Registration Dossier

Administrative data

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:
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
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 for cross-reference:
read-across source
Reason / purpose for cross-reference:
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:
body weight and weight gain
mortality
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.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference
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 for cross-reference:
read-across source
Reason / purpose for cross-reference:
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.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference
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 for cross-reference:
read-across source
Reason / purpose for cross-reference:
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
mortality
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.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from an analogue substance for which there is available data (Klimish =2).
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See cross-reference to justification of read-across.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
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:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Based on:
test mat.
Sex:
male
Basis for effect level:
other: male-rat specific nephrotoxicity at all dose levels (considered as not relevant for humans)
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day;
Key result
Dose descriptor:
LOAEL
Effect level:
1 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: mortality at 2400 mg/kg bw/day; occurrence of clinical signs of toxicity (rough hair coats, lethargy and excessive lacrimation) at 1200 and 2400 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
1 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
Key result
Critical effects observed:
not specified
Conclusions:
Based on the read-across from the analogue substance d-limonene, the NOAEL of l-limonene for male and female rats was estimated to be 600 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats could be identified in this study.
Executive summary:

A 13-week subchronic toxicity study was performed similarly to OECD Guideline 408 and in compliance with GLP with the analogue substance d-limonene which was administered through gavage to groups of 10 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 13 weeks (5 days/week).  

Five of 10 males and 9/10 female rats that received 2400 mg/kg bw/day died during week 1. Final mean body weights of male rats in 600, 1200 or 2400 mg/kg bw/day groups were 6, 12 or 23% lower than that of the vehicle controls. Final body weight of the female rat that received 2400 mg/kg bw/day and lived to end of the study was 11% lower than the mean of the vehicle controls. Rough hair coats, lethargy and excessive lacrimation were observed at 1200 or 2400 mg/kg bw/day. No treatment-related histopathologic lesions were observed in female rats. Nephropathy was identified in all groups of male rats, and there was a dose-related increased severity of the lesion in dosed groups. Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium. Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. This mechanism of nephrocarcinogenicity has been proven as being male-rat specific and not relevant for humans.

Based on the read-across from the analogue substance d-limonene, the NOAEL of l-limonene for female rats was considered to be 600 mg/kg bw/day. When considering the non relevance of the nephrotoxic effects for humans, the NOAEL for male rats would be 600 mg/kg bw/day, based on decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats was identified.

Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from an analogue substance for which there is available data (Klimish =2).
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See cross-reference to justification of read-across.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
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:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality at 2000 mg/kg bw/day; decreased bodyweight gain and occurence of clinical signs of toxicity (rough hair coats and decreased activity) at 1000 and 2000 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
Key result
Critical effects observed:
not specified
Conclusions:
Based on the read-across from the analogue substance d-limonene, the NOAEL of l-limonene was estimated to be 500 mg/kg bw/day. The LOAEL was estimated to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.
Executive summary:

A 13-week subchronic toxicity study was performed similarly to OECD Guideline 408 and in compliance with GLP with the analogue substance d-limonene which was administered through gavage to groups of 10 B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 125, 250, 500, 1000 or 2000 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all control and high dose animals scheduled to be killed at the end of the treatment period.

One of 10 males and 2/10 females that received 2000 mg/kg bw/day and 1/10 females that received 500 mg/kg bw/day died before the end of the studies. Several animals in other groups died as a result of gavage error. Clinical signs of rough hair coats and decreased activity were observed at the two highest doses. Final mean bodyweights of mice that received 1000 or 2000 mg/kg bw/day were 10% lower than that of the vehicle controls for males and 2% lower for females. An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg bw/day.

 

Based on the read-across from the analogue substance d-limonene, the NOAEL of l-limonene was estimated to be 500 mg/kg bw/day. The LOAEL was estimated to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.

Data source

Materials and methods

Results and discussion

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion