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EC number: 202-795-1 | CAS number: 99-86-5
- 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
![](https://chesar.echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Endpoint summary
Administrative data
Description of key information
Weight of evidence and read-across approach:
Some reliable experimental studies on repeated dose toxicity are available from the analogue substance d-limonene and thus a weight of evidence and read across approach was applied to fulfill this information requirement for alpha terpinene. There are subacute and subchronic studies in rats, mice and dogs.
Signs of nephrotoxicity were observed in males of all the studies conducted with rats. The absence of any d-limonene-induced renal lesions in the studies with dogs and mice provides evidence that hydrocarbon induced nephropathy in the male rat is species- and sex-specific. Therefore, the male rat response to d-limonene may not be appropriate for assessing the potential risk of a similar nephrotoxic response in any other species, including humans. According to CLP annex I 3.9.2.8.1. (e), substance-induced species-specific mechanisms of toxicity, i.e. demonstrated with reasonable certainty to be not relevant for human health, shall not justify classification.
When administered orally by gavage for at least 6 months, d-limonene induces some toxicological effects from 1000 mg/kg bw/d. At this dose level, following 90 days of exposure, d-limonene induces decreased bodyweight gain and clinical signs in mice. 180 days of exposure to d-limonene at this dose level decreased bodyweight gain and increased relative and absolute kidney weights of dogs (with protein casts in the renal tubules of females).
Thus, the key value has been selected to be the LOAEL at 1000 mg/kg bw/d. The key study was then selected to be the 180-d toxicity study by oral route in dogs (Webb, 1990) for DNEL derivation since mammalian exposure is more relevant than rodent exposure regarding human health effect assessment. Moreover effects were seen in this study on the target organ of d-limonene, kidneys.
Based on the weight of evidence and read-across approach, the key value for alpha terpinene is considered to be LOAEL = 1000 mg/kg bw from the 180-d dog study.
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- documentation insufficient for assessment
- Remarks:
- Study designed to evaluate effects of substance on kidneys of rats; dosing 5 days/week instead of 7 days/week; haematological and clinical biochemical test not followed; only liver and kidneys were weighed and processed for histological examinations.
- Principles of method if other than guideline:
- - Principle of test: Subacute toxicity study (1 or 4 weeks). Study designed to evaluate effects of substance on kidneys of rats; dosing 5 days/week instead of 7 days/week; haematological and clinical biochemical test not followed; only liver and kidneys were weighed and processed for histological examinations.
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 5 or 26 days (5 days/week)
- Frequency of treatment:
- 5 days/week
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 75 mg/kg bw/day (nominal)
- Dose / conc.:
- 150 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- Five males
- Control animals:
- yes, concurrent vehicle
- Observations and examinations performed and frequency:
- - All animals were weighed daily and feed consumption was noted weekly.
- On Days 6 and 27, approximately 24 hours after the 5th and 20th doses, respectively, animals from the appropriate groups were weighed, anaesthetized by ethyl ether inhalation, and killed by exsanguinations from the posterior vena cava. The liver and kidneys were removed and weighed. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes.
HISTOPATHOLOGY: Yes
-Transverse sections were fixed and preserved in 10% neutral buffered formalin and stained with haematoxylin/eosin. Additional kidney sections were treated with Mallory’s Heidenhain stain.
-Tissues from right kidney of three control and three d-limonene-treated (150 mg/kg bw/day) animals killed on Day 6 were processed for two-dimensional gel electrophoretic evaluation. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- daily in-life observation did not reveal any grossly evident indication of dose-related toxicity.
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Weight gains values for treatment groups were, at both time points, similar to those of the vehicle control groups.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Feed consumption values for treatment groups were, at both time points, similar to those of the vehicle control groups.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not specified
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Relative liver and kidney weights in 300 mg/kg bw/day group were significantly higher than the control in animals killed on Days 6 and 27.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Examination of animals at necropsy did not reveal any grossly evident indication of dose-related toxicity.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Dose-related hyaline droplet formation associated with renal accumulation of α2μ-globulin was observed in all rats killed on Day 6.
Chronic nephrosis, characterised by granular casts in the outer zone of the medulla and multiple cortical changes, was observed in the kidneys of rats killed on Day 27. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: nephrotoxicity and accumulation of hyaline droplets (male rat specific)
- Remarks on result:
- not determinable
- Remarks:
- no NOAEL identified
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- As nephrotoxicity and accumulation of hyaline droplets containing α2µ-globulin were observed in male rats (mechanism known to be not relevant for humans) at all dose levels, no NOAEL could be identified in this study, but mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
- Executive summary:
In a subacute toxicity study, d-limonene was administered through gavage to groups of 5 male Fisher-344 rats/dose mixed in corn oil at dose levels of 0, 75, 150 and 300 mg/kg bw/day for 1 or 4 weeks (5 days/week). Animals were weighed daily and feed consumption was recorded weekly. On Days 6 and 27, approximately 24 hours after the 5th and 20th doses, respectively, animals from the appropriate groups were subjected to gross necropsy during which weights of liver and kidneys were recorded and transverse sections were processed for histological examination. Tissues from right kidney of animals killed on Day 6 were processed for two-dimensional gel electrophoretic evaluation. Neither daily in-life observation nor examination of animals at necropsy revealed any grossly evident indication of dose-related toxicity. Weight gains and feed consumption values for treatment groups were similar to those of the vehicle control groups. Relative liver and kidney weights in 300 mg/kg bw/day group were significantly higher than the control in animals killed on Days 6 and 27. Dose-related hyaline droplet formation associated with renal accumulation of α2µ-globulin was observed in all rats killed on Day 6. Chronic nephrosis, characterised by granular casts in the outer zone of the medulla and multiple cortical changes, was observed in the kidneys of rats killed on Day 27. As nephrotoxicity and accumulation of hyaline droplets containing α2µ-globulin were observed in male rats at all dose levels, no NOAEL could be identified in this study. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- no data
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- documentation insufficient for assessment
- Remarks:
- Test method and results not sufficiently detailed. Study designed for the assessment of kidney effects of d-limonene
- Principles of method if other than guideline:
- - Principle of test: 91-day subchronic toxicity study: d-limonene (0, 150, 300, 600, 1200 and 2400 mg/kg bw/day) was administered orally (gavage) to rats for 91 days and kidney sections of the surviving male rats were processed for histological examination.
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- not specified
- Sex:
- male/female
- Route of administration:
- oral: gavage
- Vehicle:
- not specified
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 91 days
- Frequency of treatment:
- Once daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 150 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 600 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 200 mg/kg bw/day (nominal)
- Dose / conc.:
- 2 400 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- - At 0 and 1200 mg/kg bw/day: 10 males and 5 females
- At 150-600 mg/kg bw/day: 10 males
- At 2400 mg/kg bw/day: 5 males and 1 female - Control animals:
- yes, concurrent vehicle
- Observations and examinations performed and frequency:
- - Mortality and body-weight data, in-life clinical observations and renal histopathological data were derived from the Prechronic Test Phase Review and Addendum supplied by the NCI.
- Sacrifice and pathology:
- - Histopathology: Kidney sections (5 µm) of male rats were processed, stained with haematoxylin and eosin and scanned for lesion count, hyaline droplets and other more subtle alterations.
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Absolute mean bodyweight gain at 600, 1200 and 2400 mg/kg bw/day decreased by 12, 19 and 46%, respectively, relative to controls.
- Food consumption and compound intake (if feeding study):
- not specified
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not specified
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not specified
- Gross pathological findings:
- not specified
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Dose-related increase in the severity of chronic nephrosis and granular cast formation were observed in male rats at concentrations ranging from 150 to 1200 mg/kg bw/day. At 2400 mg/kg bw/day, the severity of chronic nephrosis was similar to that at 150 mg/kg bw/day and formation of granular cast was observed in 1/10 male.
- Chronic nephrosis was characterised by cytoplasmic basophilia of PCT epithelial cells, tubular hyperplasia or atrophy, fibrosis of Bowman’s capsule and an interstitial fibrolymphocytic response.
- Chronic nephrosis was much more severe in the kidneys of all the male rats treated with d-limonene than in the controls.
