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EC number: 232-433-8 | CAS number: 8028-48-6 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Citrus sinensis, Rutaceae.
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Carcinogenicity
Administrative data
Description of key information
Carcinogenic bioassay (RA from d-limonene, equivalent or similar to OECD 451):
No evidence of carcinogenic activity in mice and rats
Key value for chemical safety assessment
Carcinogenicity: via oral route
Link to relevant study records
- Endpoint:
- carcinogenicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From February 16, 1981 to February 17, 1983
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- GLP study performed similarly to OECD Guideline 451 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption and clinical biochemical tests not followed. Due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. The justification for read across is provided in the attached background material of the chapter summary.
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 451 (Carcinogenicity Studies)
- Deviations:
- yes
- Remarks:
- dosing 5 days/week instead of 7 days/week; food consumption and clinical biochemical tests not followed
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes
- 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: 8-9 weeks
- Weight at study initiation: Males: 30.2-30.3 g; females: 21.2-22.0 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): 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: 19 days
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 66-84 °F
- Humidity (%): 20-78%
- 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 - 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: After every 8 weeks
- Results: 87-110% of the target concentrations - Duration of treatment / exposure:
- 103 weeks
- Frequency of treatment:
- Once per day; 5 days/week
- Post exposure period:
- One week
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- male/female
- Dose / conc.:
- 250 mg/kg bw/day (actual dose received)
- Remarks:
- Male
- Dose / conc.:
- 500 mg/kg bw/day (actual dose received)
- Remarks:
- Male/Female
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- Female
- No. of animals per sex per dose:
- 50
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Doses were selected based on the deaths observed for both male and female mice at 2000 mg/kg bw/day during the 13-week studies and the body weight depression in male mice at 1000 mg/kg bw/day and higher.
- Rationale for animal assignment (if not random): Random - Positive control:
- No
- Observations and examinations performed and frequency:
- CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: Once per week for 12 weeks and once per month thereafter - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all animals; histologic exams performed on all animals dying during the studies, all vehicle controls and all high dose animals. Tissues examined include: adrenal glands, brain, cecum, colon, costochondral junction, duodenum, epididymis/seminal vesicles/tunica vaginalis/scrotal sac/prostate/testes or ovaries/uterus, esophagus, eyes, femur or sternebrae or vertebrae including marrow, gallbladder, gross lesions and tissue masses with regional lymph nodes, heart, ileum, jejunum, kidneys, larynx and pharynx, liver, lungs and bronchi, mammary gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, oral cavity, pancreas, parathyroids, pituitary gland, preputial or clitoral gland, rectum, salivary glands, sciatic nerve, skin, spinal cord, spleen, stomach, thigh muscle, thymus, thyroid gland, trachea, urinary bladder and Zymbal gland. Tissues examined in low dose groups include adrenal glands, kidney, liver and spleen for female mice. - Other examinations:
- None
- Statistics:
- - Survival: Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends.
- Neoplasm and nonneoplastic lesion incidences: Incidental tumor analysis, life table test (Cox, 1972; Tarone, 1975), Fisher exact test and the Cochran-Armitage trend test (Armitage, 1971; Gart et al., 1979) were used to assess neoplasm and nonneoplastic lesion prevalence. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - No treatment-related clinical signs were observed during the study.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- - Survival of the low dose group of male mice was significantly lower than that of the vehicle controls at the end of the study.
- Survival in males at week 104: 33/50, 24/50 and 39/50 animals at 0, 250 and 500 mg/kg bw/day, respectively.
- Survival in females at week 104: 43/50, 44/50 and 43/50 animals at 0, 500 and 1000 mg/kg bw/day, respectively.
- See table 1 for more data - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- - Mean bodyweights of high dose female mice were 5-15% lower than those of the vehicle controls after week 28.
- Mean bodyweights of dosed and vehicle control male mice were similar throughout the studies. - Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not specified
- Haematological findings:
- not specified
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- - No treatment-related statistically or biologically significant effects were observed during the study.
