Imidazolidine-2-thione

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No study on fertility is available on ETU.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Ethylene thiourea is teratogenic to rats and the overall NOAEL for the teratogenic effect on this species is estimated to be 5 mg per kilogram body weight per day. Rats are much more susceptible to ethylene thiourea than mice. The foetal organs primarily affected are the central nervous system, the kidney and ureter and the skeletal system. The level of exposure, method of administration and the period of dosing during pregnancy may influence the degree of teratogenicity.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline followed

Principles of method if other than guideline:

ETU was administered orally in single daily doses of 0, 5, 10, 20, 40,or 80 mg/kg to nulliparous rats

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Woodlyn Farms, Guelph, Ontario.
- Age at study initiation: no data
- Weight at study initiation: 200-225 g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS : no data

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

no

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 M / 1 F
- Length of cohabitation: overnight
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy

Duration of treatment / exposure:

3 experimentations :
- from 21-42 days before gestation to gestation day 15 (=28 days),
- from gestation days 6-15,
- from gestation days 7-20.

Frequency of treatment:

daily

Duration of test:

2 months

Remarks:

Doses / Concentrations:
5, 10, 20, 40 and 80 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

between 10 and 18 females / group

Control animals:

yes, concurrent vehicle

Details on study design:

no

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes, on gestation day 1 and 6-15, as well as before and after cesarean section
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 22
- Organs examined: their viscera (including uteri) were searched for pathological changes

Ovaries and uterine content:

no data

Fetal examinations:

The foetuses were weighed and examined for viability and external malformations.
Two-thirds of the living young from each litter of experiments I and II were processed for skeletal examination. The remaining ones from experiments I and II and ail fetuses from experiment III were fixed in Bouin's fluid for examination of viscera. Visceral examination was conducted on gross (3-4 mm) and microscopic sections cut transversely, sagittally, or horizontally.

Statistics:

yes, no details (see tables)

Indices:

no

Historical control data:

no

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
The present experiment showed that oral administration of 80 mg/kg/day ETU was lethal in 9 of 11 females after 7-8 days of administration. However, the females continually given up to 40 mg/kg from 42 days preconception until day 15 of gestation or on day 7-20 of gestation manifested no obvious signs of toxicity.

Key result

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

mortality

Dose descriptor:

LOAEL

Effect level:

80 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

mortality

Abnormalities:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Effects on prenatal survival
Doses of 80 mg/kg in experiment II and 40 mg/kg in experiments I and III reduced the mean fetal weight as compared to those in the matching control groups ( See table 1). Numbers of corpora lutea and live fetuses, and fetal death at all doses were similar to those in the controls in the three experiments. Data from experiment I (table 1) also suggested that ETU at the maximum tolerated doses administered before and during gestation had no significant effect on any reproductive parameter.