- No lesions were observed in kidneys of female rats. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Basis for effect level:
- other: chronic nephrosis and granular cast formation were observed at all dose levels
- Remarks on result:
- not determinable
- Remarks:
- no NOAEL identified
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- As chronic nephrosis and granular casts formation were observed in male rats (mechanism known to be not relevant for humans) at all dose levels, no NOAEL could be identified in this study, but mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
- Executive summary:
In a subchronic toxicity study, d-limonene was administered through gavage to groups of male and female rats at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 91 days. Surviving animals from the appropriate groups were subjected to gross necropsy and transverse sections of right and left kidneys were processed for histological examination. Absolute mean bodyweight gain at 600, 1200 and 2400 mg/kg bw/day decreased by 12, 19 and 46%, respectively, relative to controls. Dose-related increase in the severity of chronic nephrosis and granular cast formation were observed in male rats at concentrations ranging from 150 to 1200 mg/kg bw/day. At 2400 mg/kg bw/day, the severity of chronic nephrosis was similar to that at 150 mg/kg bw/day and formation of granular cast was observed in 1/10 male. Treatment-related lesions were not observed in kidneys of female rats. As chronic nephrosis and granular casts formation were observed in male rats at all dose levels, no NOAEL could be identified in this study. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- September 1979 - October 1979
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- GLP study performed similarly to OECD Guideline 407 but with deviations: study performed only for 16 days; dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- study performed only for 16 days; dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Males: 24.4-26.2 g; females: 19.6-21.9 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 13 days
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-76 °F
- Humidity (%): 80-90%
- 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 oral 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:
- not specified
- Details on analytical verification of doses or concentrations:
- Not applicable
- Duration of treatment / exposure:
- 12 doses over 16 days
- Frequency of treatment:
- Once per day; 5 days/week
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 413 mg/kg bw/day (nominal)
- Dose / conc.:
- 825 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 650 mg/kg bw/day (nominal)
- Dose / conc.:
- 3 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 6 600 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- Five
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - 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: Initially and once per week thereafter - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on six mice from survivors of highest dose groups - Other examinations:
- No
- Statistics:
- No data
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - No compound-related clinical signs were observed in mice that received 1650 mg/kg bw/day and lived to the end of the studies.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- - All but one of 20 mice that received 3300 or 6600 mg/kg bw/day died within 3 days.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- - Vehicle control mice gained little or no weight.
- Slight bodyweight loss was observed in all treated groups, but without any clear dose-response relationship. - Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- - No treatment-related histopathologic lesions were observed.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 650 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: mortality at 3300 and 6600 mg/kg bw/day
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 3 300 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- mortality
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Under the test conditions, the NOAEL in mice was considered to be 1650 mg/kg bw/day, based on mortality rates. The LOAEL for male and female mice were considered to be 3300 mg/kg bw/day, based on increased mortality rates.
- Executive summary:
In a 16-day subacute toxicity study performed similarly to OECD Guideline 407 and in compliance with GLP, d-limonene was administered through gavage to groups of five B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 413, 825, 1650, 3300 and 6600 mg/kg bw/day for 16 days (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and histological examinations were performed on six mice from survivors of highest dose groups. All but one of 20 mice that received 3300 or 6600 mg/kg bw/day died within 3 days. Vehicle control mice gained little or no weight. Slight and not treatment- related bodyweight loss was observed in all treated groups. No compound-related clinical signs were observed in mice that received 1650 mg/kg bw/day and lived to the end of the studies. No treatment-related histopathologic lesions were observed. Under the test conditions, the NOAEL in mice was considered to be 1650 mg/kg bw/day, based on mortality rates. The LOAEL for male and female mice were considered to be 3300 mg/kg bw/day, based on increased mortality rates.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- September 1979 - October 1979
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- GLP study performed similarly to OECD Guideline 407 but with deviations: study performed only for 16 days; dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- study performed only for 16 days; dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: F344/N
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: Males: 109-117 g; females: 97-103 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 12 or 13 days
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-76 °F
- Humidity (%): 80-90%
- 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 oral 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:
- not specified
- Details on analytical verification of doses or concentrations:
- Not applicable
- Duration of treatment / exposure:
- 12 doses over 16 days
- Frequency of treatment:
- Once per day; 5 days/week
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 413 mg/kg bw/day (nominal)
- Dose / conc.:
- 825 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 650 mg/kg bw/day (nominal)
- Dose / conc.:
- 3 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 6 600 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- Five
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - 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: Initially and once per week thereafter - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on seven rats from survivors of highest dose groups - Other examinations:
- No
- Statistics:
- No data
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - No treatment-related clinical signs were observed in rats that received doses of 1650 mg/kg bw/day or lower.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- - All rats in 6600 mg/kg bw/day group and 5/5 males and 3/5 females in 3300 mg/kg bw/day group died within the first 2 days.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- - Final mean body weight of male rats that received 1650 mg/kg bw/day was 10% lower than that of the vehicle controls.
