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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1975
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Original reference in Japanese language
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1975

Materials and methods

Principles of method if other than guideline:
Prenatal developmental toxicity study: Groups of pregnant Wistar rats (20/dose: 15 for teratogenicity study, 5 for postnatal development) were administered orally with d-limonene at dose levels of 0, 591 and 2869 mg/kg bw/day suspended with 1% gum-arabic solution for 7 days from Day 9 to 15 of gestation and evaluated for developmental toxicity.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): d-limonene; d-p-mentha-1,8-diene
- Boiling point: 176-177 °C
- Specific gravity (at 25 °C): 0.8340-0.8420

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
No data

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
other: 1% gum-arabic solution
Details on exposure:
Volume administered: 5 mL/kg bw for all doses
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
None
Details on mating procedure:
no data
Duration of treatment / exposure:
7 days (gestation Day 9-15)
Frequency of treatment:
Once daily
Duration of test:
Gestation Day 0 to postnatal week 7
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 591 and 2869 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
20 pregnant rats
Control animals:
yes, concurrent vehicle
Details on study design:
No data

Examinations

Maternal examinations:
See result tables
Ovaries and uterine content:
See result tables
Fetal examinations:
See result tables
Statistics:
statistical significance difference of effects from controls were calculated at 5% level.
Indices:
No data
Historical control data:
No data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Deaths (40%) and decreased bodyweight gain at 2869 mg/kg bw/day

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Prolongation of the ossification of metacarpals and proximal phalanges in fetuses, decreased bodyweight gain (male offsprings) and organ weights at 2869 mg/kg bw/day

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Maternal examinations:

- At 2869 mg/kg bw/day, maternal bodyweight decreased and several mothers (40%) died for a period of the treatment, but at 591 mg/kg bw/day, no changes were observed.

 

Fetal examinations:

- Delayed ossification of fetuses metacarpal bone and proximal phalanx at 2869 mg/kg bw/day was caused significantly, compared with the control group, but this was restored to normal within several weeks after birth.

- A decreased tendency of bodyweight was noted in postnatal male offsprings born to mothers treated at 2869 mg/kg bw/day, compared with the control group.

- Thymus, spleen and ovaries weights decreased in offsprings born to mothers treated at 2869 mg/kg bw/day.

Table 1: Body weight changes in pregnant rats treated orally with d-limonene

Dose

(mg/kg bw)

Gestational days

Gain

0

9

12

16

20

Control

214.80 ± 32.55

248.70 ± 28.92

265.60 ± 30.53

289.85 ± 35.01

325.30 ± 44.01

105.50 ± 29.67

591

221.25 ± 39.58

254.65 ± 41.16

264.80 ± 39.94

290.10 ± 38.37

325.60 ± 52.75

103.95 ± 19.38

2869

214.75 ± 4.57

258.75 ± 32.76

 

248.92 ± 26.27

263.17 ± 22.96 *

305.00 ± 27.07

90.25 ± 22.38

* Significantly different from the control, P <0.05

Table 2: Effects of d-limonene on rat fetuses

Dose (mg/kg bw)

Control

591

2869

No. of mothers

15

15

15

Mortality of mothers (%)

0

0

40

No. of total implants

12.73 ± 2.96

12.18 ± 3.65

10.44 ± 3.71

No. of dead fetuses

0

0

0

No. of resorbed fetuses

1.00 ± 1.10

1.47 ± 2.42

0.89 ± 0.73

No. of live fetuses

176

162

87

Sex ratio (Male/Female)

0.69

1.22

0.85

Fetuses body weight (g)

Male

3.71 ± 0.45

3.53 ± 0.35

3.73 ± 0.52

Female

3.46 ± 0.44

3.38 ± 0.45

3.63 ± 0.40

Placental weight(g)

Male

0.49 ± 0.07

0.49 ± 0.10

0.48 ± 0.06

Female

0.47 ± 0.07

0.46 ± 0.06

0.44 ± 0.05

Malformation

External

0

0

0

Visceral

1

0

0

Table 3: Effects of d-limonene on skeletal development of rat fetuses

Dose (mg/kg bw)

Control

591

2869

No. of examined fetuses

83

84

42

Variation

Shortness of 13th rid

1

0

0

Lumbar rid

0

1

2

Asymmetry of sternebrae

0

0

1

Ossification

Delayed ossification of parietal bone

2

0

0

Non-ossification of occipital bone

 

0

4

1

Non-ossification of parietal bone

0

3

0

No. of ossified metacarpal bone

7.69 ± 0.72

7.49 ± 0.84

6.97 ± 0.96 *

No. of ossified proximal phalanx (Forelimb)

2.48 ± 1.71

2.25 ± 1.81

0.55 ± 1.28 *

No. of ossified metatarsal bone

 

7.98 ± 0.56

8.01 ± 011

8.00 ± 0

No. of ossified

sternebraea

5.47 ± 0.98

5.60 ± 0.71

5.52 ± 0.73

No. of ossified

caudal vertebrae

3.76 ± 0.64

3.80 ± 0.57

3.95 ± 0.68

* Significantly different from the control, P <0.05

Table 4: Body weight changes of postnatal rat offsprings born to mothers treated orally with d-limonene

Postnatal

weeks

Males

Females

Dose (mg/kg bw)

Dose (mg/kg bw)

Control

591

2869

Control

591

2869

0

5.19 ± 0.55

5.46 ± 0.52

4.79 ± 0.46

4.93 ± 0.62

5.09 ± 0.68

4.89 ± 0.66

1

13.06 ± 1.50

12.49 ± 0.99

10.62 ± 1.54 *

12.82 ± 1.59

12.22 ± 1.07

10.66 ± 1.84

2

26.06 ± 3.12

24.86 ± 2.74

22.67 ± 5.05 *

25.88 ± 3.35

24.31 ± 2.42

22.72 ±.3.92

3

41.91 ± 5.89

39.55 ± 5.14

40.77 ± 5.16

41.08 ± 5.59

38.56 ± 4.37

38.39 ± 4.96

4

73.12 ± 9.89

71.33 ± 8.87

67.67 ± 8.02

69.66 ± 9.43

67.38 ± 6.71

66.01 ± 9.29

5

122.32 ± 12.25

116.47 ± 12.78

112.77 ± 12.65 *

109.57 ± 10.61

107.58 ± 7.95

107.10 ± 13.34

6

176.04 ± 15.80

164.68 ± 16.69

163.11 ± 17 .85 *

141.39 ± 10.54

140.11 ± 9.40

139.45 ± 14.22

7

235.52 ± 17.72

222.43 ± 18.57

213.64 ± 20.10 *

173.06 ± 8.89

169.65 ± 11.13

167.84 ± 15.86

* Significantly different from the control, P <0.05

Table 5: Effects of d-limonene on postnatal development of the rats

Dose

(mg/ kg bw)

Days of postnatal development

Opening of the ear-shell

Coating with
the hair

Odontiasis

Opening of the eyelid

Descending of the testis

Opening of the vaginal orifice

Control

2.55 ± 0.76

5.51 ± 0.91

10.1 ± 0.96

14.83 ± 0.55

22.5 ± 1.30

35.6 ± 2.50

591

2.09 ± 0.82

6.00 ± 0

10.4 ± 0.71

15.00 ± 0.76

21.6 ± 1.39

35.5 ± 1.75

2869

2.41 ± 0.49

8.50 ±0.50

10.4 ± 1.85

15.14 ± 0.75

21.27 ± 0.57

35.93 ± 2.20

Table 6: Effects of d-limonene on development of rat offsprings

Dose (mg/kg)

Control

591

2869

No. of mothers

5

5

5

Mortality of mothers

0

0

40

No. of offspring from birth

to the 7th week

0

61

65

33

1

53

63

30

2

53

63

30

3

53

63

28

4

53

63

28

5

53

63

28

6

53

63

28

7

53

63

28

External abnormality

0

0

0

No. of total implants

13.6 ± 3.1

14.8 ± 1.7

13.7 ± 1.7

No. of dead fetuses at birth

4

4

5

Parturient rate

95

92

93

Weaning rate

89

97

85

Table 7: Absolute organ weights of postnatal rat offsprings born to mothers treated orally with d-limonene

Sex

Dose

(mg/kg bw)

No. of

offsprings.

Final BW (g)

Pituitary (mg)

Thyroids (mg)

Thymus (g)

Lungs (g)

Heart (g)

Spleen (g)

Kidneys (g)

Liver (g)

Adrenals (g)

Testes (g) or Ovaries (mg)