Morphology of fetal anomalies
80 mg/kg. This dose was used only in experiment II. Two gravidas survived until term and provided 24 viable fetuses. External malformations in them consisted of rudimentary lower jaw and tongue, exophthalmos, coloboma of eyelids, microcephaly, fluid-filled meningoencephalocele, hemimelia, kyphoscoliosis, and short or absent tail.
Histological examination of the central nervous system from all the fetuses sectioned revealed a striking deficiency of nervous tissue together with subdural edema, occasionally in the form of cysts. The telencephalon had failed to fuse at its anterior end and was poorly differentiated into cerebral lobes. Its choroid plexus arose from the neighboring connective tissue. A part of the brain and ventricular system was exencephalic. Dorsal walls of the telencephalon and/or mesencephalon had failed to fuse and their cavities were open to the exterior. The ventricular system was distended. The diencephalon was retarded in growth and its anterior part had a large number of rosettes and ductules. The cerebellum was apparently absent. The spinal cord was devoid of dorsal and ventral fissures, neural canal, and differentiation into the ependymal, mantle, and marginal layers. The cord was atrophic and this atrophy was most marked in the scoliotic region of thoracic vertebrae 4-8 whose arches were closely applied to the centra so as to reduce the vertebral canal to a slitlike passage. The lens was dislocated and occupied the anterior chamber. In addition to the anomalies enumerated above, the stained skeletons revealed retarded ossification of skull and mandible to such an extent that most bones were unrecognizable.
40 mg/kg. Externally visible defects were fluid- or blood-filled meningocele, hydrocephalus or ectopic cranium bulging into a translucent domo, micrognathia, oligodactyly, and short and kinky tail. In addition, there was a high incidence of postural defects in the pelvic limbs characterized by abnormally increased flexion at the tibiometatarsal joint accompanied by equinovarus. Following manual manipulation to correct the postural defects at the affected joints, the original distorted posture reappeared in viable fetuses. In alizarin-stained specimens, however, the hind limbs were without skeletal anomalies.The brain appeared to be the most commonly affected organ. Gross examination revealed that the lateral, third, and fourth ventricles tended to form a single cavity. Microscopically, subdural edema of the brain was seen regularly; it was particularly marked in the regions dorsal to the fourth ventricle and medulla oblongata, and was frequently associated with meningocele. Reduction in thickness of the cerebral hemispheres accompanied enlargement of the lateral ventricles. The neuroepithelial and subependymal layers surrounding the lateral ventricles were reduced and occasionally foci of tell aggregates were seen subependymally. There was a marked con-gestion of blood vessels around the pineal body. Meningorrhagia and meningorrhea extended extracranially through the interparietal space. In cleared specimens, the meningorrheal lesion was stained with alizarin, and was surrounded by a clear area where ossification appeared to be absent. The dorsal part of the mesencephalon and cerebellum were variably reduced in size. The cell population was decreased in the mantle layer (gray) of the spinal tord as compared to that in the control. The ependymal lining in the thoracic and lumbar regions was disoriented so as to reduce or entirely obliterate the neural canal. In experiment I one fetus with a markedly reduced trunk was observed. Developmental defects in fetuses treated on days 7-20 of pregnancy were more pronounced when compared with the fetuses from the other two experimental groups. Otherwise, the types of ETU-induced lesions in all rat experiments were similar.
20 mg/kg. Moderate distension of the ventricular system, dilatation of the pineal-stalk lumen, congestion of cerebral vein surrounding the pineal gland, and hypoplasia and retarded differentiation of the cerebellum were noticed. In addition a low incidence of encephalocele occurred.
10 mg/kg. An increased incidence of arrested parietal ossification and retarded Purkinje-cell migration relative to those in the controls occurred. However, the severity and incidence of these lesions were minimal.
5 mg/kg. The incidence of abnormalities was comparable to that in controls except for a high frequency of retarded ossification of the parietal bone. This defect was limited to a few large-sized litters and involved small areas.

Incidence of fetal anomalies
The incidences of developmental defects in the external anatomy, viscera, and skeleton are listed in tables 2-4, respectively. In addition, not listed in these tables but observed only in the 80-mg/kg group, was a higher incidence of hemimelia, syndactyly, cleft palate, ectopie kidney, rudimentary calvarium and mandible, and scoliosis.
A survey of tables 2-4 reveals dose-related incidences of various anomalies, and reproducibility in all three experiments despite different treatment durations. The few instances where neither of these occurred were explainable. A low incidence of kinky tail could be due to the fact that about 80% of the fetuses had tails that were too short to develop kink.

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

visceral malformations

Dose descriptor:

LOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on central nervous system and gross developmental defects of the brain including exencephaly, dilated ventricles and hypoplastic cerebellum.

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1 : Effects of ethylenethiourea on prenatal development of rats

Exp. no.	Duration of dosing	Dose (mg/kg)	No. of dams pregnant at term	Mean no. of corpora lutea	Mean no. of live fetuses per pregnancy	% Fetal death : resorbedX100 total implants	Mean fetal wt (g)
I	from 21-42 days before gestation until day 15 of gestation	0	10	13.1	7.3	13	5.0
		40	11	14.7	10.6	11	4.5^t
		20	12	15.6	10.1	16	4.8
		10	12	14.0	9.7	11	4.9
		5	18	15.7	11.2	8	5.0
II	days 6-15 of gestation	0	13	15.5	12.8	7	4.6
		80	2	13.5	12.0	8	2.6^t
		40	14	14.1	11.6	7	4.6
		20	13	14.7	11.5	6	4.6
		10	12	15.4	11.5	9	4.5
		5	11	15.0	12.0	3	4.5
III	days 7-20 of gestation	0	17	14.8	11.9	6	4.7
		40	15	13.5	10.9	4	3.7^t
		20	17	14.8	12.8	6	4.9
		10	16	13.9	11.0	8	5.0
		5	16	13.8	10.6	9	5.0
^t P<0.025 (one-tailed t test)

Table 2 : Gross external defects in fetal rats after maternal treatment with ethylenethiourea