- Final mean body weight of female rats that received 3300 mg/kg bw/day was 8% lower than that of the vehicle controls. - Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 825 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: mortality at 3300 and 6600 mg/kg bw/day; decreased bodyweight gain at 1650 mg/kg bw/day
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 650 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- body weight and weight gain
- mortality
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 650 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: mortalities at 3300 and 6600 mg/kg bw/day; decreased bodyweight gain at 3300 mg/kg bw/day
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 3 300 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- body weight and weight gain
- mortality
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Under the test conditions, the NOAEL for male and female rats were considered to be 825 and 1650 mg/kg bw/day, respectively. The LOAEL for male and female rats were considered to be 1650 and 3300 mg/kg bw/day, respectively, based on decreased bodyweight gains.
- Executive summary:
In a 16-day subacute toxicity study performed similarly to OECD Guideline 407 and in compliance with GLP, d-limonene was administered through gavage to groups of 5 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 413, 825, 1650, 3300 and 6600 mg/kg bw/day for 16 days (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and histological examinations were performed on seven rats from survivors of highest dose groups. All rats that received 6600 mg/kg bw/day and 5/5 males and 3/5 females that received 3300 mg/kg bw/day d-limonene died within the first 2 days. The final mean body weight of male rats that received 1650 mg/kg bw/day was 10% lower than that of the vehicle controls. The final mean body weight of female rats that received 3300 mg/kg bw/day was 8% lower than that of the vehicle controls. No treatment-related clinical signs were observed in rats that received doses of 1650 mg/kg bw/day or lower. No treatment-related lesions were observed. Under the test conditions, the NOAEL for male and female rats were considered to be 825 and 1650 mg/kg bw/day, respectively. The LOAEL for male and female rats were considered to be 1650 and 3300 mg/kg bw/day, respectively, based on decreased bodyweight gains.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- January 1980 - April 1980
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- GLP study performed similarly to OECD Guideline 408 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 7-9 weeks
- Weight at study initiation: Males: 23.8-29.5 g; females: 20.2-21.5 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA) or NIH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 18 days
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 60-82 °F
- Humidity (%): 35-80%
- Air changes (per hour): 12-15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on oral 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: Once
- Results: 101-109% of the target concentrations - Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- Once per day; 5 days/week
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 125 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 250 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 500 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 2 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Doses were selected based on the mortalities observed at 3300 and 6600 mg/kg bw/day during a 16 day subacute toxicity study.
- Rationale for animal assignment (if not random): Random - Positive control:
- No
- Observations and examinations performed and frequency:
- CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: Initially and once per week thereafter - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all vehicle control and high dose animals. Tissues examined include: adrenal glands, brain, colon, esophagus, eyes (if grossly abnormal), femur or sternebrae or vertebrae including marrow, gallbladder, gross lesions and tissue masses with regional lymph nodes, heart, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular or mesenteric lymph nodes, pancreas, parathyroids, pituitary gland, prostate/testes or ovaries/uterus, salivary glands, small intestine, spinal cord (if neurologic signs present), spleen, stomach, thymus, thyroid gland, trachea and urinary bladder. - Other examinations:
- No
- Statistics:
- No data
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - Rough hair coats and decreased activity were observed at 1000 and 2000 mg/kg bw/day.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- - 1/10 male and 2/10 females died at 2000 mg/kg bw/day
- 1/10 female died at 500 mg/kg bw/day
- Several animals in other groups died as a result of gavage error. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- - Final mean bodyweights of mice that received 1000 or 2000 mg/kg bw/day were 10% lower than that of the vehicle controls for males and 2% lower for females.
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not specified
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- - An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg bw/day.
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 500 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: mortality at 2000 mg/kg bw/day; decreased bodyweight gain and occurence of clinical signs of toxicity (rough hair coats and decreased activity) at 1000 and 2000 mg/kg bw/day
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical signs
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Under the test conditions, the NOAEL was considered to be 500 mg/kg bw/day. The LOAEL was considered to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.
