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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1977

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:

1977

Materials and methods

Principles of method if other than guideline:

Prenatal developmental toxicity study: Groups of pregnant Japanese white rabbits were administered orally with d-limonene at dose levels of 250, 500 and 1000 mg/kg bw/day for 13 days from Day 6 to 18 of gestation and evaluated for teratogenicity.

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:

4-isopropenyl-1-methylcyclohexene

Test material form:

liquid

Test animals

Species:

rabbit

Strain:

other: Japanese white

Details on test animals or test system and environmental conditions:

no data

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

not specified

Details on exposure:

no data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Details on mating procedure:

No data

Duration of treatment / exposure:

13 days (gestation Day 6-18)

Frequency of treatment:

Once daily

Duration of test:

Gestatation Day 0 to postnatal Day 49

No. of animals per sex per dose:

10 (in 0-500 mg/kg bw/day groups) or 18 (in 1000 mg/kg bw/day group) pregnant females

Control animals:

yes

Details on study design:

no data

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

General behaviour observed, but no data regarding the frequency of observation

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
mean daily food consumption by treatment group is reported

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data


POST-MORTEM EXAMINATIONS: No data

OTHER:

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No data
Examinations included: Number of implantations, number of resorptions and foetus bodyweight and placental weight

Fetal examinations:

- External examinations: Yes: about 90% per litter
- Visceral examinations: Yes: about 90% per litter
- Skeletal examinations: Yes: about 90% per litter

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

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No anomalies were observed in the general behaviour of dams given 250 and 500 mg/kg of d-limonene during the gestation.

Mortality:

mortality observed, treatment-related

Description (incidence):

Treatment with the highest dose level (1000 mg/kg) of d-limonene resulted in death of dams (33% mortality).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant decrease of bodyweight gain and food consumption were temporarily observed in dams given 500 and 1000 mg/kg

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Significant decrease of bodyweight gain and food consumption were temporarily observed in dams given 500 and 1000 mg/kg

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects: The significant decrease of bodyweight gain and food consumption were temporarily observed in dams given 500 and 1000 mg/kg of d-limonene, but no anomalies were observed in the general behaviour of dams given 250 and 500 mg/kg of d-limonene during the gestation.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

mortality

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- Visceral and skeletal examinations revealed some anormalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development.
- Other non specific anormalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- Visceral and skeletal examinations revealed some anormalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development.

Other effects:

not examined

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Effect of d-limonene on prenatal development of rabbit fetuses

Dose (mg/kg bw)	Control	250	500	1000
No. of pregnant animals	10	10	10	18
No. of dead clams	0	0	0	6
(%)				33
No. of examined clams	10	10	10	10
No. of implantations	96	94	85	91
(mean ± S.E.)	9.50 ± 0.25	9.40 ± 0.21	8.50 ± 0.33	9.10 ± 0.25
No. of resorbed fetuses	5	4	4	8
No. of dead fetuses	3	5	0	3
No. of live fetuses	88	85	81	80
Sex ratio (Male/Female)	0.73 (37/51)	1.13 (45/50)	0.62 (31/50)	1.11 (38/42)
Fetus body weight (g)
Male (mean ± S.E.)	44.39 ± 1.33	48.09 ± 1.07 *	44.76 ± 1.51	43.22 ± 0.96
Female (mean ± S.E.)	45.64 ± 1.00	47.45 ± 1.08	46.14 ± 1.21	45.13 ± 1.10
Placental weight (g)
Male (mean ± S.E.)	5.76 ± 0.17	5.84 ± 0.17	5.95 ± 0.29	5.77 ± 0.19
Female (mean ± S.E.)	5.87 ± 0.19	5.70 ± 0.15	6.16 ± 0.18	5.87 ± 0.23

* Significantly different from the control at 5% level

Table 2: Prenatal examinations of rabbit fetuses

Dose (mg/kg bw)	Control	250	500	1000
External examination
No. of examined fetuses	91	90	81	83
No. of malforrned fetuses	0	0	0	0
Visceral examination
No. of examined fetuses	88	85	81	80
No. of malformed fetuses
Atrial septal defect (%)	0	2 (2.4)	0	0
No. of minor abnormality
Incomplete lobulation of lungs (%)	11 (12.5)	16 (18.8)	19 (23.5)	19 (23.8)
Enlargement of foramen ovale (%)	2 (2.3)	2 (2.4)	5 (6.2)	4 (5.0)
Skeletal examination
No. of examined fetuses	86	87	81	80
No. of malformed fetuses	0	0	0	0
No. of variation
Left lumbar rib (%)	18 (20.9)	26 (29.9)	14 (17.3)	25 (31.3)
Right lumbar rib (%)	16 (18.6)	22 (25.3)	14 (17.3)	22( 27.5)
Ossification pattern
Retarded ossification of 5th sternebrae (%)	11 (12.8)	14 (16.1)	8 (9.9)	18 (22.5)
Retarded ossification of middle phalanx of fore limbs (%)	2 (2.3)	3 (3.4)	0	6 (7.4)