- Histopathological findings: neoplastic:
- no effects observed
- Relevance of carcinogenic effects / potential:
- Not relevant as no evidence of carcinogenic activity of d-limonene for male and female B6C3F1 mice.
- Dose descriptor:
- NOAEL
- Effect level:
- >= 250 - <= 500 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: no evidence of carcinogenic activity
- Remarks on result:
- not determinable
- Remarks:
- no NOAEL identified. Effect type:carcinogenicity (migrated information)
- Dose descriptor:
- NOAEL
- Effect level:
- >= 500 - <= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: no evidence of carcinogenic activity
- Remarks on result:
- not determinable
- Remarks:
- no NOAEL identified. Effect type:carcinogenicity (migrated information)
- Conclusions:
- Under the test conditions, there was no evidence of carcinogenic activity of d-limonene for male and female B6C3F1 mice.
- Executive summary:
In a 2 -year carcinogenicity study performed similarly to OECD Guideline 451 and in compliance with GLP, d-limonene was administered through gavage to groups of 50 B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 250 and 500 mg/kg bw/day (in males) or 0, 500 and 1000 mg/kg bw/day (in females) for 103 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded once per week for 12 weeks and once per month thereafter. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all animals dying during the studies, all vehicle controls and all high dose animals at the end of the treatment period.
Survival of the low dose group of male mice was significantly lower than that of the vehicle controls at the end of the study. Survival at week 104 was 33/50 male and 43/50 female in vehicle control group; 24/50 males and 44/50 females in low dose group and 39/50 males and 43/50 females in high dose group. No treatment-related clinical signs were observed during the study. Mean bodyweights of high dose female mice were 5-15% lower than those of the vehicle controls after week 28. Mean bodyweights of dosed and vehicle control male mice were similar throughout the studies. No treatment-related statistically or biologically significant histologic effects were observed during the study.
Under the test conditions, there was no evidence of carcinogenic activity of d-limonene for male and female B6C3F1 mice.
- Endpoint:
- carcinogenicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From February 02, 1981 to February 11, 1983
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- GLP study performed similarly to OECD Guideline 451 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption and clinical biochemical tests not followed. Due to the read-across purpose it was given a Klimisch 2 rating, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID. The justification for read across is provided in the attached background material of the chapter summary.
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 451 (Carcinogenicity Studies)
- Deviations:
- yes
- Remarks:
- dosing 5 days/week instead of 7 days/week; food consumption and clinical biochemical tests not followed
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes
- 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: 183-187 g; females: 132-133 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): 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: 19 days
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 66-84 °F
- Humidity (%): 20-78%
- 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 - 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: After every 8 weeks
- Results: 87-110% of the target concentrations - Duration of treatment / exposure:
- 103 weeks
- Frequency of treatment:
- Once per day; 5 days/week
- Post exposure period:
- 1 week in males; 1-2 weeks in females
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Male/Female
- Dose / conc.:
- 75 mg/kg bw/day (actual dose received)
- Remarks:
- Male
- Dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- Remarks:
- Male
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Remarks:
- Female
- Dose / conc.:
- 600 mg/kg bw/day (actual dose received)
- Remarks:
- Female
- No. of animals per sex per dose:
- 50
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Doses were selected based on compound-related, potentially life-threatening kidney lesions observed in males at 300 mg/kg bw/day and higher and on the large number of deaths of female rats at 2400 mg/kg bw/day during the 13-week study.
- Rationale for animal assignment (if not random): Random - Positive control:
- No
- Observations and examinations performed and frequency:
- CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: Once per week for 12 weeks and once per month thereafter - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all animals; histologic exams performed on all animals dying during the studies, all vehicle controls, all low dose female rats and all high dose animals. Tissues examined include: adrenal glands, brain, cecum, colon, costochondral junction, duodenum, epididymis/seminal vesicles/tunica vaginalis/scrotal sac/prostate/testes or ovaries/uterus, esophagus, eyes, femur or sternebrae or vertebrae including marrow, gross lesions and tissue masses with regional lymph nodes, heart, ileum, jejunum, kidneys, larynx and pharynx, liver, lungs and bronchi, mammary gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, oral cavity, pancreas, parathyroids, pituitary gland, preputial or clitoral gland, rectum, salivary glands, sciatic nerve, skin, spinal cord, spleen, stomach, thigh muscle, thymus, thyroid gland, trachea, urinary bladder and Zymbal gland. Tissues examined in low dose groups include adrenal glands, kidney, liver, spleen and testis for male rats. - Other examinations:
- None
- Statistics:
- - Survival: Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends.
- Neoplasm and nonneoplastic lesion incidences: Incidental tumor analysis, life table test (Cox, 1972; Tarone, 1975), Fisher exact test and the Cochran-Armitage trend test (Armitage, 1971; Gart et al., 1979) were used to assess neoplasm and nonneoplastic lesion prevalence. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - No treatment-related clinical signs were observed during the study.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- - Survival of the high dose female rats after week 39 and of the vehicle control male rats after week 81 was significantly reduced.
- Survival in males at week 104: 29/50, 33/50 and 40/50 animals at 0, 75 and 150 mg/kg bw/day, respectively.
- Survival in females at week 104: 42/50, 40/50 and 26/50 animals at 0, 300 and 600 mg/kg bw/day, respectively.
- Several animals died accidentally. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- - Mean body weights of high dose rats were generally 4-7% lower than those of the vehicle controls from week 2 (in males) or 28 (in females) to the end of the studies.
- 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:
- no effects observed
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Kidney:
- Non-neoplastic effects: No lesions were observed in female rats. For males, the nonneoplastic lesions included exacerbation of the age-related nephropathy, linear deposits of mineral in the renal medulla and papilla (in 7/50, 43/50 and 48/50 rats at 0, 75 and 150 mg/kg bw/day, respectively), and focal hyperplasia of the transitional epithelium overlying the renal papilla (in 0/50, 35/50 and 43/50 rats at 0, 75 and 150 mg/kg bw/day, respectively).
Uterus, testis, hematopoietic system, skin, subcutaneous tissue and eye:
- No treatment-related statistically or biologically significant effects were observed during the study. - Histopathological findings: neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Kidney:
- Neoplastic effects: Uncommon tubular cell adenomas (in 0/50, 4/50 and 8/50 rats at 0, 75 and 150 mg/kg bw/day, respectively) and adenocarcinomas (in 0/50, 4/50 and 3/50 rats at 0, 75 and 150 mg/kg bw/day, respectively) of the kidney occurred in dosed male rats, and this effect was supported by a dose-related increased incidence of tubular cell hyperplasia (in 0/50, 4/50 and 7/50 rats at 0, 75 and 150 mg/kg bw/day, respectively) - Relevance of carcinogenic effects / potential:
- This mechanism of nephrocarcinogenicity has been proven as being nale-rat specific and not relevant for humans.
- Dose descriptor:
- NOAEL
- Effect level:
- >= 75 - <= 150 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: clear evidence of carcinogenic activity based on the increased incidences of tubular cell hyperplasia, adenomas and adenocarcinomas of the kidney
- Remarks on result:
- not determinable
- Remarks:
- no NOAEL identified. Effect type:carcinogenicity (migrated information)
- Dose descriptor:
- NOAEL
- Effect level:
- >= 300 - <= 600 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: no evidence of carcinogenic activity
- Remarks on result:
- not determinable
- Remarks:
- no NOAEL identified. Effect type:carcinogenicity (migrated information)
- Conclusions:
- Under the test conditions, there was clear evidence of carcinogenic activity of d-limonene for male F344/N rats, as shown by increased incidences of tubular cell hyperplasia, adenomas, and adenocarcinomas of the kidney. There was no evidence of carcinogenic activity of d-limonene for female F344/N rats.
- Executive summary:
In a 2 year carcinogenicity study performed similarly to OECD Guideline 451 and in compliance with GLP, d-limonene was administered through gavage to groups of 50 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 75 and 150 mg/kg bw/day (in males) or 0, 300 and 600 mg/kg bw/day (in females) for 103 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded once per week for 12 weeks and once per month thereafter. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all animals dying during the studies, all vehicle controls, all low dose female rats and all high dose animals at the end of the treatment period.