- Executive summary:
In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene was administered through gavage to groups of 10 B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 125, 250, 500, 1000 or 2000 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all control and high dose animals scheduled to be killed at the end of the treatment period. One of 10 males and 2/10 females that received 2000 mg/kg bw/day and 1/10 females that received 500 mg/kg bw/day died before the end of the studies. Several animals in other groups died as a result of gavage error. Clinical signs of rough hair coats and decreased activity were observed at the two highest doses. Final mean bodyweights of mice that received 1000 or 2000 mg/kg bw/day were 10% lower than that of the vehicle controls for males and 2% lower for females. An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg bw/day. Under the test conditions, the NOAEL was considered to be 500 mg/kg bw/day. The LOAEL was considered to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- January 1980 - April 1980
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- GLP study performed similarly to OECD Guideline 408 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: F344/N
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: 136-153 g; females: 101-120 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA) or NIH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 18 or 19 days
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 60-82 °F
- Humidity (%): 35-80%
- Air changes (per hour): 12-15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on oral 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: Once
- Results: 101-109% of the target concentrations - Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- Once per day; 5 days/week
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 600 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 200 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 2 400 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Doses were selected based on the mortalities observed at 3300 and 6600 mg/kg bw/day during a 16 day subacute toxicity study.
- Rationale for animal assignment (if not random): Random - Positive control:
- No
- Observations and examinations performed and frequency:
- CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: Initially and once per week thereafter - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all vehicle control and high dose animals and all female rats in the 1200 mg/kg bw/day group. Tissues examined include: adrenal glands, brain, colon, esophagus, eyes (if grossly abnormal), femur or sternebrae or vertebrae including marrow, gross lesions and tissue masses with regional lymph nodes, heart, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular or mesenteric lymph nodes, pancreas, parathyroids, pituitary gland, prostate/testes or ovaries/uterus, salivary glands, small intestine, spinal cord (if neurologic signs present), spleen, stomach, thymus, thyroid gland, trachea, and urinary bladder. Kidneys examined for all male rats. - Other examinations:
- No
- Statistics:
- No data
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - Rough hair coats, lethargy and excessive lacrimation were observed for rats that received 1200 or 2400 mg/kg bw/day.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- - 5/10 males and 9/10 females at 2400 mg/kg bw/day died during week 1.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- - Final mean body weights of male rats in 600, 1200 or 2400 mg/kg bw/day groups were 6, 12 or 23% lower than that of the vehicle controls.
- Final body weight of the female rat that received 2400 mg/kg bw/day and lived to end of the study was 11% lower than the mean of the vehicle controls. - Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not specified
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- - Nephropathy was identified in all groups of male rats, and there was a dose-related increased severity of the lesion in dosed groups.
- Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium.
- Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOAEL
- Sex:
- male
- Basis for effect level:
- other: male-rat specific nephrotoxicity at all dose levels (considered as not relevant for humans)
- Remarks on result:
- other: no NOAEL identified
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 600 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day;
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 200 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- body weight and weight gain
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 600 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: mortality at 2400 mg/kg bw/day; occurrence of clinical signs of toxicity (rough hair coats, lethargy and excessive lacrimation) at 1200 and 2400 mg/kg bw/day
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 200 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- clinical signs
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Under the test conditions, the NOAEL for male and female rats was considered to be 600 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats could be identified in this study.
- Executive summary:
In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene was administered through gavage to groups of 10 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all control and high dose animals scheduled to be killed at the end of the treatment period. Five of 10 males and 9/10 female rats that received 2400 mg/kg bw/day died during week 1. Final mean body weights of male rats in 600, 1200 or 2400 mg/kg bw/day groups were 6, 12 or 23% lower than that of the vehicle controls. Final body weight of the female rat that received 2400 mg/kg bw/day and lived to end of the study was 11% lower than the mean of the vehicle controls. Rough hair coats, lethargy and excessive lacrimation were observed at 1200 or 2400 mg/kg bw/day. No treatment-related histopathologic lesions were observed in female rats. Nephropathy was identified in all groups of male rats, and there was a dose-related increased severity of the lesion in dosed groups. Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium. Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. This mechanism of nephrocarcinogenicity has been proven as being male-rat specific and not relevant for humans.
Under the test conditions, the NOAEL for female rats was considered to be 600 mg/kg bw/day. When considering the non relevance of the nephrotoxic effects for humans, the NOAEL for male rats would be 600 mg/kg bw/day, based on decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats had primarily been identified in this study.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- no data
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- documentation insufficient for assessment
- Remarks:
- Well-conducted study but reported in Japanese language
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- - Principle of test:
A combination of rules from OECD Guidelines 409 and 452 were followed (by anticipation as thoses guidelines did not exist when the study was conducted).
- Short description of test conditions: d-limonene was administered by gavage to dogs for 6 months. This duration was chosen as usual when developing new medicinal products (d-limonene was explored in this study as a possible gallstone solubiliser). - GLP compliance:
- no
- Limit test:
- no
- Species:
- dog
- Strain:
- other: Japanese beagle
- Sex:
- male/female
- Route of administration:
- oral: gavage
- Vehicle:
- not specified
- Details on oral exposure:
- no data
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 6 months
- Frequency of treatment:
- once a day
- Dose / conc.:
- 0.4 other: mL/kg-bw/day
- Remarks:
- Equivalent to 340 mg/kg-bw/day
- Dose / conc.:
- 1.2 other: mL/kg-bw/day
- Remarks:
- Equivalent to 1000 mg/kg-bw/day
- Dose / conc.:
- 3.6 other: mL/kg-bw/day
- Remarks:
- Equivalent to 3000 mg/kg-bw/day
- No. of animals per sex per dose:
- 3/sex/dose
- Control animals:
- yes
- Observations and examinations performed and frequency:
- DETAILED CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes
FOOD CONSUMPTION AND COMPOUND INTAKE: yes
OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:
HAEMATOLOGY: Yes
CLINICAL CHEMISTRY: Yes
URINALYSIS: Yes
- Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Frequent vomiting and nausea were caused, which appeared to depend on the dose used.
- Mortality:
- not specified
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- All males in the high dose group, all females in the intermediate group and 2/3 females in the high dose group lost weight over the 6 month-study. Not dose-related in females.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Not affected by treatment except in the intermediate dose group females. Not dose-related.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Total cholesterol and glucose decrease in blood in the high dose group. The initial cholesterol level was recovered at the end of the 6-month treatment period in both sexes (when the level had increased in other control and treated groups).
- Urinalysis findings:
- effects observed, non-treatment-related
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- The relative to body weight kidney and liver weights were slightly higher in the high dose group males than in other groups.
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- At 1.2 and 3.6 mL/kg bw/day, protein casts were observed in the renal tubule of most animals. No remarkable treatment-related change was observed in other organs.
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Decreased body weight and protein casts observed in the renal tubule at 3000 mg/lg bw/d.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 3 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Decreased body weight and protein casts observed in the renal tubule at 3000 mg/lg bw/d.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 340 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: Decreased body weight and protein casts observed in the renal tubule at 1000 mg/lg bw/d.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: Decreased body weight and protein casts observed in the renal tubule at 1000 mg/lg bw/d.
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- The NOAEL in this study is 1.2 mL/kg bw/day (equivalent to 1000 mg/kg bw/day) in males and 0.4 mL/kg bw/day (equivalent to 340 mg/kg bw/day) in females on the basis of decreased body weight and protein casts observed in the renal tubule at the next dose level. Food consumption was also decreased in the intermediate dose females.
- Executive summary:
Three dogs per sex and per dose group were administered d-limonene by gavage once per day for 6 months at the dose level of 0, 0.4, 1.2 or 3.6 mL/kg bw/day. All 6 animals (males and females) from the high dose group except one female and all females in the intermediate dose group lost weight between the first and the last day of study. Food consumption only decreased in the intermediate dose group females. Urinalysis and hematology were not affected by treatment. The glucose and total cholesterol levels in blood decreased in the high dose group males and females when compared to the pre-treatment levels; the total cholesterol level recovered the pre-test level by the end of the 6 -month treatment period. The relative to body weight kidney and liver weights were slightly higher in the high dose group males than in other groups. A dose-related increased incidence of protein casts were observed in the renal tubule: all males in the high dose group and all females in the intermediate and high dose groups showed this effect.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1989
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Remarks:
- Study designed to evaluate effects of substance on kidneys of rats: dosing 5 days/week instead of 7 days/week; haematological and clinical biochemical test not followed
- Principles of method if other than guideline:
- - Principle of test: Study designed to evaluate effects of substance on kidneys of rats: dosing 5 days/week instead of 7 days/week; haematological and clinical biochemical test not followed
- GLP compliance:
- not specified
- Limit test:
- no
- Specific details on test material used for the study:
- Additional information: D-limonene used for preliminary acute study:
Radiolabelled d-limonene [9-14C]
- Source: Wizard Laboratories (Davies, CA, USA)
- Radiochemical purity (if radiolabelling): 99% (GC)
- Specific activity (if radiolabelling): 8.7 mCi/mmol - Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- 5 days/week
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 2 mg/kg bw/day (nominal)
- Dose / conc.:
- 5 mg/kg bw/day (nominal)
- Dose / conc.:
- 10 mg/kg bw/day (nominal)
- Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Dose / conc.:
- 75 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- - At 0, 10 and 75 mg/kg bw/day: 5 or 10 males
- At 2, 5 and 30 mg/kg bw/day: 10 males - Control animals:
- yes, concurrent vehicle
- Observations and examinations performed and frequency:
- Preliminary acute toxicity study:
- D-limonene (200 mg/kg bw; 200 µCi/kg bw in corn oil) was administered to a group of male and female Fischer 344 rats by oral gavage. After 24 hours, all animals were sacrificed and kidney tissues were processed for histological examinations and 2D-gel electrophoresis.
Subchronic toxicity study:
- Rats were observed daily during the experimental period for signs of toxicity.
- Body weights were recorded before each daily administration of d-limonene and at the time of sacrifice.
- Feed consumption values were recorded weekly throughout the study and were used to determine weekly body-weight gain and feed efficiency values.
- Interim necropsies were conducted on 5 animals/group on Days 8 and 15 (group 4), and Days 8, 15, 22 and 29 (groups 1 and 6). All remaining rats (10/group) were sacrificed at the end of the 91-day study. Liver and kidneys were isolated and weighed. All tissues were collected and fixed in 10% neutral-buffered formalin for routine histological processing and light microscopic evaluation of sections stained with H&E. An additional kidney section was also stained with Mallory's Heidenhain stain and examined for hyaline droplets under light microscopy. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes - Clinical signs:
- not specified
- Mortality:
- not specified
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- The cumulative body-weight gain for treated males did not differ significantly from those of the control males.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Feed consumption for treated males did not differ significantly from those of the control males.
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- Feed efficiency for treated males did not differ significantly from those of the control males.
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not specified
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Linear regression analyses indicated a dose-related trend in the increased relative weights of the kidney and liver at 30 and 75 mg/kg bw/day.
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Incidence and type of gross pathological lesions observed at necropsy for treated males did not differ significantly from those of the control males.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- At the earliest necropsy, 8 days after the start of the treatment, it was evident that d-limonene exacerbated the hyaline droplets at 10 mg/kg bw/day.
Histological examination of kidney tissue confirmed that d-limonene induced changes characterized by hyaline droplets, granular casts at the corticomedullary junction and multiple cortical changes collectively classified as chronic nephrosis. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 5 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: chronic nephrosis and a dose-related trend in the increased relative weights of the kidney and liver were observed at 30 and 75 mg/kg bw/day
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 30 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: chronic nephrosis and a dose-related trend in the increased relative weights of the kidney and liver were observed at 30 and 75 mg/kg bw/day
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Under the test conditions, the NOAEL and LOAEL were considered to be 5 and 30 mg/kg bw/day, respectively, based on observation of chronic nephrosis. Mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
- Executive summary:
In a subchronic toxicity study, d-limonene was administered through gavage to groups of 5 or 10 male Fisher-344 rats/dose mixed in corn oil at dose levels of 0, 2, 5, 10, 30 and 75 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed and weighed daily, and feed consumption was recorded weekly. Rats from selected dose groups received interim necropsies from Days 8-29, while all groups were necropsied at the end of the study. In the preliminary acute toxicity study, d-limonene (200 mg/kg bw; 200 µCi/kg bw in corn oil) was administered to a group of male and female Fischer 344 rats by oral gavage. After 24 hours, an increase in the incidence and severity of hyaline droplets containing alpha-2µ-globulin was observed in the kidneys of males only. In the main study, incidence and type of gross pathological lesions observed at necropsy, the cumulative body-weight gain, feed consumption and feed efficiency for treated males did not differ significantly from those of the control males. Linear regression analyses indicated a dose-related trend in the increased relative weights of the kidney and liver at 30 and 75 mg/kg bw/day. Histological examination of kidney tissue confirmed induction of chronic nephrosis characterized by hyaline droplets, granular casts at the corticomedullary junction and multiple cortical changes. At the earliest necropsy, 8 days after the start of the treatment, it was evident that d-limonene exacerbated the hyaline droplets at the 10 mg/kg body weight dose.
