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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
publication
Title:
Ethylenethiourea : teratogenicity study in rats and rabbits.
Author:
Khera KS.
Year:
1973
Bibliographic source:
Teratology, 7 : 243-252.

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Other name = Ethylenethiourea (ETU)
ETU : 100 % purity

Test animals

Species:
rat
Strain:
Wistar
Details on test animals 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

Administration / exposure

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
Doses / concentrations
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

Examinations

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

open allclose all
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

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

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.

Effect levels (fetuses)

open allclose all
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.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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

4.5t

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

2.6t

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

3.7t

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 4thventricule

41

21

100

50

23

100

-cytic aqueduct and 4thventricle

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

Applicant's summary and conclusion

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).