Survival of the high dose female rats after week 39 and of the vehicle control male rats after week 81 was significantly reduced. Survival at week 104 was 29/50 male and 42/50 female in vehicle control group; 33/50 males and 40/50 females in low dose group and 40/50 males and 20/50 females in high dose group. Mean body weights of high dose rats were generally 4-7% lower than those of the vehicle controls from week 2 (in males) or 28 (in females) to the end of the studies. No treatment-related clinical signs were observed during the study. Kidney was confirmed as the primary target organ for chemically related lesions. No lesions were observed in female rats. For males, the nonneoplastic lesions included exacerbation of the age-related nephropathy, linear deposits of mineral in the renal medulla and papilla, and focal hyperplasia of the transitional epithelium overlying the renal papilla. Uncommon tubular cell adenomas and adenocarcinomas of the kidney also occurred in dosed male rats and this effect was supported by a dose-related increased incidence of tubular cell hyperplasia.
Under the test conditions, there was clear evidence of carcinogenic activity of d-limonene for male F344/N rats, as shown by increased incidences of tubular cell hyperplasia, adenomas, and adenocarcinomas of the kidney. There was no evidence of carcinogenic activity of d-limonene for female F344/N rats. This mechanism of nephrocarcinogenicity has been proven as being nale-rat specific and not relevant for humans.
- Endpoint:
- carcinogenicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- See attached justification
- Reason / purpose for cross-reference:
- read-across source
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - No treatment-related clinical signs were observed during the study.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- - Survival of the low dose group of male mice was significantly lower than that of the vehicle controls at the end of the study.
- Survival in males at week 104: 33/50, 24/50 and 39/50 animals at 0, 250 and 500 mg/kg bw/day, respectively.
- Survival in females at week 104: 43/50, 44/50 and 43/50 animals at 0, 500 and 1000 mg/kg bw/day, respectively.
- See table 1 for more data - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- - Mean bodyweights of high dose female mice were 5-15% lower than those of the vehicle controls after week 28.
- Mean bodyweights of dosed and vehicle control male mice were similar throughout the studies. - Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not specified
- Haematological findings:
- not specified
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- - No treatment-related statistically or biologically significant effects were observed during the study.
- Histopathological findings: neoplastic:
- no effects observed
- Relevance of carcinogenic effects / potential:
- Not relevant as no evidence of carcinogenic activity of d-limonene for male and female B6C3F1 mice.
- Dose descriptor:
- NOAEL
- Effect level:
- >= 250 - <= 500 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: no evidence of carcinogenic activity
- Remarks on result:
- not determinable
- Remarks:
- no NOAEL identified. Effect type:carcinogenicity (migrated information)
- Dose descriptor:
- NOAEL
- Effect level:
- >= 500 - <= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: no evidence of carcinogenic activity
- Remarks on result:
- not determinable
- Remarks:
- no NOAEL identified. Effect type:carcinogenicity (migrated information)
- Conclusions:
- Under the test conditions, there was no evidence of carcinogenic activity of d-limonene for male and female B6C3F1 mice.
- Executive summary:
In a 2 -year carcinogenicity study performed similarly to OECD Guideline 451 and in compliance with GLP, d-limonene was administered through gavage to groups of 50 B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 250 and 500 mg/kg bw/day (in males) or 0, 500 and 1000 mg/kg bw/day (in females) for 103 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded once per week for 12 weeks and once per month thereafter. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all animals dying during the studies, all vehicle controls and all high dose animals at the end of the treatment period.
Survival of the low dose group of male mice was significantly lower than that of the vehicle controls at the end of the study. Survival at week 104 was 33/50 male and 43/50 female in vehicle control group; 24/50 males and 44/50 females in low dose group and 39/50 males and 43/50 females in high dose group. No treatment-related clinical signs were observed during the study. Mean bodyweights of high dose female mice were 5-15% lower than those of the vehicle controls after week 28. Mean bodyweights of dosed and vehicle control male mice were similar throughout the studies. No treatment-related statistically or biologically significant histologic effects were observed during the study.
