Semicarbazide hydrochloride

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the fact that most of the available studies have been performed using juvenile animals (i.e. not matures), and no literature data have demonstrated effects on humans.

Based on results showing effects on juveniles but not on mature animals, leading us to conclude that no effect have been observe on mature animals.

Based on the 90-days study on mature rats did not observe any effects on animals.

As major effects have been observed in reproductive organs from juvenile animals (rats), suggesting teratogenesis effects, we propose to classify the substance as Reproductive toxicity 2.

Link to relevant study records

Reference

Reference 1

Endpoint:

reproductive toxicity, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Study published in 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:

Juvenile rats have been treated as it is a relevant population regarding exposure scenario.

GLP compliance:

not specified

Remarks:

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

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

Vehicle:

not specified

Details on exposure:

Orally by a gastric tube.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Once daily

Details on study schedule:

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.

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-Al
drich, 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 I
I, then left untreated and maintained on basal diet for another 2 weeks as recovery groups. Body we
ight 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 tes
tes 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 w
ashed 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 counte
rstained 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, Zaga
zig 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-stainedsections at a magnification of 400 of ten seminiferous tubules from five sections of each rat in r
andomly chosen five rats of each group. In addition, the mean number of positive Fas Ligand sperma
togenic 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

Parental animals: Observations and examinations:

Observations and examinations performed and frequency
At the end of the experiment, animals were weighed and anesthetized with diethyl ether inhalation. T
he 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.

Postmortem examinations (parental animals):

At the end of the experiment, animals were weighed and anesthetized with diethyl ether inhalation. T
he 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 sta
tistically 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 va
lue <0.001 and non significant when P value >0.05 (Dean et al., 2000).

Clinical signs:

not examined

Dermal irritation (if dermal study):

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Table 1

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

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)

Key result

Dose descriptor:

LOAEL

Effect level:

<= 40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

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

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

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:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Dose descriptor:

other: Not the goal of the study

Remarks on result:

other: Not the goal of the study

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

other: Not the goal of the study

Remarks on result:

other: Not the goal of the study

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

other: Not the goal of the study

Remarks on result:

other: Not the goal of the study

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

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

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 pace 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 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(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,in bleaching steps and flour treatment. A relatively high consumption of 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:

reproductive toxicity, other

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

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:

The aim of this study was to evaluate the effects of semicarbazide on the testicular morphology of juvenile Wistar rats because young people is a relevant population for exposure scenario.

GLP compliance:

not specified

Remarks:

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

Limit test:

no

Justification for study design:

The aim of this study was to evaluate the effects of semicarbazide on the testicular morphology of juvenile Wistar rats because young people is a relevant population for exposure scenario.

Specific details on test material used for the study:

Sigma-Aldrich Ref S2201
Purity : 99%
Test material form : solid: particulate/powder

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

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 with wood ship bedding junder a 12h light/dark cycle and room temperature of 22-24”C, with water and food ad libitum.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

The animals of the control group (G0) recieved the standard diet
The animals of the G3 and G6 groups received the semicarbazide hydrochloride in the diet at a concentration of 3g/kg and 6g/kg, respectively.

Details on mating procedure:

NA

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

30 days

Frequency of treatment:

once daily

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 j uvenile Wistar rats.

The animals of the control group (G0) recieved the standard diet, the animals of he G3 and G6 groups recieved the semicarbazide hydrochloride in the diet at a concentration of 3g/ 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 phosphatebuffered (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 semine ferous 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.

Parental animals: Observations and examinations:

Body weight changes were recorded and the testes were excised for analyzing

Sperm parameters (parental animals):

The testes were excised for analyzing

Litter observations:

NA

Postmortem examinations (parental animals):

The testes were excised for analyzing

Postmortem examinations (offspring):

NA

Statistics:

NA

Reproductive indices:

NA

Offspring viability indices:

NA

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

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

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 specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

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:

LOAEL

Effect level:

<= 3 000 mg/kg diet

Based on:

test mat.

Sex:

male

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Critical effects observed:

yes

Lowest effective dose / conc.:

3 000 mg/kg diet

System:

male reproductive system

Organ:

germ cells

seminiferous tubules

testes

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

yes

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

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:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Dose descriptor:

other: Not the goal of this study

Remarks on result:

other: Not the goal of this study

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

other: Not the goal of this study

Remarks on result:

other: Not the goal of this study

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

other: Not the goal of this study

Remarks on result:

other: vNot the goal of this study

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

3 000 mg/kg diet

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

not specified

Relevant for humans:

yes

Conclusions:

Semicarbazide 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.

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 (ten animals/group) and kept in polupropylene cages with wood ship bedding junder a 12h light/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 of 3g/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.

Semicarbazide 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.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

40 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Correct

Effects on developmental toxicity

Description of key information

2 published studies for developmental and teratogenicity toxicity are available, secondary litterature, Klimisch score 4.

Information from each single source alone is not sufficient to cover the endpoint. However, they can be used both in a weight of evidence as they followed strong scientific relevant protocoles.

Moreover, these 2 independant studies from different research teams showed significant teratogenic effects on rats and mice, following administration of Semicarbazide by oral route or intraperitoneal injection in a relevant day of gestation for this endpoint.

Therefore, based on expert judgment and these two presents studies, we can conclude that SC produced signs clear teratogenic effects, in rats and mice dosed with SC either by oral route and intraperitoneal injection at differents relevant days of gestation ( resorptions, cleft palate, skeletal anomalies (skull, a sternuum, ribs), abnormalities appeared in the brain, kidney, liver and intestines).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1986

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

SC hydrochloride (Supplier Merck) was dissolved in 1 mL of saline solution at a concentration of 0,9% NaCl and a final pH of 7,2. The animals were weighed at the moment of injection in order to calculate the mg of SC to be added to the mL of saline solution necessary for the dosis indicated by kg of weight.

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Female rats : Virgin
Weight at the beginning of the study : 200-250g

Food : standard rat died ad libitum
Water :ad libitum

Room temperature : 25+/-2°C

Light cycles : 12 hours light/12 hours dark, automatically regulated

Route of administration:

intraperitoneal

Vehicle:

other: Saline solution of NaCl

Analytical verification of doses or concentrations:

no

Details on mating procedure:

Virgin female Wistar rats were bred in estrus during the early afternoon and remainded caged with fertile male Wistar rats overnight (4 females to 2 males)
Pregnancy diagnosis was determined by vaginal smears.

Duration of treatment / exposure:

One group is injected with 17 mg/kg/day C throughout gestation
Another group on day 5, 7, 10, 13 or 15 of gestation with 50, 75, 100 or 150 mg/kg SC respectively

Frequency of treatment:

One group is injected with 17 mg/kg/day C throughout gestation
Another group on day 5, 7, 10, 13 or 15 of gestation with 50, 75, 100 or 150 mg/kg SC respectively

No. of animals per sex per dose:

All the experimental (single and coninuous dose) and control animals were divided into 2 groups : A and B.
Group A was made of 190 experimental animals.
Group B was made of 105 experimental pregant animal and 30 controls.

Control animals:

yes, concurrent vehicle

Details on study design:

Control animals received the same volume of saline solution intraperitoneally on a similar schedule i e on the day of gestation corresponding to that of the treated (experimental) animals.
10 pregnants females were used as controls for each of the days (5, 7, 10, 13 or 15) of gestation in which SC was administered.
Another 10 females controls received 1 mL saline solution daily during the whole gestation.

The animals of the group A were sacrified on day 21 of gestation. A midline abdominal incision was performed and both uterine horns were exposed.
Live and dead fetuses were removed and counted together with the number of resoprtions and implantations.
All fetuses were carefully examined for signs of externals malformations and their sex and weight noted down.
The live fetuses were then subdivided at random into 2 subgroups, a and b, in order to carry out a specific study in each of the subgroups.
In subgroup a, organic anomalies were studied. The fetuses were placed in Bouin's fluid, and their internal organs were then examined by sectioning with a razor blade following Wilson's technique..
The fetuses in subgroup b, were used to study the skeleton anomalies. They were fixed in 70% ethanol and then cleared with 1% OHK and stained with alizarin red S.

Group B: all animals from this group were allowed to deliver, and the mortality rate of their offspringwas studied during the first month of life.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number resorptions: Yes

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: No data
- Skeletal examinations: Yes:
- Head examinations: No data
- Internal organs : Yes

Statistics:

The mean +/-SEM values for treated and non-treated (control) fetuses were statistically compared with a 2-tail Student's t test. p<0.05 being the minimum significant value.

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

Some maternal deaths ensued after treatment with the 50, 100 and especially 150 mg/kg SC regimen.

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

There were several degrees of resoprtion rates as well deaths almost every day with all the doses studied.
The resroption rate were directly correlated with the dose; while the number of live fetuses was inversely related to it.
Treatment with SC was most embryotoxic onday 7 abd 10 of gestation, with statistically significant differences in the number of live fetuses with respect to controls with all the doses used.
The administration of SC on day 5 produced a significant decrease in the number od live fetuses only with the 100 mg/kg dose while on day 13 and 15 this occured with the 100 and 150 mg/kg doses.

The percentage of resorptions, expressed as small, medium or large in size and appaearing on the different days of treatment, were for days 5, 7, 10, 13 and 15 : 48, 27, 25, 3 and 0%, for the samll resorptions; 52, 68, 39, 38 and 32% respectively for the medium resorptions and 0, 4, 36, 52 and 68% respectively for the large resorptions.

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not specified

Key result

Dose descriptor:

conc. level:

Effect level:

>= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

mortality

Remarks on result:

other: NA

Abnormalities:

not examined

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Fetal weight is significantly decreased with all the doses used when these were admninistered on days 5, 7 or 10 of gestation.
With the 75, 100 and 150 mg/kg doses, fetal weight was significantly decreased on all the days of gestation in which SC was administered.

Reduction in number of live offspring:

effects observed, treatment-related

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

SC produced a high mortality rate with high statistical differences as compared to controls on all the days and with all the doses studied.
Mortality was highest when SC was administered on day 10 of gestation (63+/-3% beign the highest value found and corresponding to the 100 mg/kg dose).

External malformations:

not specified

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

The most frequent skeletal lesions found were an incomplete ossification of the skull and sternum, especially when SC was administered during the implantation period.
A small percentage of lesions was also found in the ribs. An incomplete ossification of the limbs also occured.
The malformations found in the fetuses whose mothers were injected with 17 mg/kg/day SC during the entire course of pregnancy were similar to the ones mentioned for the single-dose treatment; although the hemorrhages in the liver and hydronephroses were less frequent.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

The fetuses showed some abnormalities in the brain, especially when the rats were treated during the differentiation period.
These abnormalities were mainly internal hydrocephaly, and in somes cases, exencephaly and meningocele.
Severe hemorrhages in liver and intentines were also observed in the fetuses treated during this period.
A high percentage of hydronephroses appeared in the fetuses, regardless of the period in which their mothers were treated, although the percentage was higher during the developmental period.
A small percentage of anophtalmia on one side, cleft palate or an absence of testes was also observed in the fetuses which came to term.

Other effects:

not specified

Key result

Dose descriptor:

LOEL

Effect level:

< 17 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Remarks on result:

other: NA

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: limb

skeletal: skull

skeletal: sternum

skeletal: rib

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

17 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

not specified

Conclusions:

SC produced a significantly smaller number of live fetuses with respect to controls.
This toxic effect after injection at days 7 and 10 of gestation was highest for all single doses.
The mean of fetal weight decreased with respect to controls with almost all of the SC treatment studied.
The number of implantations and live fetuses was significantly decreased in the rats receiving the continuous treatment.
Most abnormalities in the 21-day-old fetuses wre found in the brain, kidney, intestines and liver; skeletal anomalies were seen in the skull, sternuum and ribs.
SC also produced high postnatal mortality rates during the first month of life with the single doses as well as the continuous treatment.
Thus, SC produced signs of toxicity and/or teratogenic effects in rats with all doses administered.

Executive summary:

In a developmental and teratogenicity toxicity study (published), 5 groups of pregnant Wistar rats were injected intraperitoneally with a single dose of SemiCarbazide (SC) on days 5, 7, 10, 13 or 15 of gestation. The lots in each group received either 50, 75, 100 or 150 mg/kg SC. A sixth group received 17 mg/kg of the drug during ther entire course of pregnancy.

Results indicate that SC has an embryotoxic effect on Wistar rats when it was administered intraperitoneally in single doses of 50, 75, 100 pr 150 mg/kg on the different gestation days studied or in daily injections of 17 mg/kg throughout the entire course of pregnancy.

One of the most notable effect of this substance wasfetal death and resorption, resulting in a significant decrease in the number of live fetuses with respect to controls.

The embryotoxicity of SC as seen from the number of resorbed or dead conceptuses may provide an interesting correlation as to the extent of its teratogenicity.

The dosage levels producing these effects also caused some maternal toxicity. However, some pregnant females who did not show anu overt signs of toxicity had litters with malformed embryos. Thus, the embryotoxic consequences of SC treatment were due to a direct action on the embryo (and not to an indirect effect through the mother).

Intrauterine growth (weight of the 21 -day old fetuses) was decreased with the single dose injections but not with the continuous treatment. The decrease in the number of live fetuses and implantations per litter, however, wss more notable with the coninuous treatment.

Thus from the results foundon the relationship between resorption sizes and days injected, it is possible to infer taht SC acts on intrauterine life, stopping its growth one the drug is administered.

A slight increase in the number of organic lesions when SC was injected during the differentiation period (which corresponds to the teratogenic susceptibility othe rat during these days 9 and 10 of gestation) has been observed. Abnormatlities in the brain, the kidney, intestines and liver ; skeletal aomalies were seen in the skull, sternuum and ribs. SC produced also high postnatal mortality rates during the first month of life with the single doses as well as with the continuous treatment.

Tus, SC produced signs of toxicity and/or teratogenic effects in rats with all doses administered.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

no guideline followed

Principles of method if other than guideline:

Ingestion of diets containing 50% ground sweet pea seeds (Lathyrus odoratus) by pregnant Sprague-Dawley rats on days 10-20 of gestation resulted in high frequencies of fetal resorption and surviving young with various degrees of edema, forelimb flexure, angulation of the vertebral column, micrognathia, cleft palate, and other defects.
Three other lathyrogenic agents produced high frequencies of cleft palate in rats: 50 mg aminoacetonitrile on day 15, 400 mg D-penicillamine on days 10-15, and 50 mg
semicarbazide on days 10-16.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Not available

Species:

rat

Strain:

Sprague-Dawley

Remarks:

A second study conducted on mice

Details on test animals or test system and environmental conditions:

Female Sprage-Dawley rats weighing 230-260g
Female A/J mice weighing 25g

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Test items were administered in aqueous solutions

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Female Sprague-Dawley rats were mated with fertile males overnight and checked by vaginal smears for insemination the following morning. Rats with smears positive for sperm were considered to be at day 0 of gestation.

Likewise, female A/J mice weighing about 25 g were placed with males in late afternoon and removed the following morning. Copulation was ascertained by the presence of a vaginal plug and the time was designated day 0 of pregnancy.

Duration of treatment / exposure:

SC has been administered on days 10-16 of gestation

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6, 8, 3, 11 and 4 female pregnant rats at respectively 100, 50, 25, 10 and 5 mg/kg

Control animals:

no

Details on study design:

Nb of females treated Dose Days of gestation adm.

6 100 12-15
8 50 10-16
3 25 10-16
11 10 12-15
4 5 12-15

Maternal examinations:

Not available

Ovaries and uterine content:

Not available

Fetal examinations:

- External examinations: yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes:
- Head examinations: Yes:

Statistics:

Not available

Indices:

Not available

Historical control data:

Not available

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

3 of 6 pregnants rats treated daily with 100 mg of SC on days 12-15 of gestation died

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Nb of females treated Dose (mg/day) Days of gestation adm. Resorption
No %
6 100 12-15 28/50 56
8 50 10-16 26/68 38
3 25 10-16 1/29 3
11 10 12-15 0/107 0
4 5 12-15 1/33 3

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not specified

Dose descriptor:

conc. level:

Effect level:

>= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

mortality

Remarks on result:

other: NA

Abnormalities:

no effects observed

Fetal body weight changes:

not specified

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

effects observed, treatment-related

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Nb of females treated Dose (mg/day) Days of gestation adm. Cleft palate
No %
6 100 12-15 22/22 100
8 50 10-16 40/42 95
3 25 10-16 12/28 43
11 10 12-15 0/107 0
4 5 12-15 0/32 0

Visceral malformations:

not specified

Other effects:

not specified

Key result

Dose descriptor:

LOAEL

Effect level:

ca. 25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

skeletal malformations

Remarks on result:

other: NA

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: skull sutures

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

not specified

Relevant for humans:

no

Conclusions:

The four lathyrogenic compounds tested in this study had a considerable teratogenic effect.
Semicarbazide produced cleft palate.
A high frequency of cleft palate was produced after acute administration of the four lathyrogens tested.
(The period of greatest susceptibility for cleftpalate produclion with these lathyrogens was day 15, since multiple doses of these agents produced the highest incidence of cleft palate when this day was involved).

Executive summary:

In a teratogenicity study (published), rats and mice were administered SemiCarbazide by oral intubation in aqueous solutions.

Pregnant rat were administered at dose of 5, 10, 25, 50 and 100 mg/kg day on days of gestation12 -15, 12 -15, 10 -16, 10 -16 and 12 -15 respectively. Resorptions and Clef palate have been studied.

Three of six pregnant rats treated daily with 100 mg of semicarbazide on days 12-15 of gestation died.

The surviving animals produced litters with many resorptions and 100% cleft palate.

Cleft palate and resorption were also produced by 25 or 50 mg of this lathyrogen on days 10-16 of gestation, but not by 5 or 10 mg on days 12-15.

Thus, as Semicarbazide produced cleft palate, a terotogenic effect.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

17 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Correct

Justification for classification or non-classification

Based on the fact that most of the available studies have been performed using juvenile animals (i.e. not matures), and no literature data have demonstrated effects on humans.

Based on results showing effects on juveniles but not on mature animals, leading us to conclude that no effect have been observe on mature animals.

Based on the 90-days study on mature rats did not observe any effects on animals.

As major effects have been observed in reproductive organs from juvenile animals (rats), suggesting teratogenesis effects (effects showed with the studies on developmental), we propose to classify the substance as Reproductive toxicity 2.