Registration Dossier
Registration Dossier
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EC number: 297-672-2 | CAS number: 93686-22-7 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Citrus reticulata, x C. sinensis, Rutaceae.
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on developmental toxicity
Description of key information
Developmental toxicity study in rats: not teratogenic, NOAELmaternal and developmental 591 mg/kg bw/day
Developmental toxicity study in rabbits: not teratogenic, NOAELmaternal 250 mg/kg bw/day, NOEALdevelopmental 1000 mg/kg bw/day
Devlopmental toxicity study in mice: not teratogenic, NOAELmaternal and developmental 591 mg/kg bw/day
Link to relevant study records
- 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:
- read-across: supporting information
- 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
- Remarks:
- Doses / Concentrations:
0, 591 and 2869 mg/kg bw/day
Basis:
actual ingested - 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
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
Deaths (40%) and decreased bodyweight gain at 2869 mg/kg bw/day - Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
Prolongation of the ossification of metacarpals and proximal phalanges in fetuses, decreased bodyweight gain (male offsprings) and organ weights at 2869 mg/kg bw/day - Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- skeletal malformations
- other: decreased bodyweight gain
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- Under the test conditions, 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.
Under the test conditions, 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.
- Endpoint:
- developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- other: Original reference in Japanese language
- Justification for type of information:
- The read across justification is attached below
- Reason / purpose for cross-reference:
- read-across source
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
Treatment with the highest dose level (1000 mg/kg) of d-limonene resulted in death of dams with less than 40% mortality. The significant decrease of 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. - Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
- External examination of fetuses showed no anormalies.
- Visceral and skeletal examinations revealed some anormalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development.
- Other non specific anormalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene. - Dose descriptor:
- NOAEL
- Effect level:
- > 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: fetotoxicity
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- Under the test conditions, 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 anormalies 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 anormalies. Visceral and skeletal examinations revealed some anormalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development. Other non specific anormalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene.
Under the test conditions, 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:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- other: Original reference in Japanese language
- Justification for type of information:
- The read across justification is attached below
- Reason / purpose for cross-reference:
- read-across source
- Details on maternal toxic effects:
- Details on maternal toxic effects:
See results tables - Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
See results tables - Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- skeletal malformations
- other: A significant decrease of bodyweight gain was observed in male offsprings born to dams given drug orally at 2363 mg/kg bw/day
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- Under the test conditions, 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.
Under the test conditions, 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:
- 1977
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- other: Original reference in Japanese language
- Reason / purpose for cross-reference:
- read-across: supporting information
- 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
- Remarks:
- Doses / Concentrations:
0, 250, 500 or 1000 mg/kg bw/day
Basis:
actual ingested - 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
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
Treatment with the highest dose level (1000 mg/kg) of d-limonene resulted in death of dams with less than 40% mortality. The significant decrease of 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. - Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
- External examination of fetuses showed no anormalies.
- Visceral and skeletal examinations revealed some anormalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development.
- Other non specific anormalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene. - Dose descriptor:
- NOAEL
- Effect level:
- > 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: fetotoxicity
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- Under the test conditions, 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 anormalies 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 anormalies. Visceral and skeletal examinations revealed some anormalies such as incomplete lobulation of the lungs, enlargement of the foramen ovale and retarded ossification of the middle phalanx of fore limbs in addition to the 5th sternebrae. These did not appear to be dose-dependent and restored to normal during the postnatal development. Other non specific anormalies involved the lumber ribs in fetuses and offsprings, formation of the accessory ossification center of the 5th sternebrae in offsprings and the atrial septal defect detected in only 2 fetuses of a litter from dams treated with 250 mg/kg bw/day of d-limonene.
Under the test conditions, 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 for cross-reference:
- read-across: supporting information
- 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
- Remarks:
- Doses / Concentrations:
0, 591 and 2363 mg/kg bw/day
Basis:
actual ingested - 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
- Details on maternal toxic effects:
- Details on maternal toxic effects:
See results tables - Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
See results tables - Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- skeletal malformations
- other: A significant decrease of bodyweight gain was observed in male offsprings born to dams given drug orally at 2363 mg/kg bw/day
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- Under the test conditions, 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.
Under the test conditions, 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:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- other: Original reference in Japanese language
- Justification for type of information:
- The read across justification is attached below
- Reason / purpose for cross-reference:
- read-across source
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
Deaths (40%) and decreased bodyweight gain at 2869 mg/kg bw/day - Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
Prolongation of the ossification of metacarpals and proximal phalanges in fetuses, decreased bodyweight gain (male offsprings) and organ weights at 2869 mg/kg bw/day - Dose descriptor:
- NOAEL
- Effect level:
- 591 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- skeletal malformations
- other: decreased bodyweight gain
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- Under the test conditions, 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.
Under the test conditions, 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.
Referenceopen allclose all
Maternal examinations:
- 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.
Fetal examinations:
- 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 bw/day.
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 |
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
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) |
Maternal examinations:
- Significant decrease of bodyweight gain in pregnant mice was observed at 2363 mg/kg bw/day.
- No anomalies were observed in the general behavior of dams during the period of gestation.
Fetal examinations:
- 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.
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) |
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) |
Maternal examinations:
- Significant decrease of bodyweight gain in pregnant mice was observed at 2363 mg/kg bw/day.
- No anomalies were observed in the general behavior of dams during the period of gestation.
Fetal examinations:
- 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.
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) |
Maternal examinations:
- 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.
Fetal examinations:
- 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 bw/day.
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 |
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
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 591 mg/kg bw/day
- Study duration:
- subacute
- Species:
- other: rat, rabbit, mouse
Additional information
Three developmental toxicity studies with main constituent limonene are available in three species (rats, rabbits and mice), which together were used in a weight of evidence approach.
In the developmental toxicity study in rats, 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.
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.
Under the test conditions, 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.
In the prenatal developmental toxicity study with rabbits, 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.
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.
Under the test conditions, 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.
In the prenatal developmental toxicity study in mice, 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. 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.
Under the test conditions, 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 at 2363 mg/kg bw/day.
Justification for classification or non-classification
Based on the available information, there is not enough evidence to classify Tangerine oil for developmental toxicity, in accordance with the criteria outlined in Annex I of 1272/2008/EC (CLP/EU-GHS).
Additional information
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