Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No study on toxicity to reproduction was available for l-limonene. No effects were observed on sexual organs in the NTP 90-d study conducted in rats and mice exposed to d-limonene (gross pathology and histopathology examination of prostate/testes or ovaries/uterus).

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because a pre-natal developmental toxicity study is available
other:
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No study on toxicity to reproduction was available for l-limonene. In developmental toxicity studies, with detailed results but limited information on test conditions, d-limonene did not ellicit any teratogenic effects in tested rabbits, mice or rats. Based on the absence of observed teratogenic effects and on the absence of effects on sexual organs in the NTP 90-d study conducted in rats and mice (gross pathology and histopathology examination of prostate/testes or ovaries/uterus), a toxicity to reproduction study is deemed not to be scientifically justified. Moreover, European Food Safety Authority made a review of the toxicologic properties of d-limonene in 2010, and concluded on the absence of safety concern due to to d-limonene intake, with no specific concern related to the absence of any study for toxicity to reproduction for this substance (EFSA Journal 2010; 8(5): 1334).

For further information on read-across justification, see section 13: point "read-across approach"

Effects on developmental toxicity

Description of key information

Weight of evidence: d-limonene was not teratogenic in rabbit fetuses and the NOAEL for fetal toxicity was considered to be greater than the highest dose tested. In two developmental toxicity studies in rats and mice, slight ossification delays/malformations and organ weights changes were observed but not dose-related and/or observed at doses where maternal toxicity was identified.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1977
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Read-across from an analogue substance for which there is available information (Klimish=4)
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See cross-reference to justification of read-across.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Treatment with the highest dose level (1000 mg/kg) of d-limonene resulted in death of dams with less than 40% mortality. The significant decrease of body- weight 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.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality
body weight and weight gain
Remarks on result:
other: Based on the read-across from an analogue substance.
Key result
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- External examination of fetuses showed no anormalies.
- 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.
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

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)

Conclusions:
Based on the read-across from the analogue substance d-limonene, l-limonene is not considered to have teratogenic potential in rabbit, the NOAEL for fetal toxicity was estimated to be greater than 1000 mg/kg bw/day. The NOAEL for maternal toxicity was estimated to be 250 mg/kg bw/day based on the decreased bodyweight gain.
Executive summary:

Based on the read-across from the analogue substance d-limonene, l-limonene is not considered to have teratogenic potential in rabbit, the NOAEL for fetal toxicity is estimated  to be greater than 1000 mg/kg bw/day. The NOAEL for maternal toxicity is estimated to be 250 mg/kg bw/day based on the decreased bodyweight gain.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1977
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Read-across from an analogue substance for which there is available information (Klimish=4)
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See cross-reference to justification of read-across.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Based on the read-across from an analogue substance.
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increased incidences of abnormal skeletal formation in fetuses and decreased bodyweight gain in male offsprings born to dams administered with 2363 mg/kg-bw/day
Remarks on result:
other: Based on the read-across from the analogue substance d-limonene.
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: rib
Description (incidence and severity):
Lumber rib and fused rib in the fetuses
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
2 363 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
no
Relevant for humans:
not specified
Conclusions:
Based on the read-across from the analogue substance d-limonene, the NOAEL for maternal and fetal toxicity of l-limonene was estimated to be 591 mg/kg bw/day based on the decreased bodyweight gain in dams and increased incidences of abnormal bone formation in fetuses.
Executive summary:

Based on the read-across from the analogue substance d-limonene, the NOAEL for maternal and fetal toxicity of l-limonene was estimated to be 591 mg/kg bw/day based on the decreased bodyweight gain in dams and increased incidences of abnormal bone formation in fetuses.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Read-across from an analogue substance for which there is available information (Klimish=4)
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See cross-reference to justification of read-across.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
mortality
body weight and weight gain
Remarks on result:
other: Based on the read-across from an analogue substance.
Key result
Abnormalities:
no effects observed
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
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 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
Remarks on result:
other: Based on the read.across from an analogue substance.
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: 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
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
2 869 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
not specified
Dose response relationship:
no
Relevant for humans:
not specified
Conclusions:
Based on the read-across from the analogue substance d-limonene, the NOAEL for maternal toxicity of l-limonene was estimated to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain and the NOAEL for fetal toxicity was estimated to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.
Executive summary:

Based on the read-across from the analogue substance d-limonene, the NOAEL for maternal toxicity of l-limonene was estimated to be 591 mg/kg bw/day based on the deaths and decreased bodyweight gain and the NOAEL for fetal toxicity was estimated to be 591 mg/kg bw/day based on the delayed skeletal formation and decreased bodyweight gain.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
591 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Read-across from the analogue substande d-limonene for which there are available information which have detailed results but limited information on test conditions (Klimish =4).
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Develomental toxicity studies, with detailed results but limited information on test conditions (studies in Japanese), did not ellicit any teratogenic effects of d-limonene on tested rabbits, mice or rats.

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. External examination of fetuses showed no anormalies. 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 but these effects were not dose-dependent. 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.

In a prenatal developmental toxicity study, d-limonene was administered orally to groups of pregnant ICR mice (20/dose: 15 for teratogenicity study, 5 for postnatal development) at dose levels of 0, 591 and 2363 mg/kg bw/day for 6 days from Day 7 to 12 of gestation. A significant decrease of bodyweight gain in pregnant mice was observed at 2363 mg/kg bw/day. An incidence of lumber rib and fused rib in the fetuses increased significantly at 2363 mg/kg bw/day compared with those of control. In the observation of skeletal development in fetuses, retarded ossification of proximal phalanx of fore limb, metatarsal bone and proximal phalanx of hind limb were observed but were not dose-dependent or only observed at the highest dose, associated with maternal toxicity.

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 for 7 days from Day 9 to 15 of gestation. At 2869 mg/kg bw/day, maternal bodyweight decreased and several mothers (40%) died during treatment. Delayed ossification of fetuses metacarpal bone and proximal phalanx at 2869 mg/kg bw/day was increased and 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.

For further information on read-across justification, see section 13: point "read-across approach"

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available according to the Regulation (EC) No 1272/2008 .

Self classification:

All potential effects of d-limonene observed on reproduction and development were either associated with maternal toxicity, or not dose-related. In conclusion, based on the read-across approach, l-limonene should not be classified for toxicity to reproduction or develomental toxicity.