Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No effects were observed on reproductive organs in the 90-day repeated dose toxicity study conducted in rats and mice (gross pathology and histopathology examination of prostate/testes or ovaries/uterus). Therefore no effects on reproductive peformance are anticipated.

Link to relevant study records

Referenceopen allclose all

Endpoint:
reproductive toxicity, other
Remarks:
data from a 90-day repeated dose toxicity study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From January 28 to April 30, 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study performed similarly to OECD Guideline 408 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 7-9 weeks
- Weight at study initiation: Males: 23.8-29.5 g; females: 20.2-21.5 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA) or NIH 07 Rat and
Mouse Ration (Zeigler Bros., Inc., Gardners, PA, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 60-82 °F
- Humidity (%): 35-80%
- Air changes (per hour): 12-15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amount of test substance was weighed and mixed with corn oil by shaking in a volumetric flask.
VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw
Details on mating procedure:
Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Apparatus: Periodic analysis for d-limonene in dose preparations was determined by extraction with methanol followed by gas chromatographic analysis of the extract with a 6-foot 3% OV-17 glass column, a nitrogen carrier at a flow rate of 30 mL/min, and a flame ionization detector.
- Sampling frequency: Once
- Results: 101-109% of the target concentrations
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once per day; 5 days/week
Details on study schedule:
Not applicable
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the mortalities observed at 3300 and 6600 mg/kg bw/day during a 16 day subacute toxicity study.
- Rationale for animal assignment (if not random): Random
Positive control:
None
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Initially and once per week thereafter
Oestrous cyclicity (parental animals):
Not applicable
Sperm parameters (parental animals):
Not applicable
Litter observations:
Not applicable
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all vehicle control and high dose animals and all female rats in the 1200 mg/kg bw/day group. Tissues examined include: adrenal glands, brain, colon, esophagus, eyes (if grossly abnormal), femur or sternebrae or vertebrae including marrow, gross lesions and tissue masses with regional lymph nodes, heart, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular or mesenteric lymph nodes, pancreas, parathyroids, pituitary gland, prostate/testes or ovaries/uterus, salivary glands, small intestine, spinal cord (if neurologic signs present), spleen, stomach, thymus, thyroid gland, trachea, and urinary bladder.
Postmortem examinations (offspring):
Not applicable
Statistics:
No data
Reproductive indices:
Not applicable
Offspring viability indices:
Not applicable
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Rough hair coats and decreased activity were observed at 1000 and 2000 mg/kg bw/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
- 1/10 male and 2/10 females died at 2000 mg/kg bw/day
- 1/10 female died at 500 mg/kg bw/day
- Several animals in other groups died as a result of gavage error.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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
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 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
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Key result
Critical effects observed:
no
Remarks on result:
not measured/tested
Critical effects observed:
not specified
Key result
Reproductive effects observed:
no
Conclusions:
The NOAEL was considered to be 500 mg/kg bw/day. The LOAEL was considered 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. No effects in reproductive organs were observed at either the macroscopic or the microscopic levels therefore no effects on reproductive performance are anticipated.
Executive summary:

In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene 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.

Therefore, the NOAEL was considered to be 500 mg/kg bw/day. The LOAEL was considered 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. No effects in reproductive organs were observed at either the macroscopic or the microscopic levels therefore no effects on reproductive performance are anticipated.

