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

Endpoint summary

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

Reference

Reference 1

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 for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

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 or test system 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.

Reference 2

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 for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

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 or test system 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

Reference

Reference 1

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

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 or test system 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:

body weight and weight gain

mortality

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.

Reference 2

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

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 or test system 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.

Reference 3

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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

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 or test system 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 developmental toxicity.