils and lymphocytes, respectively, in the 1000 ppm group. At the same dose, WBC counts were decreased in males and increased in females, although not statistically significant. 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):

In the serum biochemical analysis, significant alterations of CRN, ALT, A/G, total Bil and K were found in males at 500 and/or 1000 ppm (Table 2). In 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

Description (incidence and severity):

Final body weights were significantly decreased in both sexes of the 1000 ppm group (Table 4). Absolute weights of the lungs in both sexes and the thymus, heart and liver in males were statistically lowered at 1000 ppm. In 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. In 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):

In the macroscopic examination, bowing of the tibia was apparent in males and females at 500 and 1000 ppm. Also, 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 examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings for bones and joints
In the 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 pp). In the metaphysis, spongy bones were decreased and irregularly-branched. The compact bones in the metaphysis became thin with a rugged surface, and deformed to expand outward, resulting in enlargement of the marrow cavities. Also in the 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. In the animals with deformation of the wrist joint macroscopically, the epiphyseal plates of the radius and ulna exhibited similar histological lesions with the tibia. Deformation and fissures of articular cartilage with disarrangement of chondrocytes were observed in the knee joints and the intervertebral joints (from cervical to caudal) at all doses tested, and 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. Additionally, thinning of bone in the vertebral body was obvious in the 1000 ppm group, suggesting loss of bone mass. Overall, 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.

Histopathological findings for the thoracic aorta
In both sexes, although the number of elastic laminae was unchanged, 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.
In other organs, 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 (Table 8). 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.

Key result

Dose descriptor:

NOAEL

Effect level:

< 250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

250 ppm

System:

musculoskeletal system

Organ:

aorta

bone

cartilage

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Table 3 Findings of clinical observation at week 13 in GALAS rats fed diet containing SEM-HCl for 90 days

Sites and findings	Sex	Males				Females
	Dose (ppm)	0	250	500	1000	0	250	500	1000
	No. of animals examined	10	10	10	10	10	10	10	10
Hindlimb	Enlargement and deformation of the knee joint	0	1	10	10	0	0	10	10
Forelimb	Enlargement and deformation of the wrist joint	0	0	0	6	0	0	0	8
Thorax	Prominence	0	0	3	10	0	0	1	9
Tail	Stiff flexion	0	1	4	9	0	0	0	0

Table 4 Final body and organ weights of GALAS rats fed diet containing SEM-HCl for 90 days

	SEM-HCl (ppm)
	0	250	500	1000
No. of animals examined	10	10	10	10
Males Body weight (g)	409.3 ± 18.6^a	407.8 ± 26.6	396.9 ± 27.3	323.8 ± 36.0^**
Brain (g)	2.06 ± 0.08	2.04 ± 0.08	2.00 ± 0.08	2.00 ± 0.08
(g%)	0.50 ± 0.02	0.50 ± 0.03	0.51 ± 0.03	0.63 ± 0.06^**
Thymus (g)	0.30 ± 0.06	0.32 ± 0.08	0.31 ± 0.06	0.21 ± 0.04^**
(g%)	0.07 ± 0.02	0.08 ± 0.02	0.08 ± 0.01	0.06 ± 0.01
Lungs (g)	1.25 ± 0.13	1.25 ± 0.10	1.15 ± 0.12	0.96 ± 0.12^**
(g%)	0.31 ± 0.02	0.31 ± 0.02	0.29 ± 0.04	0.30 ± 0.03
Heart (g)	1.05 ± 0.12	1.01 ± 0.10	1.06 ± 0.06	0.89 ± 0.11^**
(g%)	0.26 ± 0.02	0.25 ± 0.02	0.27 ± 0.02	0.27 ± 0.02
Spleen (g)	0.66 ± 0.08	0.66 ± 0.05	0.62 ± 0.07	0.61 ± 0.10
(g%)	0.16 ± 0.02	0.16 ± 0.01	0.16 ± 0.02	0.19 ± 0.02^**
Liver (g)	10.62 ± 0.99	9.70 ± 0.81	10.28 ± 1.04	8.51 ± 1.43^**
(g%)	2.59 ± 0.19	2.38 ± 0.07^*	2.58 ± 0.10	2.61 ± 0.20
Adrenals (mg)	52.40 ± 7.17	51.30 ± 5.66	55.50 ± 5.40	56.10 ± 10.88
(mg%)	12.83 ± 1.92	12.62 ± 1.45	13.99 ± 1.05	17.34 ± 2.78^**
Kidneys(g)	2.28 ± 0.12	2.38 ± 0.20	2.35 ± 0.20	2.08 ± 0.28
(g%)	0.56 ± 0.03	0.58 ± 0.03	0.59 ± 0.04^*	0.64 ± 0.03^**
Testes (g)	3.38 ± 0.34	3.33 ± 0.27	3.37 ± 0.30	3.56 ± 0.43
(g%)	0.83 ± 0.07	0.82 ± 0.05	0.85 ± 0.08	1.10 ± 0.08^**
Females Body weight (g)	220.9 ± 13.6	229.2 ± 22.4	225.2 ± 11.6	189.4 ± 7.8^**
Brain (g)	1.83 ± 0.09	1.82 ± 0.08	1.87 ± 0.09	1.82 ± 0.08
(g%)	0.83 ± 0.05	0.80 ± 0.09	0.83 ± 0.04	0.97 ± 0.06^**
Thymus (g)	0.26 ± 0.05	0.31 ± 0.11	0.25 ± 0.05	0.20 ± 0.04
(g%)	0.12 ± 0.03	0.13 ± 0.04	0.11 ± 0.02	0.11 ± 0.02
Lungs (g)	0.86 ± 0.06	0.85 ± 0.07	0.88 ± 0.11	0.75 ± 0.06^*
(g%)	0.39 ± 0.02	0.37 ± 0.03	0.39 ± 0.04	0.40 ± 0.03
Heart (g)	0.63 ± 0.06	0.67 ± 0.08	0.66 ± 0.06	0.61 ± 0.05
(g%)	0.29 ± 0.02	0.29 ± 0.03	0.30 ± 0.02	0.32 ± 0.03^**
Spleen (g)	0.47 ± 0.04	0.44 ± 0.06	0.45 ± 0.04	0.48 ± 0.05
(g%)	0.21 ± 0.02	0.19 ± 0.01^*	0.20 ± 0.02	0.25 ± 0.03
Liver (g)	5.40 ± 0.37	5.84 ± 0.87	5.57 ± 0.55	4.77 ± 0.50
(g%)	2.45 ± 0.11	2.55 ± 0.29	2.47 ± 0.17	2.52 ± 0.23
Adrenals (mg)	69.10 ± 10.34	65.30 ± 12.48	68.90 ± 10.21	60.00 ± 7.09
(mg%)	31.27 ± 4.22	28.52 ± 4.94	30.54 ± 3.65	31.73 ± 3.94
Kidneys (g)	1.42 ± 0.17	1.38 ± 0.16	1.47 ± 0.15	1.33 ± 0.11
(g%)	0.64 ± 0.04	0.60 ± 0.05	0.65 ± 0.04	0.70 ± 0.05^*

 

Conclusions:

Taken together, toxicological effects of subchronic exposure to SEM-HCI were mainly observed in the bones, cartilages and aorta. From the histopathological examination, since disarrangement, fissures and deformation of the cartilages were found in both sexes from 250 ppm, the no-observed-adverse-effect-levels (NOAELs) estimated from present study were less than 250 ppm in both sexes, equivalent to 18.1 and 21.1 mg/kg/day in males and females

Executive summary:

A ninety-day toxicity study of semicarbazide hydrochloride (SEM-HCl) was conducted in male and female Wistar Hannover GALAS rats fed diet containing the compound at concentration of 0, 250, 500 and 1000 ppm. Suppression of body weight gain and food consumption was found in both sexes at 1000 ppm throughout the study. 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. The severity of these lesions increased dose-dependently, accompanied by increased connective tissues and bone deformation at high doses. Additionally, 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 together, toxicological effects of subchronic exposure to SEM-HCI were mainly observed in bone, cartilage and the aorta, with the no-observedadverse-effect-level estimated from the present histopathological examination of less than 250 ppm in both sexes

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

18.1 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Organ:

aorta

bone

cartilage

Additional information

In the 30-day repeat dose toxicity study by Ramos et al. 2012, Semicarbazide treatment induced testicular atrophy accompanied by degeneration of germ cells within the seminiferous tubules, and the tubules were shrunken and greatly depleted of germ cells.

In the 4-week repeat dose toxicity study by Fayza El-Sayed et al. 2016, the results showed that oral administration of semicarbazide induced important changes during juvenile period in rat testicular morphology in the form of testicular damage and germ cell apoptosis which still present after withdrawal and probably may affect reproductive functions. This can be considered relevant for food safety in particular for children who represent a group of major exposure and susceptibility to semicarbazide.

The 28-day repeat dose toxicity study by Maranghi et al. 2009 indicates that the NOAEL in juvenile rats is lower than 40mg/kg for Semicarbazide oral administration.

Conclusion from the 90-days repeat dose toxicity study by Takahashi et al. 2009: Taken together, toxicological effects of sub-chronic exposure to SEM-HCI were mainly observed in the bones, cartilages and aorta. From the histopathological examination, since disarrangement, fissures and deformation of the cartilages were found in both sexes from 250 ppm, the no-observed-adverse-effect-levels (NOAELs) estimated from present study were less than 250 ppm in both sexes, equivalent to 18.1 and 21.1 mg/kg/day in males and females

Justification for classification or non-classification

STOT RE category 2 based classification criteria in the CLP regulation Table 3.9.3.