Table 3: Absolute organ weights of rabbit offsprings

	Male				Female
	Control	250	500	1000	Control	250	500	1000
No. of offsprings	13	12	8	13	10	13	16	9
Final body weight (g)	893.0 ± 45.3	1021.2 ± 45.4 **	931.9 ± 55.5	957.3 ± 52.4	1005.5 ± 53.7	1093.1 ± 46.6	860.0 ± 31.6 *	1071.7 ± 58.1
Liver (g)	36.49 ± 2.52	45.08 ± 1.52 *	38.67 ± 3.10	34.91 ± 2.76	41.79 ± 3.39	13.95 ± 3.18	34.68 ± 1.90	48.30 ± 6.14
Lungs (g)	5.53 ± 0.35	6.25 ± 0.26	5.98 ± 0.36	5.46 ± 0.18	5.91 ± 0.25	5.94 ± 0.28	5.72 ± 0.18	5.93 ± 0.45
Heart (g)	2.63 ± 0.17	3.50 ± 0.16 **	2.86 ± 0.17	3.04 ± 0.19	3.19 ± 0.17	3.38 ± 0.21	2.72 ± 0.12 *	3.34 ± 0.20
Spleen (g)	0.71 ± 0.05	0.77 ± 0.04	0.71 ± 0.08	0.81 ± 0.04	0.64 ± 0.06	0.74 ± 0.05	0.74 ± 0.04	0.78 ± 0.07
Thymus (g)	2.31 ± 0.20	2.51 ± 0.23	2.11 ± 0.38	1.96 ± 0.11	2.40 ± 0.30	2.77 ± 0.19	1.671.14 *	2.36 ± 0.31
Kidneys (g)	7.67 ± 0.42	9.80 ± 0.46 **	8.29 ± 0.29	8.58 ± 0.52	8.82 ± 0.46	8.40 ± 0.30	7.86 ± 0.37	9.56 ± 0.55
Thyroids (mg)	75.89 ± 8.35	102.68 ± 4.18*	86.74 ± 10.97	80.93 ± 7.41	82.78 ± 8.00	89.90 ± 4.11	75.75 ± 5.43	96.30 ± 9.67
Adrenals (mg)	69.65 ± 6.02	9.1.93 ± 1.06 **	78.79 ± 5.89	71.36 ± 6.00	82.23 ± 4.37	94.24 ± 5.07	87.64 ± 4.18	105.39 ± 15.11
Testes or Ovaries (mg)	180.42 ± 17.15	272.86 ± 16.46 **	185.62 ± 23.78	162.84 ± 20.59	46.31 ± 7.90	46.10 ± 2.80	43.53 ± 2.69	47.77 ± 3.59

* Significantly different from the control at 5% level

** Significantly different from the control at 1% level

Table 4: Relative organ weights per 100 g of rabbit offsprings

	Male				Female
	Control	250	500	1000	Control	250	500	1000
No. of offsprings	13	12	8	13	10	13	16	9
Final body weight (g)	893.0 ± 45.3	1021.2 ± 45.4 **	931.9 ± 55.5	957.3 ± 52.4	1005.5 ± 53.7	1093.1 ± 46.6	860.0 ± 31.6 *	1071.7 ± 58.1
Liver (g/100 g)	4.08 ± 0.25	3.99 ± 0.22 *	4.16 ± 0.26	3.52 ± 0.10	4.25 ± 0.17	4.03 ± 0.21	4.02 ± 0.16	4.04 ± 0.32
Lungs (g/100 g)	0.61 ± 0.03	0.55 ± 0.03	0.65 ± 0.04	0.58 ± 0.03	0.62 ± 0.02	0.55 ± 0.02 *	0.67 ± 0.03	0.51 ± 0.02 **
Heart (g/100 g)	0.29 ± 0.01	0.31 ± 0.01	0.31 ± 0.01	0.31 ± 0.02	0.33 ± 0.01	0.31 ± 0.01	0.32 ± 0.01	0.29 ± 0.01 *
Spleen (g/100 g)	0.08 ± 0.01	0.07 ± 0.01	0.08 ± 0.01	0.08 ± 0	0.07 ± 0.01	0.07 ± 0.01	0.09 ± 0	0.07 ± 0.01
Thymus (g/100 g)	0.25 ± 0.02	0.22 ± 0.02	0.25 ± 0.03	0.20 ± 0.01 *	0.24 ± 0.03	0.25 ± 0.01	0.195 ± 0.01	0.20 ± 0.02
Kidneys (g/100 g)	0.85 ± 0.04	0.86 ± 0.04	0.90 ± 0.03	0.88 ± 0.02	0.91 ± 0.02	0.80 ± 0.01	0.91 ± 0.01	0.83 ± 0.03 *
Thyroids (mg/100 g)	8.21 ± 0.69	9.10 ± 0.51	9.21 ± 0.87	8.18 ± 0.42	8.40 ± 0.56	8.41 ± 0.45	8.81 ± 0.57	8.13 ± 0.41
Adrenals (mg/100 g)	7.82 ± 0.60	8.37 ± 0.40	8.58 ± 0.77	7.18 ± 0.28	8.61 ± 0.51	8.79 ± 0.57	9.15 ± 0.42	9.04 ± 0.14
Testes or Ovaries (mg/100 g)	23.68 ± 1.31	19.68 ± 0.94 *	19.48 ± 1.69	16.35 ± 1.56	5.15 ± 1.19	4.33 ± 0.32	5.19 ± 0.43	3.84 ± 0.32

* Significantly different from the control at 5% level

** Significantly different from the control at 1% level

Table 5: Effects of d-limonene on gross differentiations of rabbit offsprings

	Control	250	500	1000
No. of examined offsprings	23	25	24	22
Days of gross differentiation after birth Opening of the ear-shell
6th day (%)	0	0	1 (4.2)	0
7th day (%)	23 (100)	25 (100)	23 (95.8)	22 (100)
Coating with the hair
2nd day (%)	7 (30.4)	0	0	0
3rd day (%)	16 (69.6)	25 (100)	24 (100)	22 (100)
Odontiasis
At birth (%)	23 (100)	25 (100)	24 (100)	22 (100)
Opening of the eyelids
9th day (%)	0	0	0	3 (13.6)
10th day (%)	11 (47.8)	4 (16.0)	13 (54.2)	5 (22.7)
11th day (%)	4 (17.4)	15 (60.0)	10 (41.7)	12 (54.5)
12th day (%)	3 (13.0)	5 (20.0)	1 (4.2)	2 (9.1)
13th day (%)	5 (21.7)	1 (4.0)	0	0

Table 6: Effects of d-limonene on postnatal development of rabbit offsprings

	Control	250	500	1000
No of dams	3	3	3	3
No. of still-birth (Male/Female)	1 (1/0)	0	0	1 (0/1)
No. of offsprings (Male/Female) At birth	28 (15/13)	27 (14/13)	26 (8/18)	27 (15/12)
1st week	28 (15/13)	27 (14/13)	25 (8/17)	26 (14/12)
2nd week	26 (14/12)	27 (14/13)	25 (8/17)	26 (14/12)
3rd week	24 (14/10)	27 (14/13)	25 (8/17)	25 (14/11)
4th week	23 (13/10)	26 (13/13)	25 (8/17)	22 (13/ 9)
5th week	23 (13/10)	25 (12/13)	25 (8/17)	22 (13/ 9)
6th week	23 (13/10)	25 (12/13)	25 (8/17)	22 (13/ 9)
7th week	23 (13/10)	25 (12/13)	24 (8/16)	22 (13/ 9)
Weanling rate (%)	79.3 (81.2/76.9)	92.6 (85.7/100)	92.3 (100/88.9)	78.6 (86.7/69.2)

Table 7: Postnatal examinations of rabbit offsprings

	Control	250	500	1000
No. of dams	3	3	3	3
No. of examined offsprings	23	25	24	22
Sensory function	Normal	Normal	Normal	Normal
External examination No. of malformed offsprings	0	0	0	0
Visceral examination No. of malformed offsprings	0	0	0	0
No. of minor abnormality Incomplete lobulation of lungs (%)	2 (8.7)	1 (4.0)	0	0
Accessory spleen (%)	2 (8.7)	0	0	0
Protrusion of gall bladder (%)	1 (4.3)	1 (4.0)	0	0
Skeletal examination No. of malformed offsprings	0	0	0	0
No. of variation Left lumbar rib (%)	4 (17.4)	4 (16.0)	4 (16.7)	4 (18.2)
Right lumbar rib (%)	2 (8.7)	6 (24.0)	6 (25.0)	4 (18.2)
Translocation of caudal vertebrae (%)	1 (4.3)	0	1 (4.2)	0
Ossification pattern Retarded ossification of 5th sternebrae (%)	0	2 (8.0)	0	1 (4.5)
Accessory ossification center of 5th sternebrae (%)	1 (4.3)	2 (8.0)	0	3 (13.6)

Applicant's summary and conclusion

Conclusions:

d-Limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be greater than 1000 mg/kg bw/day. The NOAEL for maternal toxicity was considered to be 250 mg/kg bw/day based on the decreased bodyweight gain.

Executive summary:

In a prenatal developmental toxicity study, d-limonene was administered orally to groups of pregnant Japanese white rabbits at dose levels of 250, 500 and 1000 mg/kg bw/day for 13 days from Day 6 to 18 of gestation. Food consumption and bodyweights of pregnant rabbits were recorded 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.

 

Treatment with the highest dose level (1000 mg/kg bw/day) of d-limonene resulted in death of 6/18 dams (33% mortality). The significant decrease of bodyweight gain and food consumption were temporarily observed in dams given 500 and 1000 mg/kg bw/day of d-limonene, but no anomalies were observed in the general behavior of dams given 250 and 500 mg/kg bw/day of d-limonene during the gestation. External examination of fetuses showed no anomalies. Visceral and skeletal examinations revealed some anomalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development. Other non specific anomalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene.

 

Therefore, d-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be higher than 1000 mg/kg bw/day. The NOAEL for maternal toxicity was considered to be 250 mg/kg bw/day based on the decreased bodyweight gain.