Semicarbazide hydrochloride

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Semicarbazide hydrochloride

EC Number:

209-247-0

EC Name:

Semicarbazide hydrochloride

Cas Number:

563-41-7

Molecular formula:

CH5N3O.ClH

IUPAC Name:

semicarbazide hydrochloride

Specific details on test material used for the study:

Semicarbazide hydrochloride (Sigma-Aldrich, Ref S2201)

Test animals

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.

Administration / exposure

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.

Doses / concentrations

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

Examinations

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).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

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)

Effect levels (P0)

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

Target system / organ toxicity (P0)

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

Results: P1 (second parental generation)

General toxicity (P1)

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 / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Effect levels (P1)

Dose descriptor:

other: Not the goal of the study

Remarks on result:

other: Not the goal of the study

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

other: Not the goal of the study

Remarks on result:

other: Not the goal of the study

Results: F2 generation

General toxicity (F2)

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

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Dose descriptor:

other: Not the goal of the study

Remarks on result:

other: Not the goal of the study

Overall reproductive toxicity

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

Any other information on results incl. tables

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).

Applicant's summary and conclusion

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.