Under the test conditions, the NOAEL and LOAEL were considered to be 5 and 30 mg/kg bw/day, respectively, but mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1990
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Study conducted similarly to OECD Guideline 409 with deviations: age of animals > 9 months; no data on initial bodyweights; only two dose levels studied; ophthalmological examination not followed; individual animal data not reported
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
- Deviations:
- yes
- Remarks:
- age of animals > 9 months; no data on initial bodyweights; only two dose levels studied; ophthalmological examination not followed; individual animal data not reported
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- dog
- Strain:
- Beagle
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 10-15 months
- Housing: Housed in runs with outdoor access
- Diet (e.g. ad libitum): Meals provided for only 1 hour
- Water (e.g. ad libitum): Ad libitum - Route of administration:
- oral: gavage
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- - PREPARATION OF DOSING SOLUTIONS:
The test item was directly dosed by gavage in doses of 0.12 and 1.2 ml/kg body weight/day. - Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- Not applicable
- Duration of treatment / exposure:
- 180 days
- Frequency of treatment:
- Each daily dose was divided into two equal amounts, administered at approximately 9.30 am and 2.30 pm.
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- (1.2 ml tap-water/kg bw/day)
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- (0.12 ml test item/kg bw/day)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- (1.2 ml test item/kg bw/day)
- No. of animals per sex per dose:
- Five
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- - Dose selection rationale: Highest daily dose was determined in a pilot 28-day study conducted in 5 male and 5 female beagles. Kidney weights for the d-limonene-treated animals were unaffected, but absolute and relative liver weights were slightly increased. Based on these findings, the 1.2 mL/kg bw/day treatment was chosen.
- Positive control:
- No
- Observations and examinations performed and frequency:
- CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily for at least 1 hour after dosing
BODY WEIGHT: Yes
- Time schedule for examinations: At study initiation, weekly during the study and at the time of sacrifice
FOOD CONSUMPTION: Yes
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 2 week pre-study (baseline) and then 1, 3 and 6 months during the study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- Parameters examined: White cell count, red blood cell count, haemoglobin, haematocrit, platelet count, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At 2 week pre-study (baseline) and then 1, 3 and 6 months during the study
- Animals fasted: No data
- Parameters examined: Blood urea nitrogen (BUN), BUN/creatinine, creatinine, aspartate amino transferase, alanine amino transferase, phosphorus, glucose, albumin, total protein, globulins, albumin/globulin, alkaline phosphatase, cholesterol, triglycerides, sodium, potassium, calcium and chloride
URINALYSIS: Yes
- Time schedule for collection of urine: 24-hour urine samples were collected at approximately 2 week pre-study and again at 6 months.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: Colour and appearance, specific gravity, occult blood, protein, pH, glucose, ketones, bilirubin and urobilinogen and urinary sediment - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; each animal anaesthetized with pentobarbital, killed by exsanguination and observed for gross post-mortem examinations
HISTOPATHOLOGY: Yes; samples of the following tissues were collected and fixed in 10% buffered formalin for routine histological processing, and light microscopical evaluation of haematoxylin and eosin stained sections: lungs, bronchial lymph node, heart, thoracic aorta, tongue, oesophagus, trachea, thyroid, parathyroid, submandibular lymph node, mesenteric lymph node, stomach, parotid salivary gland, palatine tonsil, liver, gall bladder, duodenum, jejunum, ileum, colon, rectum, urinary bladder, kidneys, testicles with epididymis, prostate, ovaries, uterus, vagina, cervix, adrenals, thymus, psoas muscle, spleen, pancreas, bone/marrow, skin, brain, spinal cord, sciatic nerve, pituitary gland, and eyes. Mallory-Heidenhain-stained kidney sections were also prepared and evaluated for protein accumulation. Kidney weights were determined immediately on removal from the animal. Absolute organ weights were calculated as percentages of the body weights (relative weights). - Other examinations:
- None
- Statistics:
- - