Mandarin orange, ext.

Developmental toxicity / teratogenicity

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-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

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

Test material information

Constituent 1

Reference substance name:

(R)-p-mentha-1,8-diene

EC Number:

227-813-5

EC Name:

(R)-p-mentha-1,8-diene

Cas Number:

5989-27-5

Molecular formula:

C10H16

IUPAC Name:

(4R)-1-methyl-4-(prop-1-en-2-yl)cyclohexene

Constituent 2

Reference substance name:

Dipentene

EC Number:

205-341-0

EC Name:

Dipentene

Cas Number:

138-86-3

IUPAC Name:

4-isopropenyl-1-methylcyclohexene

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 or test system 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)

1

Dose descriptor:

NOAEL

Effect level:

591 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

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.