Under the test conditions, there was no evidence of carcinogenic activity of d-limonene for male and female B6C3F1 mice.
- Endpoint:
- carcinogenicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- See attached justification
- Reason / purpose for cross-reference:
- read-across source
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - No treatment-related clinical signs were observed during the study.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- - Survival of the high dose female rats after week 39 and of the vehicle control male rats after week 81 was significantly reduced.
- Survival in males at week 104: 29/50, 33/50 and 40/50 animals at 0, 75 and 150 mg/kg bw/day, respectively.
- Survival in females at week 104: 42/50, 40/50 and 26/50 animals at 0, 300 and 600 mg/kg bw/day, respectively.
- Several animals died accidentally. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- - Mean body weights of high dose rats were generally 4-7% lower than those of the vehicle controls from week 2 (in males) or 28 (in females) to the end of the studies.
- 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:
- no effects observed
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- effects observed, treatment-related
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Kidney:
- Non-neoplastic effects: No lesions were observed in female rats. For males, the nonneoplastic lesions included exacerbation of the age-related nephropathy, linear deposits of mineral in the renal medulla and papilla (in 7/50, 43/50 and 48/50 rats at 0, 75 and 150 mg/kg bw/day, respectively), and focal hyperplasia of the transitional epithelium overlying the renal papilla (in 0/50, 35/50 and 43/50 rats at 0, 75 and 150 mg/kg bw/day, respectively).
Uterus, testis, hematopoietic system, skin, subcutaneous tissue and eye:
- No treatment-related statistically or biologically significant effects were observed during the study. - Histopathological findings: neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Kidney:
- Neoplastic effects: Uncommon tubular cell adenomas (in 0/50, 4/50 and 8/50 rats at 0, 75 and 150 mg/kg bw/day, respectively) and adenocarcinomas (in 0/50, 4/50 and 3/50 rats at 0, 75 and 150 mg/kg bw/day, respectively) of the kidney occurred in dosed male rats, and this effect was supported by a dose-related increased incidence of tubular cell hyperplasia (in 0/50, 4/50 and 7/50 rats at 0, 75 and 150 mg/kg bw/day, respectively) - Relevance of carcinogenic effects / potential:
- This mechanism of nephrocarcinogenicity has been proven as being nale-rat specific and not relevant for humans.
- Dose descriptor:
- NOAEL
- Effect level:
- >= 75 - <= 150 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: clear evidence of carcinogenic activity based on the increased incidences of tubular cell hyperplasia, adenomas and adenocarcinomas of the kidney
- Remarks on result:
- not determinable
- Remarks:
- no NOAEL identified. Effect type:carcinogenicity (migrated information)
- Dose descriptor:
- NOAEL
- Effect level:
- >= 300 - <= 600 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: no evidence of carcinogenic activity
- Remarks on result:
- not determinable
- Remarks:
- no NOAEL identified. Effect type:carcinogenicity (migrated information)
- Conclusions:
- Under the test conditions, there was clear evidence of carcinogenic activity of d-limonene for male F344/N rats, as shown by increased incidences of tubular cell hyperplasia, adenomas, and adenocarcinomas of the kidney. There was no evidence of carcinogenic activity of d-limonene for female F344/N rats.
- Executive summary:
In a 2 year carcinogenicity study performed similarly to OECD Guideline 451 and in compliance with GLP, d-limonene was administered through gavage to groups of 50 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 75 and 150 mg/kg bw/day (in males) or 0, 300 and 600 mg/kg bw/day (in females) for 103 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded once per week for 12 weeks and once per month thereafter. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all animals dying during the studies, all vehicle controls, all low dose female rats and all high dose animals at the end of the treatment period.