Male

Control

27

235.5 ± 17.7

10.04 ± 1.95

14.06 ± 2.52

0.77 ± 0.08

1.15 ± 0.10

0.82 ± 0.08

0.75 ± 0.11

2.18 ± 0.36

11.55 ± 1.14

38.85 ± 7.37

2.15 ± 0.17

591

29

222.4 ± 18.6

9.58 ± 4.84

13.12 ± 2.77

0.72 ± 0.09

1.10 ± 0.11

0.81 ± 0.08

0.69 ± 0.08

2.10 ± 0.20

11.24 ± 1 .49

36.77 ± 6.93

2.13 ± 0.28

2869

11

213.6 ± 20.1

9.69 ± 0.65

14.41 ± 3.60

0.66 ± 0.08 *

1.19 ± 0.17

0.80 ± 0.07

0.63 ± 0.07 *

2.23 ± 0.38

11.64 ± 1.64

43.23 ± 10.91

2.18 ± 0.14

Female

Control

25

173.1 ± 8.9

11.18 ± 3.15

12.26 ± 1.32

0.59 ± 0.08

0.97 ± 0.11

0.66 ± 0.07

0.52 ± 0.06

1.72 ± 0.22

8.88 ± 0.85

45.51 ± 8.01

77.24 ± 22.01

591

34

169.7 ± 11.7

10.05 ± 3.34

11.57 ± 1.62

0.54 ± 0.07

0.96 ± 0.07

0.67 ± 0.05

0.50 ± 0.06

1.60 ± 0.15 *

8.16 ± 0.85

46.56 ± 8.45

85.84 ± 42.52 *

2869

17

167.8 ± 15.9

9.95 ± 1.87

12.31 ± 1.80

0.51 ± 0.07 *

0.94 ± 0.10

0.62 ± 0.06

0.44 ± 0.04 *

1.62 ± 0.17 *

8.50 ± 0.58

47.15 ± 6.63

63.15 ± 7.99

* Significantly different from the control, P <0.05

Table 8: Relative organ weights per 100 g body weights of postnatal rat offsprings born to mothers treated orally with d-limonene

Sex

Dose

(mg/kg bw)

No. of

offsprings.

Final BW (g)

Pituitary (mg/100 g)

Thyroids (mg/100 g)

Thymus (mg/100 g)

Lungs (mg/100 g)

Heart (mg/100 g)

Spleen (mg/100 g)

Kidneys (g/100 g)

Liver (g/100 g)

Adrenals (mg/100 g)

Testes or Ovaries (mg/100 g)

Male

Control

27

235.5 ± 17.7

4.31 ± 0.78

5.98 ± 0.99

0.33 ± 0.04

0.48 ± 0.04

0.36 ± 0.03

0.31 ± 0.05

0.93 ± 0.08

4.89 ± 0.36

16.49 ± 2.71

0.90 ± 0.04

591

29

222.4 ± 18.6

4.02 ± 0.50

5.93 ± 0.86

0.33 ± 0.04

0.49 ± 0.04

0.37 ± 0.04

0.33 ± 0.09

0.95 ± 0.07

5.10 ± 0.37

16.39 ± 2.53

0.95 ± 0.08 *

2869

11

213.6 ± 20.1

4.23 ± 0.43

5.93 ± 0.66

0.28 ± 0.03 *

0.51 ± 0.05

0.35 ± 0.02

0.27 ± 0.02 *

0.96 ± 0.06

5.03 ± 0.27

17.23 ± 2.26

0.95 ± 0.09

Female

Control

25

173.1 ± 8.9

6.09 ± 1.08

7.08 ± 0.85

0.34 ± 0.05

0.54 ± 0.04

0.38 ± 0.04

0.30 ± 0.04

0.99 ± 0.12

5.14 ± 0.42

26.38 ± 4.71

47.94 ± 9.78

591

34

169.7 ± 11.7

5.82 ± 0.81

6.85 ± 0.96

0.32 ± 0.04

0.54 ± 0.12

0.40 ± 0.03

0.30 ± 0.03

0.95 ± 0.07

4.83 ± 0.37

27.61 ± 3.76

46.74 ± 10.76

2869

17

167.8 ± 15.9

6.27 ± 1.90

7.31 ± 0.90

0.31 ± 0.03 *

0.57 ± 0.08

0.37 ± 0.03

0.27 ± 0.04 *

0.94 ± 0.06

5.14 ± 0.33

26.75 ± 2.93

36.89 ± 4.25 *

* Significantly different from the control, P <0.05

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.
Executive summary:

In a developmental toxicity study, d-limonene was administered orally to groups of pregnant Wistar rats (20/dose: 15 for teratogenicity study, 5 for postnatal development) at dose levels of 0, 591 and 2869 mg/kg bw/day suspended with 1% gum-arabic solution for 7 days from Day 9 to 15 of gestation. Bodyweight of pregnant rats were recorded on Days 0, 9, 12, 16 and 20 during organogenesis. Caesarean sections were performed and the number of dead, live or resorbed fetuses, sex ratio and number of implantation sites were recorded. Fetuses were weighed and examined for external, visceral and skeletal malformations. Number of live offsprings, gross differentiation and organ weights of offsprings were recorded until postnatal week 7.

 

At 2869 mg/kg bw/day, maternal bodyweight decreased and several mothers (40%) died for a period of the treatment, but at 591 mg/kg bw/day, no changes were observed. Delayed ossification of fetuses metacarpal bone and proximal phalanx at 2869 mg/kg bw/day was caused significantly, compared with the control group, but this was restored to normal within several weeks after birth. A decreased tendency of bodyweight was noted in postnatal male offsprings born to mothers treated at 2869 mg/kg bw/day, compared with the control group. Thymus, spleen and ovaries weights decreased in offsprings born to mothers treated at 2869 mg/kg.

 

Under the test conditions, the NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.