Endpoint:
reproductive toxicity, other
Remarks:
data from a 90-day repeated dose toxicity study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From January 28 to April 30, 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study performed similarly to OECD Guideline 408 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: 136-153 g; females: 101-120 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA) or NIH 07 Rat and
Mouse Ration (Zeigler Bros., Inc., Gardners, PA, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 18 or 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 60-82 °F
- Humidity (%): 35-80%
- Air changes (per hour): 12-15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amount of test substance was weighed and mixed with corn oil by shaking in a volumetric flask.
VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw
Details on mating procedure:
Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Apparatus: Periodic analysis for d-limonene in dose preparations was determined by extraction with methanol followed by gas chromatographic analysis of the extract with a 6-foot 3% OV-17 glass column, a nitrogen carrier at a flow rate of 30 mL/min, and a flame ionization detector.
- Sampling frequency: Once
- Results: 101-109% of the target concentrations
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once per day; 5 days/week
Details on study schedule:
Not applicable
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Dose / conc.:
1 200 mg/kg bw/day (actual dose received)
Dose / conc.:
2 400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected based on the mortalities observed at 3300 and 6600 mg/kg bw/day during a 16 day subacute toxicity study.
- Rationale for animal assignment (if not random): Random
Positive control:
None
Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Initially and once per week thereafter
Oestrous cyclicity (parental animals):
Not applicable
Sperm parameters (parental animals):
Not applicable
Litter observations:
Not applicable
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all vehicle control and high dose animals and all female rats in the 1200 mg/kg bw/day group. Tissues examined include: adrenal glands, brain, colon, esophagus, eyes (if grossly abnormal), femur or sternebrae or vertebrae including marrow, gross lesions and tissue masses with regional lymph nodes, heart, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular or mesenteric lymph nodes, pancreas, parathyroids, pituitary gland, prostate/testes or ovaries/uterus, salivary glands, small intestine, spinal cord (if neurologic signs present), spleen, stomach, thymus, thyroid gland, trachea, and urinary bladder. Kidneys examined for all male rats.
Postmortem examinations (offspring):
Not applicable
Statistics:
No data
Reproductive indices:
Not applicable
Offspring viability indices:
Not applicable
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Rough hair coats, lethargy and excessive lacrimation were observed for rats that received 1200 or 2400 mg/kg bw/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
5/10 males and 9/10 females at 2400 mg/kg bw/day died during week 1.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 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.
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 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
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 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.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
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:
body weight and weight gain
other: if male-rat specific nephrotoxicity (not relevant for humans) is not considered
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:
clinical signs
mortality
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Remarks on result:
not measured/tested
Critical effects observed:
not specified
Key result
Reproductive effects observed:
no
Conclusions:
The NOAEL for male and female rats was considered 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 o
bserved in male rats at all dose levels, no NOAEL for male rats could be identified in this study. No effects in reproductive organs were observed at either the macroscopic or the microscopic levels therefore no effects on reproductive performance are anticipated.
Executive summary:

In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene 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). 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.

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.

Therefore, the NOAEL 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 had primarily been identified in this study. No effects in reproductive organs were observed at either the macroscopic or the microscopic levels therefore no effects on reproductive performance are anticipated.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
2 000 mg/kg bw/day
Study duration:
subchronic
Species:
other: rats and mice
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity studies, with detailed results but limited information on test conditions, did not elicit any teratogenic effects of d-limonene on tested rabbits, mice or rats. Based on the absence of observed teratogenic effects and on the absence of effects on reproductive organs in the 90-day repeated dose toxicity study conducted in rats and mice (gross pathology and histopathology examination of prostate/testes or ovaries/uterus), no effects on reproductive performance are anticipated. Moreover, European Food Safety Authority made a review of the toxicological 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).


Effects on developmental toxicity

Description of key information

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:
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
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
Species:
rabbit
Strain:
other: Japanese white
Details on test animals and environmental conditions:
no data
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
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
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
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.
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
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
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
Abnormalities:
no effects observed
Developmental effects observed:
no

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:
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.

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
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Principles of method if other than guideline:
Prenatal developmental toxicity study: Groups of pregnant ICR mice (20/dose: 15 for teratogenicity study, 5 for postnatal development) were administered orally with d-limonene at dose levels of 0, 591 and 2363 mg/kg bw/day for 6 days from Day 7 to 12 of gestation and evaluated for developmental and postnatal development toxicity.
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
ICR
Details on test animals and environmental conditions:
No data
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:
6 days (gestation Day 7-12)
Frequency of treatment:
Once daily
Duration of test:
Gestatation Day 0 to postnatal week 7
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on study design:
No data
Maternal examinations:
See results tables
Ovaries and uterine content:
See results tables
Fetal examinations:
See results tables
Statistics:
statistical significance difference of effects from controls were calculated at 5% and 1% levels.
Indices:
No data
Historical control data:
No data
Clinical signs:
no effects observed
Description (incidence and severity):
No anomalies were observed in the general behavior of dams during the period of gestation.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Significant decrease of bodyweight gain in pregnant mice was observed at 2363 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
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
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:
no effects observed
Details on maternal toxic effects:
Details on maternal toxic effects:
See results tables
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
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, treatment-related
Description (incidence and severity):
- An incidence of lumber rib and fused rib in the fetuses increased significantly at 2363 mg/kg bw/day comparing 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. However, these retarded ossifications were restored to normal during postnatal development.
Visceral malformations:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
A significant decrease of bodyweight gain was observed in male offsprings born to dams given drug orally at 2363 mg/kg bw/day, but there were not differences in weaning rate, sensory function, organ weight and histological findings of the testis and ovary comparing with those of control.
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1: Effects of d-limonene on development of mouse fetuses.

