Bergamot, 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:

supporting study

Study period:

1975

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Original reference in Japanese language

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 animals/dose: 15 animals for teratogenicity study, 5 animals for postnatal development) were administered with d-limonene at dose levels of 0, 591 and 2869 mg/kg bw/day suspended with 1% gum-arabic solution (via oral route) for 7 days during gestation day 9 to 15. Maternal toxicity and developmental toxicity were evaluated.

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

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:
Maternal 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:
Significantly delayed ossification of metacarpal bones and proximal phalanxes in fetuses, significantly decreased bodyweight gain (male offspring) 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 (significant at GD 16) and several mothers (40%) died during the treatment period. At 591 mg/kg bw/day no maternal effects were observed.

 

Fetal examinations:

- Significantly delayed ossification of fetal metacarpal bone and proximal phalanx was found in the 2869 mg/kg bw/day dose group, compared to the control group. This effect was restored to normal within several weeks after birth.

- A decreased tendency of bodyweight was noted in postnatal male offspring of dams administered with 2869 mg/kg bw/day, compared to the control group.

- Relative weights of thymus, spleen and ovaries were significantly decreased in offspring of dams administered with 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
Parturition 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 maternal deaths (40%) and decreased bodyweight gain. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on significantly delayed ossification and decreased bodyweight gain in male offspring.

Executive summary:

In this developmental toxicity study in rats, d-limonene was administered to pregnant Wistar rats (20 animals/dose: 15 animals for teratogenicity study, 5 animals for postnatal development) at dose levels of 0, 591 and 2869 mg/kg bw/day (via oral route, suspended with 1% gum-arabic solution) for 7 days during gestation day 9-15.

At 2869 mg/kg bw/day, maternal bodyweight decreased (significant at GD 16) and several mothers (40%) died during the treatment period. At the 591 mg/kg bw/day dose level no maternal effects were observed. Significantly delayed ossification of fetal metacarpal bone and proximal phalanx was found in the 2869 mg/kg bw/day dose group, compared to the control group. This effect was restored to normal within several weeks after birth. A decreased tendency of bodyweight was noted in postnatal male offspring of dams administered with 2869 mg/kg bw/day, compared to the control group.

Under the test conditions, the NOAEL for maternal toxicity was considered to be 591 mg/kg bw/day based on maternal deaths (40%) and decreased bodyweight gain at 2869 mg/kg bw/day. The NOAEL for fetal toxicity was considered to be 591 mg/kg bw/day based on significantly delayed ossification in fetuses, significantly decreased bodyweight gain (male offspring) and organ weights at 2869 mg/kg bw/day