Survival of the high dose female rats after week 39 and of the vehicle control male rats after week 81 was significantly reduced. Survival at week 104 was 29/50 male and 42/50 female in vehicle control group; 33/50 males and 40/50 females in low dose group and 40/50 males and 20/50 females in high dose group. Mean body weights of high dose rats were generally 4-7% lower than those of the vehicle controls from week 2 (in males) or 28 (in females) to the end of the studies. No treatment-related clinical signs were observed during the study. Kidney was confirmed as the primary target organ for chemically related lesions. No lesions were observed in female rats. For males, the nonneoplastic lesions included exacerbation of the age-related nephropathy, linear deposits of mineral in the renal medulla and papilla, and focal hyperplasia of the transitional epithelium overlying the renal papilla. Uncommon tubular cell adenomas and adenocarcinomas of the kidney also occurred in dosed male rats and this effect was supported by a dose-related increased incidence of tubular cell hyperplasia.
Under the test conditions, there was clear evidence of carcinogenic activity of d-limonene for male F344/N rats, as shown by increased incidences of tubular cell hyperplasia, adenomas, and adenocarcinomas of the kidney. There was no evidence of carcinogenic activity of d-limonene for female F344/N rats. This mechanism of nephrocarcinogenicity has been proven as being nale-rat specific and not relevant for humans.
Referenceopen allclose all
Table 1: Survival of mice in the 2-year gavage studies of d-limonene
|
Vehicle Control |
250 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
MALE (a) |
||||
Animals initially in study |
50 |
50 |
50 |
|
Nonaccidental deaths before termination (b) |
14 |
24 |
9 |
|
Accidentally killed |
2 |
2 |
2 |
|
Animals missing |
1 |
0 |
0 |
|
Killed at termination |
33 |
24 |
38 |
|
Died during termination period |
0 |
0 |
1 |
|
Survival P values (c) |
0.361 |
0.048 |
0.348 |
|
FEMALE (a) |
||||
Animals initially in study |
50 |
|
50 |
50 |
Nonaccidental deaths before termination (b) |
7 |
|
5 |
7 |
Accidentally killed |
0 |
|
1 |
0 |
Killed at termination |
42 |
|
44 |
42 |
Died during termination period |
1 |
|
0 |
1 |
Survival P values (c) |
1 |
|
0.757 |
0.995 |
(a) Termination period: week 104
(b) Includes animals killed in a moribund condition
(c) The result of the life table trend test is in the vehicle control column, and the results of the life table pairwise comparisons with the vehicle controls are in the dosed columns.
Table 1: Survival of rats in the 2-year gavage studies of d-limonene
|
Vehicle Control |
Low Dose |
High Dose |
MALE (a) |
|
75 mg/kg bw/day |
150 mg/kg bw/day |
Animals initially in study |
50 |
50 |
50 |
Nonaccidental deaths before termination (b) |
20 |
16 |
5 |
Accidentally killed |
1 |
1 |
5 |
Killed at termination |
29 |
33 |
40 |
Survival P values (c) |
0.001 |
0.497 |
0.001 |
FEMALE (a) |
|
300 mg/kg bw/day |
600 mg/kg bw/day |
Animals initially in study |
50 |
50 |
50 |
Nonaccidental deaths before termination (b) |
5 |
8 |
16 |
Accidentally killed |
3 |
2 |
8 |
Killed at termination |
42 |
39 |
24 |
Died during termination period |
0 |
1 |
2 |
Survival P values (c) |
0.003 |
0.571 |
0.006 |
(a) Termination period: male--week 104; female--weeks 104-105
(b) Includes animals killed in a moribund condition
(c) The result of the life table trend test is in the vehicle control column, and the results of the life table pairwise comparisons with the vehicle controls are in the dosed columns.
Table 2: Incidences of male rats with renal lesions in the 2-year gavage study of d-limonene
Site/Lesion |
Vehicle Control |
75mg/kg bw/day |
150 mg/kg bw/day |
Renal papilla |
|||
Mineralization |
7/50 |
43/50 |
48/50 |
Epithelial hyperplasia |
0/50 |
35/50 |
43/50 |
Kidney |
|||
Tubular cell hyperplasia |
0/50 |
4/50 |
7/50 |
Tubular cell adenoma |
0/50 |
4/50 |
8/50 |
Tubular cell adenocarcinoma |
0/50 |
4/50 |
3/50 |
Table 1: Survival of mice in the 2-year gavage studies of d-limonene
|
Vehicle Control |
250 mg/kg bw/day |
500 mg/kg bw/day |
1000 mg/kg bw/day |
MALE (a) |
||||
Animals initially in study |
50 |
50 |
50 |
|
Nonaccidental deaths before termination (b) |
14 |
24 |
9 |
|
Accidentally killed |
2 |
2 |
2 |
|
Animals missing |
1 |
0 |
0 |
|
Killed at termination |
33 |
24 |
38 |
|
Died during termination period |
0 |
0 |
1 |
|
Survival P values (c) |
0.361 |
0.048 |
0.348 |
|
FEMALE (a) |
||||
Animals initially in study |
50 |
|
50 |
50 |
Nonaccidental deaths before termination (b) |
7 |
|
5 |
7 |
Accidentally killed |
0 |
|
1 |
0 |
Killed at termination |
42 |
|
44 |
42 |
Died during termination period |
1 |
|
0 |
1 |
Survival P values (c) |
1 |
|
0.757 |
0.995 |
(a) Termination period: week 104
(b) Includes animals killed in a moribund condition
(c) The result of the life table trend test is in the vehicle control column, and the results of the life table pairwise comparisons with the vehicle controls are in the dosed columns.
Table 1: Survival of rats in the 2-year gavage studies of d-limonene
|
Vehicle Control |
Low Dose |
High Dose |
MALE (a) |
|
75 mg/kg bw/day |
150 mg/kg bw/day |
Animals initially in study |
50 |
50 |
50 |
Nonaccidental deaths before termination (b) |
20 |
16 |
5 |
Accidentally killed |
1 |
1 |
5 |
Killed at termination |
29 |
33 |
40 |
Survival P values (c) |
0.001 |
0.497 |
0.001 |
FEMALE (a) |
|
300 mg/kg bw/day |
600 mg/kg bw/day |
Animals initially in study |
50 |
50 |
50 |
Nonaccidental deaths before termination (b) |
5 |
8 |
16 |
Accidentally killed |
3 |
2 |
8 |
Killed at termination |
42 |
39 |
24 |
Died during termination period |
0 |
1 |
2 |
Survival P values (c) |
0.003 |
0.571 |
0.006 |
(a) Termination period: male--week 104; female--weeks 104-105
(b) Includes animals killed in a moribund condition
(c) The result of the life table trend test is in the vehicle control column, and the results of the life table pairwise comparisons with the vehicle controls are in the dosed columns.
Table 2: Incidences of male rats with renal lesions in the 2-year gavage study of d-limonene
Site/Lesion |
Vehicle Control |
75mg/kg bw/day |
150 mg/kg bw/day |
Renal papilla |
|||
Mineralization |
7/50 |
43/50 |
48/50 |
Epithelial hyperplasia |
0/50 |
35/50 |
43/50 |
Kidney |
|||
Tubular cell hyperplasia |
0/50 |
4/50 |
7/50 |
Tubular cell adenoma |
0/50 |
4/50 |
8/50 |
Tubular cell adenocarcinoma |
0/50 |
4/50 |
3/50 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
Carcinogenicity: via inhalation route
Endpoint conclusion
- Endpoint conclusion:
- no study available
Carcinogenicity: via dermal route
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Based on the results of the key studies, orange oil does not need to be classified for carcinogenicity when considering the criteria outlined in Annex I of 1272/2008/EC .
Additional information
One carcinogenicity study (equivalent or similar to OECD 451, read-across from d-limonene to orange oil) considering two species was available. Both mice and rats were tested.
In the experiment with B6C3F1 mice there was found no evidence of carcinogenic activity of d-limonene for both males and females. The experiment with F344/N rats found clear evidence of carcinogenic activity of d-limonene for males, as shown by increased incidences of tubular cell hyperplasia, adenomas, and adenocarcinomas of the kidney. However, the observed effect was considered to be male rat specific (nephrocarcinogenicity) and therefore not relevant for human. There was no evidence of carcinogenic activity of d-limonene for female F344/N rats. In conclusion, both experiment indicate that there is no evidence for carcinogenicity of orange oil.
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