Experiment no.	I					II						III
Dose (mg/kg)	0	40	20	10	5	0	80	40	20	10	5	0	40	20	10	5
Fetus examined	66	127	121	100	156	167	24	178	81	138	132	191	163	203	176	158
Numbers of litter	10	12	11	10	14	13	2	14	8	12	11	17	15	16	16	15
% anomalies
exencephaly	a	20	1				75	16	1				32	1	3
hydrocephalus													25	3
Coloboma of eyelids							58
Micrognathia		19	3				75	20					7
Oligodactyly (forepaw)							58	3
Adnormal flexion at tibiotarsus joint and equines foot		41	2					42	6				30	8
Hemimelia, partial short tail		42					80	33					43
Hemimelia, partial kinky or twisted tail		57	11				13	42	15	1			34	3
% total malformed fetuses		83	11				100	80	17	1			91	12	3
a Empty cells denote 0 incidence

Table 3 : Effects of ethylenethiourea on fetal rat skeleton

Experiment no.	I					II
Dose (mg/kg)	0	40	20	10	5	0	80	40	20	10	5
Fetus examined	40	68	71	723	131	95	9	88	79	78	72
Numbers of litter	10	11	10	12	18	13	2	14	12	11	11
% anomalies
Ectopic tissue in interparietal space	a	31					100	43	1
Retarded ossification
- parietal	3	52	11	42	33		100	67	20	12	9
- interparietal		25					100	11	1
- occipital							100	9
Kyphoscoliosis							45
Spontaneous anomalies*	13	16	4	9	5	9	100	8	14	3	10
a Empty cells denote 0 incidence * Wavy, fused, or extra ribs, missing or non-aligned sternebrae, fused vertebrae

Table 4 : Effect of maternal treatment with ETU on development of fetal brain and viscera

Experiment no.	I					II						III
Dose (mg/kg)	0	40	20	10	5	0	80	40	20	10	5	0	40	20	10	5
Fetus examined	24	46	42	35	69	24	6	38	29	20	28	182	140	206	176	149
Numbers of litter	10	11	11	11	7	11	2	13	11	10	11	17	15	17	16	16
% anomalies
exencephaly	a	24					100	21
Encephalocele or meningocele		7	7				100	21	3				32
Dilatation of :
-lateral ventricles		84	45	3			100	95	23				100	19	1
-mesencephalic cavity		52	29	6			100	42	23				100	20
-aqueduc and 4^thventricule		41	21				100	50	23				100
-cytic aqueduct and 4^thventricle		19						21					100
Hypoplastic cerebellum		98	64				100	70	23	10			100	41	2
Other defects *							50	5	3	3
% total malformed fetuses		98	69	6			100	95	38	3			100	41	2
a Empty cells denote 0 incidence * Cleft palate, distorted course of aortic arch, ectopic kidney

Conclusions:

ETU was teratogenic in rats at doses that produced no apparent maternal toxicity or fetal lethality. Lesions were consistently seen in the central nervous system

Executive summary:

In a developmental toxicity study, ethylene thiourea was administered to Wistar rats by gavage at doses of 0, 5, 10, 20, 40, or 80 (group II only) mg/kg/day. There were 3 treatment groups: Group I was treated from 21-42 days before mating until gestation day (GD) 15, Group II was treated from GD 6-15, and Group III from GD 7-20. Maternal toxicity was noted at the dose level of 80 mg/kg/da. The NOAEL for maternal toxicity is 40 mg/kg/day.Similar developmental defects occurred at generally the same doses in groups which were treatedat different times of gestation. At 10 mg/kg/day and above, defects included: exencephaly, dilated ventricles, and hypoplastic cerebellum; at 20 mg/kg/day and above: hydrocephalus, encephalocele, meningocele, micrognathia, abnormal flexion of ankle, kinky or twisted tail; at 40 mg/kg/day and above: oligodactyl, domed head, retarded ossification of the skull occurred; and at 80 mg/kg/day: coloboma of the eyelids, hemimelia, syndactyl, cleft palate, ectopic kidney, rudimentary calvarium, short tail, scoliosis and several types of skeletal anomalies occurred. Decreased fetal weight was noted at 40 mg/kg/day in groups I (90% of controls) and group III (79% of controls) and at 80 mg/kg/day in group II (56% of controls). Fetuses at 40 mg/kg/day in group II had weights comparable to controls. Number of live fetuses and corpora lutea were comparable to controls.The NOAEL for developmental toxicity is 5 mg/kg/day and the LOAEL is 10 mg/kg/day based on developmental defects of the brain (exencephaly, dilated ventricles, and hypoplastic cerebellum).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

All key studies are reliable with a klimisch score of 2.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Humans

 

A retrospective study of women who had been employed in the manufacture of rubber containing ethylenethiourea was reported (Smith, 1976). The potential participants were all 699 women of child-bearing age who had left employment at the factory between 1963 and 1971. Of these, 255 who had given birth to 420 children were traced. Of these women, 59 had been employed in the rubber plant at the time of their first pregnancy, and none had given birth to an abnormal child. Of the 420 children, 11 had malformations. Three of these had been born before the employment of their mother and eight had been born more than 1 year after their mothers’ employment.

 

Experimental systems

 

(a) General developmental toxicity

 

ETU (100% purity) was administered orally at doses of 0, 5, 10, 20, 40 and 80 mg/kg bw in distilled water to nulliparous rats (Wistar) (10-17 pregnant dams per dose). Treatment was made from 21-42 days before conception to pregnancy day 15, and on days 6-15 or 720 of pregnancy. All pups were delivered via C-section and examined for skeletal and visceral anomalies. Microscopic examinations were performed on brains. Doses of 40 mg/kg were not toxic to rats; however, 80 mg/kg was lethal to 9 of 11 female rats. Mean foetal weight was reduced at 40 mg/kg compared to control. Measurements of sterility, pre-implantation loss and post-implantation survival were comparable to controls. The brain was the most commonly affected organ. ETU induced meningoencephalocele, meningorrhagia, meningorrhea, hydrocephalus, obliterated neural canal, abnormal pelvic limb posture with equinovarus, and short or kinky tail at 10 mg/kg in all phases of the rat studies. Although no abnormalities were reported in rats at 5 mg/kg, there was a higher frequency of delayed ossification of the parietal bone, compared to controls (Khera, 1973).

 

ETU (100% purity) was administered via oral gavage at 40 mg/kg bw from days 7 to 15 of gestation to pregnant CR rats (10-12 rats/group). Rats were hypothyroid and euthyroid. There was a problem, however, in maintaining the euthyroid state in rats given T₄supplement. Rats were also given thyroxine to determine if ETU teratogenicity occurred through alterations of maternal thyroid function. ETU was found to be teratogenic in the rat but not through alteration of maternal thyroid status. It was also demonstrated that ETU lowered serum [T₄]; that hypothyroidismper seincreased the background level of malformations in the rat; that T₄alone was embryotoxic but not teratogenic; and that hypothyroidism altered the spectrum of malformations in response to ETU both quantitatively and qualitatively (Lu & Staples, 1978).

 

The teratogenic effects of ethylenethiourea were evaluated in groups of 12–29 Sprague-Dawley rats, 31–33 CD-1 mice, 15–19 golden hamsters and three to five Hartley guinea-pigs exposed daily by oral gavage on days 7–21, 7–16, 5–10 and 7–25 of gestation, respectively, to a dose of 0, 5, 10, 20, 30, 40 or 80 mg/kg bw per day, 0, 100 or 200 mg/kg bw per day, 0, 75, 150 or 300 mg/kg bw per day and 0, 50 or 100 mg/kg bw per day, respectively. The fetuses were examined at the end of gestation for external, internal and skeletal malformations. Ethylenethiourea was toxic to the pregnant rats at 80 mg/kg bw per day, while a variety of malformations (e.g., hydrocephalus, encephalocoele, cleft palate, kyphosis and limb and digital defects) were observed at doses >=20 mg/kg bw per day; fetal body weights were reduced at doses as low as 10 mg/kg bw per day. In mice, the maternal liver weights were increased at the two highest doses; the only significant fetal effect was an increased incidence of supernumerary ribs at 200 mg/kg bw per day. No significant maternal or fetal effects were seen in hamsters or guinea-pigs. Other groups of 11–13 rats received 0, 20, 25 or 30 mg/kg bw per day ethylenethiourea on gestation day 7; they delivered their offspring, and exposure was continued until lactation day 15. The offspring were tested for a variety of indicators of reflex development, and the motor activity of males was recorded in an open-field device for 4 min over 2 consecutive days at 6 weeks of age. There were no effects on litter size at birth, but 6/13 litters of dams at the highest dose failed to nurse, and 40% of the surviving offspring had developed hydrocephaly by day 45. There were no treatment-related effects on the offspring body weights, startle or righting reflex development or eye opening, but there was a dose-related increase in defaecation in the openfield test on days 1 and 2 and in activity on day 2 (Chernoff et al., 1979).

Teramoto et al. (1978) investigated the teratogenicity of ETU in rats, mice, and hamsters. It was teratogenic when given orally to rats at 20 - 50 mg/kg body weight per day on days 6 - 15 of pregnancy and to hamsters at 270 - 810 mg/kg body weight per day on days 6 - 13 of pregnancy. However, no malformations were induced in mice up to a daily oral dose of 800 mg/kg body weight when given on days 7 - 15 of pregnancy. In hamsters, cleft palate, kinky tail, oligodactyly, and anal atresia were noted as gross external malformations. Skeletal examination revealed a high incidence of defects in the ribs and vertebral column, but no apparent defect was observed during visceral examination.

In a screening assay for developmental toxicity, 600 mg/kg bw ethylenethiourea were given by oral gavage to 35 CD-1 mice on days 7–14 of gestation, and the growth and viability of the offspring were evaluated after birth for 3 days and compared with those of a group of 45 untreated controls. A significant increase in the frequency of litters that were completely resorbed was found, but there were no effects on postnatal growth or viability (Plasterer et al., 1985).

 

Groups of 20–23 Sprague-Dawley rats were given 0, 15, 25 or 35 mg/kg bw per day ethylenethiourea by oral gavage on gestation days 6–20. There were no signs of maternal toxicity at any dose. The fetal body weights were reduced at the highest dose, which also caused malformations such as cranial meningocoele and meningorrhoea, severe hind limb talipes and short and/or kinky tails. Rats at the two higher doses had higher incidences of dilated brain ventricles and hydroureter than controls (Saillenfait et al., 1991).

 

ETU (100% purity) was administered orally at doses of 0, 5, 10, 20, 40 and 80 mg/kg bw in distilled water to nulliparous rabbits (New Zealand white). There were 5-7 pregnant does per group. Treatment was made from days 7 to 20 of pregnancy. All pups were delivered via C-section and examined for skeletal and visceral anomalies. Microscopic examinations were performed on brains. No toxicity was apparent in rabbits given 80 mg/kg bw. Foetal weights were not affected. Measurements of sterility, pre-implantation loss and post-implantation survival were comparable to controls. Rabbits presented no evidence of malformations at the doses administered. However, there was an increase in resorption sites, decreased brain weight, and degeneration of the proximal convoluted tubules in the kidneys of foetuses at 80 mg/kg bw (Khera, 1973).

Pregnant rats were exposed whole-body 3 hours daily to 27.2, 55.5 and 120.4 mg/m3 of aerosols of ethylenethioureaduring Days 7 through 14 of gestation. All animals were sacrificed on Day 20 of gestation, and the dams and fetuses were examined for gross changes. Fetuses were fixed in Bouin's solution or alcohol and examined later for teratology. Ethylenethiourea caused increased fetal mortality and decreased fetal weight gain at 120.4 mg/m3. Ethylenethioureawas not teratogenic (Newell et al., 1978; Dilley et al., 1977).

 

(b) Phase specificity

 

The stage-dependence of the teratogenic effects of ethylenethiourea was demonstrated in Wistar rats exposed by gavage to 40–480 mg/kg bw on one of days 6–21 of gestation. The earliest teratogenic effects were seen after treatment on day 10, and included failure of coccygeal growth, spina bifida, ectopic genitalia and nephrosis. The incidence of defects peaked after exposure on days 12-15, but eefects such as hydrancephaly, hydronephrosis and subcutaneous oedema were seen after exposure as late as day 21 (Ruddick & Khera, 1975).

 

Ethylenethiourea was given orally to Wistar rats at a single dose of 1–50 mg/kg bw in aqueous suspension on day 17, 18, 19 or 20 of gestation. The incidence of stillbirths was increased at doses of 30 and 50 mg/kg bw on day 18, 19 or 20. Regardless of the age at exposure, doses as low as 10 mg/kg bw were associated with reduced offspring viability due to hydrocephaly (Lewerenz & Bleyl, 1980).

 

Ethylenethiourea has been used as a prototype teratogen to study postnatal functional development of the kidney. Prenatal exposure of Sprague-Dawley rats to 0–160 mg/kg bw ethylenethiourea on day 11 of gestation produced dose-related increases in the incidence of enlarged renal pelvis in the fetuses on day 21 of gestation. Further studies were conducted to explore the postnatal consequences on renal development and function after exposure to 0, 20, 40 or 60 mg/kg bw on day 11 of gestation. The incidence of hydronephrosis after birth was lower than anticipated from the study of prenatal exposure, probably as a result of increased postnatal mortality. The severity of the hydronephrosis, however, increased with postnatal age. The hydonephrotic animals had impaired concentrating ability, but cortical function (proximal tubule transport) was unaffected. Rats exposed to ethylenethiourea prenatally had grossly normal kidneys but showed suppressed electrolyte clearance early in life. The latter effect was no longer apparent by postnatal day 27 (Daston et al., 1988).