 

Control

591

2363

No. of mothers

15

15

15

No. of implantations

162

179

154

(mean ± S.E.)

(10.80 ± 0.20)

(11.93 ± 0.13)

(10.27 ± 0.21)

No. of dead fetuses

9

1.1

6

(mean ± S.E.)

(0.60 ± 0.05)

(0.73 ± 0.10)

(0.40 ± 0.05)

No. of resorbed fetuses

18

20

20

(mean ± S.E.)

(1.20 ± 0.05)

(1.33 ± 0.10)

(1.33 ± 0.10)

No. of live fetuses

135

148

125

Sex ratio (Male/Female)

1.33

0.83

1.13

Fetuses Body weight (g)

Male (mean ± S.E.)

1.34 ± 0.02

1.24 ± 0.01

1.28 ± 0.02

Female (mean ± S.E.)

1.28 ± 0.02

1.22 ± 0.01

1.19 ± 0.02

Placental weight (mg)

Male (mean ± S.E.)

94 (2 ± 2.0)

86 ( 0 ± 1.7)

89 (3 ± 1.7)

Female (mean ± S.E.)

87 (8 ± 2.6)

81 (5 ± 1.9)

83 (5 ± 2.2)

External observation

No. of fetuses examined

135

148

128

No. of fetuses malformed

0

4

0

Cleft palate

0

4

0

Incidence (%)

0

2.7

0

Visceral observation

No. of fetuses examined

71

76

68

No. of fetuses malformed

4

4

3

Enlargement of foramen ovale

4

4

3

Incidence (%)

5.6

5.3

4.4

Table 2: Effects of d-limonene on skeletal development of mouse fetuses

 Dose (mg/kg bw)

Control 

591

2363

No. of fetuses examined

64

72

61

Variation

Lumbar rib (%)

17 (26.6)

12 (16.7)

28 (46.7) *

Cervical rib (%)

1 (1.6)

(5.6)

1 (1.7)

Fused rib (%)

0 (0)

0 (1)

5 (8.3) *

Crooked rib (%)

2 (3.1)

0 (1)

0 (0)

Asymmetry of sternebrae (%)

2 (1.7)

7 (9.7)

7 (11.7)

Fused sternebrae

0 (0)

1 (1.4)

1 (1.7)

No. of ossification

Sternebrae

5.99 ± 0.01

5.97 ± 0.02

5.94 ± 0.03

Fore limb

Metacarpal bone

8.00 ± 0

7.97 ± 0.03

8.00 ± 0

Proximal phalanx

7.50 ± 0.13

7.67 ± 0.16

6.87 ± 0.30 *

Middle phalanx

1.16 ± 0.23

2.14 ± 0.29 **

2.18 ± 0.29 **

Distal phalanx

9.08 ± 0.32

9.72 ± 0.20

9.13 ± 0.36

Hind limb

Metatarsal bone

10.00 ± 0

9.92 ± 0.05

9.80 ± 0.09 *

Proximal phalanx

8.12 ± 0.23

8.03 ± 0.23

7.23 ± 0.39 *

Middle phalanx

0.09 ± 0.09

0.33 ± 0.18

0.20 ± 0.11

Distal phalanx

9.47 ± 0.24

9.72 ± 0.20

9.30 ± 0.31

Caudal vertebrae

7.12 ± 0.95

6.50 ± 0.21

7.00 ± 0.25

* Significantly different from the control at 5% level.

** Significantly different from the control at 1% level.

Table 3: Effects of d-limonene on postnatal development of mouse offsprings

 

 

d-Limonene (mg/kg bw)

 

Control

591

2363

No. of mothers

5

5

5

No. of implantations

51

52

50

(mean ± S.E.)

(10.80 ± 0.33)

(10.41 ± 0.96)

(10.03 ± 0.63)

No. of offsprings

50

46

39

No. of dead offsprings at birth

0

0

0

Sensory function

Normal

Normal

Normal

No. of live offsprings

At birth

50

46

39

1st week

50

46

39

2nd week

50

46

39

3rd week

50

46

39

4th week

50

46

39

5th week

50

46

39

6th week

50

46

39

7th week

50

46

39

Weanling rate (%)

100

100

100

Table 4: Effects of d-limonene on gross differentiation of mouse offsprings

 

 

d-Limonene (mg/kg bw)

Gross differentiation

Control

591

2363

Opening of the ear-shell

3.5 ± 0.07

3.6 ± 0.08

4.1 ± 0.08

Coating with the hair

5.0 ± 0.00

4.11 ± 0.08

5.2 ± 0.06

Odontiasis

9.8 ± 0.07

9.3 ± 0.07

9. 1 ± 0.04

Opening of the eyelid

13.3 ± 0.08

12.9 ± 0.06

13.6 ± 0.09

Descending of the testis

23.0 ± 0.20

23.3 ± 0.11

25.0 ± 0.27

Opening of the vaginal orifice

29.5 ± 0.26

30.5 ± 0.16

30.6 ± 0.15

Table 5: Absolute organ weights of postnatal mouse offsprings born to mothers given d-limonene

Sex

Dose

(mg/kg bw)

No. of

offsprings

Final BW (g)

Thyroids (mg)

Thymus (g)

Lungs (g)

Heart (g)

Spleen (g)

Kidneys (g)

Liver (g)

Adrenals (g)

Testes (g) or Ovaries (mg)

Female

Control

27

34.2 ± 0.50

5.43 ± 0.18

75.72 ± 4.14

199.26 ± 4.70

158.22 ± 2.13

131.54 ± 9.96

0.63 ± 0.02

2.06 ± 0.09

8.39 ± 0.49

217.52 ± 1.65

591

27

34.9 ± 0.44

6.04 ± 0.68

65.26 ± 4.26

203.76 ± 2.95

166.66 ± 7.58

121.66 ± 4.74

0.63 ± 0.02

2.08 ± 0.07

8.29 ± 0.67

209.76 ± 9.23

2363

23

32.2 ± 0.77

5.41 ± 0.62

64.08 ± 3.64

189.60 ± 6.69

158.96 ± 3.12

125.52 ± 3.04

0.58 ± 0.02

2.14 ± 0.04

8.53 ± 0.86

200.20 ± 0.39

Male

Control

23

27.3 ± 0.50

4.80 ± 0.22

81.47 ± 4.38

172.80 ± 7.20

118.98 ± 3.91

121.76 ± 6.48

0.37 ± 0.01

1.37 ± 0.02

11.71 ± 0.40

13.38 ± 0.68

591

19

28.8 ± 0.45

4.29 ± 0.20

69.44 ± 5.52

174.26 ± 5.66

129.96 ± 3.62

112.68 ± 2.79

0.38 ± 0.01

1.37 ± 0.04

11.36 ± 0.43

16.98 ± 1.71

2363

16

28.1 ± 0.34

3.53 ± 0.41 *

63.95 ± 9.72

171.75 ± 5.70

135.15 ± 6.89

116.15 ± 5.78

0.38 ± 0.01

1.46 ± 0.03 *

11.61 ± 0.30 *

17.93 ± 1.30 *

* Significantly different from the control at 5% level.

Table 6: Relative organ weights per 100 g body weights of postnatal mouse offsprings born to mothers given d-limonene

Sex

Dose

(mg/kg bw)

No. of

offsprings

Final BW (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)

Female

Control

27

34.2 ± 0.50

15.80 ± 0.44

220.36 ± 10.67

580.50 ± 13.59

461.18 ± 8.65

386.55 ± 39.35

1.83 ± 0.07

6.01 ± 0.26

24.48 ± 1.55

634.84 ± 19.06

591

27

34.9 ± 0.44

17.20 ± 1.93

185.29 ± 8.48 *

581.21 ± 9.42

474.60 ± 717.10

346.85 ± 12.29

1.80 ± 0.05

5.93 ± 0.14

23.55 ± 1.48

596.69 ± 14.95

2363

23

32.2 ± 0.77

16.55 ± 2.64

191.59 ± 6.25

568.43 ± 13.67

477.39 ± 11.90

376.91 ± 10.23

1.73 ± 0.03

6.44 ± 0.29

25.74 ± 2.82

602.30 ± 34.26

Male

Control

23

27.3 ± 0.50

17.13 ± 18.47

291.98 ± 34.66

619.50 ± 8.54

425.00 ± 32.37

437.67

1.31 ± 0.04

4.89 ± 0.08

41.89 ± 1.41

47.85 ± 2.42

591

19

28.8 ± 0.45

15.06 ± 0.61 *

242.35 ± 13.57

611.98 ± 17.12

456.02 ± 4.93 *

397.44 ± 20.40

1.33 ± 0.02

4.81 ± 0.11

39.96 ± 1.73

59.59 ± 5.78

2363

16

28.1 ± 0.34

12.60 ± 1.44 *

229.38 ± 35.46

612.74 ± 20.32

482.14 ± 24.21 *

414.30 ± 17.34

1.37 ± 0.04

5.23 ± 0.09 *

41.45 ± 1.27

64.33 ± 5.38 *

* Significantly different from the control at 5% level.

Table 7: Summaried data on postnatal development of mouse offsprings

 

 

d-limonene (mg/kg bw)

 

Control

591

2363

External observation

 

No. of offsprings examined

50

46

39

No. of offsprings malformed

0

0

0

Visceral observation

 

No. of offsprings examined

50

46

39

No. of offsprings malformed

0

0

0

Skeletal observation

 

No. of offsprings examined

50

46

39

No. of offsprings malformed

0

0

0

Skeletal variation

 

Lumbar rib (%)

17 (34.0)

23 (50.0)

20 (51.3)

Fusion of 13th and lumbar rib (%)

0

0

1 (2.6)

Fusion of lumbar vertebra (%)

1 (2.0)

1 (2.2)

1 (2.6)

Crooked tail (%)

0

0

1 (2.6)

Conclusions:
The NOAEL for maternal and fetal toxicity was considered 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:

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. Bodyweights of pregnant mice 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. Number of live offsprings, sensory functions, gross differentiation and organ weights of offsprings were recorded until postnatal week 7.

 

A significant decrease of bodyweight gain in pregnant mice was observed at 2363 mg/kg bw/day. However, no anomalies were observed in the general behavior of dams during the period of gestation. An incidence of lumber rib and fused rib in the fetuses increased significantly at 2363 mg/kg bw/day comparing 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. However, these retarded ossifications were restored to normal during postnatal development. A significant decrease of bodyweight gain was observed in male offsprings born to dams given drug orally at 2363 mg/kg bw/day, but there were not differences in weaning rate, sensory function, organ weight and histological findings of the testis and ovary comparing with those of control.

Therefore, the NOAEL for maternal and fetal toxicity was considered to be 591 mg/kg bw/day based on the decreased bodyweight gain in dams and increased incidences of abnormal skeletal formation in fetuses at 2363 mg/kg bw/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1975
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Original reference in Japanese language
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
No data
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
No. of animals per sex per dose:
20 pregnant rats
Control animals:
yes, concurrent vehicle
Details on study design:
No data
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
Clinical signs:
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
Deaths (40%) and decreased bodyweight gain at 2869 mg/kg bw/day
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Deaths (40%) and decreased bodyweight gain at 2869 mg/kg bw/day
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
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:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
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:
See results tables
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
no effects observed
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:
effects observed, treatment-related
Description (incidence and severity):
Decreased number of live fetuses due to the increased mortality of dams at the highest dose tested
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, treatment-related
Description (incidence and severity):
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.
Visceral malformations:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
A tendency of decreased 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.
Key result
Dose descriptor:
NOAEL
Effect level:
591 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
other: decreased bodyweight gain (male offsprings) and organ weights at 2869 mg/kg bw/day
Key result
Abnormalities:
no effects observed
Developmental effects observed:
not specified

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

Conclusions:
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.

 

Therefore, 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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
591 mg/kg bw/day
Study duration:
subacute
Species:
rat
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 elicit 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 comparing 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.


Justification for classification or non-classification

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