 

The effect of prenatal exposure to ethylenethiourea on the development of the posterior gut was studied in 28 Wistar-Imamichi rats treated with 100, 125, 150 or 200 mg/kg bw ethylenethiourea by intragastric administration on day 11 of gestation. Another four pregnant rats were available as controls. Fetuses were examined on day 20 of gestation. The dose-related malformations included absent or kinked tails, spina bifida and myeloschisis. The incidences of malformations were significantly higher in male than female fetuses. Histological examination of 57 fetuses exposed to 125 mg/kg bw revealed an incidence of anorectal malformations in 92% of males and 41% of females (Hirai & Kuwabara, 1990).

 

The phase specificity of ethylenethiourea was studied in Sprague-Dawley rats exposed by oral gavage to 0, 60, 120 or 240 mg/kg bw on one day of gestation between days 8 and 19. The number of litters per group was not specified, but there were 113 females in the experiment and 717 fetuses (16–86 per group). Fetuses were examined on day 20 for soft-tissue anomalies by histological procedures. A high rate of mortality was seen after exposure on days 8–10. Exposure to the two higher doses resulted in a variety of central nervous system malformations (e.g., spinal raphism, exencephaly, hydranencephaly and hydrocephaly) after exposure on one of days 11–18 of gestation, and the specific malformations showed phase sensitivity. Thus, short tail was observed after exposure on one of days 11–14, spinal raphism after exposure on day 11, exencephaly after exposure on day 12 or 13, microencepahly after exposure on day 14 and hydranencephaly after exposure on day 15 or 16 (Hung et al., 1986).

 

The effects of ethylenethiourea on prenatal brain development were further studied in 20 pregnant Sprague-Dawley rats that were exposed to 60, 120, 240 or 360 mg/kg bw ethylenethiourea by gavage on day 11 of gestation (Hung, 1992). A total of 155 fetuses from the treated groups and 38 fetuses from three controls were examined on day 20 of gestation. Dose-related incidences of malformations, which reached 100% at the highest dose, were observed. The most prominent defects included omphalocoele, lumbosacral myeloschisis and imperforate anus. No malformations were observed in the fetuses of control dams. The author noted that the effects were consistent with an early alteration of mesodermal development (Hung et al., 1986).

In studies by Khera & Tryphonas (1977), groups of pregnant rats were administered ETU at dose levels of 0, 15, 30, or 45 mg/kg body weight on day 15 of gestation and then subjected to a variety of test conditions to evaluate pre- and postnatal effects. Postnatal mortality occurred in pups from mothers treated with dose levels exceeding 15 mg/kg or pups cross-fostered to evaluate lactation exposure. All pups from mothers treated with 45 mg/kg died within 4 weeks of birth. A high incidence of hydrocephalus and microphthalmia was observed in pups of mothers treated with 30 mg/kg and these pups died within 6 weeks of birth. Motor defects observed in some survivors (16/65) of this group, were shown to result from the hydrocephalic condition, which was accompanied by atrophy of the cerebral cortex and subcortical white matter. These defects were found to be a direct result of in utero exposure to ETU and not of exposure during lactation (cross-fostered pups showed the same effects as pups weaned from treated dams). When mated with normal male rats, all female offspring of rats administered 30 mg/kg gave birth to normal offspring. The F2 generation was

not impaired, though some of the parents had neurological defects. In these studies, no effects on the parameters examined were observed at 15 mg/kg body weight.

An oral dose of 100 or 200 mg ETU/kg body weight given to pregnant rats consistently produced brain abnormalities in the fetuses, when given on day 12 or 13 of pregnancy, and forelimb abnormalities, when given on day 13 of pregnancy (Teramoto et al., 1978). Histological studies revealed extensive cell necrosis in the brain and forelimbs of embryos 24 h after the treatment. These lesions were considered to be the main cause of the abnormalities observed.

(c) Mode of action in vitro

 

The direct effect of ethylenethiourea on rodent embryo development was studied in whole-embryo cultures. Addition of 40–200 µg/mL ethylenethiourea to 10-day-old Sprague-Dawley rat embryos and culturing for 48 h in vitro resulted in dose-related inhibition of growth and differentiation and increased incidences of malformations. The authors attributed the findings to altered osmotic fluid balance in the embryo, as the osmolality of the exocoelomic fluid was reduced after 48 h in culture (Daston et al., 1987).

 

The development of 10-day-old Sprague-Dawley rat embryos exposedin vitro to ethylenethiourea by direct addition of 0–2.0 mmol/L (0–204 µg/mL) ethylenethiourea to the growth medium of whole-embryo culture or exposedin uteroto 0, 60 or 120 mg/kg bw ethylenethiourea by oral gavage was examined to assess the similarity of the two approaches in inducing central nervous system defects (Khera, 1989). In culture, the embryos showed hydrocephalus after 26 h of exposure to 1.5 or 2.0 mmol/L ethylenethiourea. No hydrocephaly was observed in embryos exposed in vivo. The lack of consistency in results obtained in vitro and in vivo may be due to differences in kinetics and in the critical period of exposure. It has been pointed out that the concentrations and the areas under the curve of concentration–time used in vitro are substantially higher than those obtained for teratogenic exposures in vivo (Daston, 1990).

 

The sensitivity of comparably staged Sprague-Dawley rats (gestation day 10.5) and CD-1 mice (gestation day 8.5) in whole-embryo culture was evaluated after a 48-h exposure to ethylenethiourea (at 0, 80, 120 or 160 µg/mL for rats and at 0, 80, 160, 240 or 320 µg/mL for mice). The teratogenic effects were qualitatively similar in the two species and were characterized by excessive accumulation of fluid in structures, particularly in the neural tube, but the potency was approximately twice as great in rats. When an exogenous metabolic activation system from a 9000×g supernatant of liver from Arochlor 1254-induced rats and mice (S9 mix) was added to the treatment protocol, rat S9 had virtually no effect on the embryonic effects typical of ethylenethiourea, but these were virtually eliminated by mouse S9 in both species. Of note, however, was that addition of mouse S9 and ethylenethiourea to mouse embryos in culture resulted in the induction of abnormalities (mainly open neural tube) not seen in rat or mouse embryos exposed in vitro to ethylenethiourea alone, or in mouse embryos exposed in vivo (Daston et al., 1989).

 

The effects were studied of direct addition of ethylenethiourea to 11-day-old Wistar-Imamichi rat embryos cultured for 48 h and to midbrain and limb bud cells removed from 11-day-old embryos. Malformations in cultured embryos were observed at concentrations >=30 µg/mL ethylenethiourea. Consistent with the predilection for neural tube defects over limb defects, when the cells were exposed to ethylenethiourea in culture, the median concentration for inhibition of differentiation of midbrain cells was 2.3- and > 14-fold lower than that for limb bud cells on days 11 and 12 of gestation, respectively (Tsuchiya et al., 1991a).

 

Using micromass cultures of midbrain or limb bud cells from 10-day-old JcL/ICR mice or 11-, 12- or 13-day-old Wistar-Imamichi rats exposed either directly to ethylenethiourea in culture (0–600 µg/mL) or using serum of rats and mice treatedin vivo(collected 2 h after exposure to 200 mg/kg bw), it was demonstrated that the species difference is at least partially intrinsic to the embryo. That is, the concentration of ethylenethiourea required to affect midbrain cell cultures from 10-day-old mouse embryos directly was 11-fold greater than that required for cultures from 12- and 13-day-old rat embryos. In addition, rat, but not mouse, midbrain cell differentiation was affected when serum from treated rats or mice was used in the culture medium. In the rat cell culture, the midbrain was affected more than limb bud cells, in parallel with effects noted in embryos treated in vivo. The concentration of ethylenethiourea in rat sera was only twofold higher than that in mouse sera. The study indicates that the species difference is likely to be due to differences in both kinetics and dynamics between rats and mice (Tsuchiya et al., 1991b).

 

(d)Altered thyroid function

 

A teratogenic dose (40 mg/kg bw per day) of ethylenethiourea was given by gavage once daily on days 7–15 of gestation to hypothyroid and euthyroid Charles River rats, and the fetuses were examined on day 20 of gestation. Additional euthyroid groups received subcutaneous injections of ethylenethiourea with or without thyroxine (5 µg/0.1 mL per 100 g bw/day) on days 7–15 of gestation. Hypothyroidism was induced by surgical removal of the thyroparathyroid gland at 75 days of age, 3 weeks before breeding. As expected, the serum concentration of T4 was reduced by this surgery (2.3 versus 6.2 µg/mL). The endogenous concentrations of T4 were further reduced by ethylenethiourea in the thyroparathyroidectomized groups (1.4 versus 2.3ìg/mL, compared with 4.8 versus 5.9 µg/mL in the sham-operated controls). Malformations were present in 100% of the fetuses regardless of thyroid status, although some different malformations (e.g., oedema, micrognathia, cleft palate and micromelia) were seen in the thyroparathyroidectomized females given ethylenethiourea. The only increase in the incidence of malformations in control groups was a 10.3% incidence in the thyroparathyroidectomized animals not treated with ethylenethiourea. The results did not support a role of altered thyroid function in ethylenethiourea-induced teratogenesis in rats (Lu & Staples, 1978).

 

The teratogenic potential of ethylenethiourea was compared with that of the thyroid antagonist methimazole in rat embryo cultures. Exposure of 9.5-day-old Wistar rat embryos to ethylenethiourea at a concentration of 50 µmol/L to 1 mmol/L for 48 h resulted in dose-related reductions in embryonic growth and differentiation; the effects were significant at concentrations of 500 µmol/L and 1 mmol/L. The commonest anomaly was abnormal development of the caudal region of the neural tube. While some similarities in embryonic responses were noted, reductions and swellings of the caudal region in many embryos exposed to ethylenethiourea was not seen in embryos exposed to methimazole, and other effects seen in methimazoleexposed embryos were not seen in ethylenethiourea-treated embryos (Stanisstreet et al., 1990).

 

(e) Other aspects of developmental toxicity

 

In order to study the potential of nitrites to activate ethylenethiourea by nitrosation, the teratogenic effects of ethylenethiourea were studied in SLC-ICR mice exposed to either ethylenethiourea alone or ethylenethiourea in combination with sodium nitrite. The authors hypothesized that ethylenethiourea would react with nitrite at the low pH in the stomach and form a reactiveN-nitroso compound. Ethylenethiourea was administered by oral gavage at 400 mg/kg bw with or without 200 mg/kg bw sodium nitrite on day 6, 8, 10 or 12 of gestation. There were 12–29 dams in each group, and the fetuses were examined on gestation day 18. When nitrite was administered 2 h after treatment with ethylenethiourea, no teratogenic effects were seen in mouse embryos. Concomitant treatment on day 6 was most effective for induction of fetal death and growth retardation, while various malformations were present after exposure on day 6, 8 or 10. Exposure on day 12 did not adversely effect embryonic development. In particular, treatment on day 6 or 8 caused abnormal lobation of the left and right lung, respectively. Some of the observed defects resembled those observed in ethylenethiourea-treated rats (Teramoto et al., 1980).

 

The role of altered hepatic function in ethylenethiourea-induced teratogenicity was studied in Swiss-Webster mice that received 0, 1600, 2000 or 2400 mg/kg bw ethylenethiourea by oral gavage on day 12 of gestation. The modulating treatments included phenobarbital (at 60 mg/kg bw per day by subcutaneous injection on days 7–10 of gestation), SKF-525A (at 40 mg/kg bw by intraperitoneal injection on day 12) and 3-methylcholanthrene (at 20 mg/kg bw per day on days 10–12). Ethylenethiourea alone induced dose-related incidences of hindpaw ectrodactyly and syndactyly and low incidences of cleft palate and hindpaw polydactyly. The incidence of defects was altered only by 3-methylcholanthrene, which reduced the incidences of hindpaw ectrodactyly, syndactyly and cleft palate at the two higher doses of ethylenethiourea (Khera, 1984).

 

Histological changes to the central nervous system were studied after exposure of Wistar rats to 0, 15 or 30 mg/kg bw ethylenethiourea by oral gavage on day 13 of gestation. Four to six females per dose were killed 12, 24, 48 and 72 h after exposure. Other dams were allowed to litter, and their offspring were followed to postnatal day 80. Within 12 h of receiving the higher dose, karyorrhexis was evident in the germinal layer of the basal lamina of the central nervous system, extending from the spinal cord to the telencephalon. By 48 h, rosettes were present in the neuroepithelium and there was extensive disorganization of the germinal and mantle layers. Similar, but less severe responses were seen at the lower dose. During the postnatal phase, 50% of the offspring of dams at the higher dose had died by 80 days after birth, and hydrocephaly was invariably present. There were no postnatal effects at the lower dose (Khera & Tryphonas, 1985).

 

Justification for selection of Effect on developmental toxicity: via oral route:
Several studies are chosen to evaluate the reprotoxic potential of ETU. Khera's study showed the smallest developmental NOAEL in rats.

Justification for classification or non-classification

According to the findings in the rat, the hamster and the mouse, ETU is teratogenic at doses that not maternally toxic and also impairs post-natal development in the rat. The rat is the most sensitive species. The marked species difference in the effect of ETU on reproduction in rats and mice has been attributed to differences in biotransformation and differing sensitivities of the target tissues. A definitive statement on the teratogenicity of ETU in guinea pigs or rabbits is not possible.

ETU are classified on category 1B for reproductive toxicity by Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures. Because there are some evidence from experimental animals of an adverse effect on development in rat.

Regulation (EC) No 1272/2008:Repr. Category 1B -H360D: May damage the unborn child.

 

Directive 67/548/EEC:Carc. Category 2 – R61 "May cause harm to the unborn child.".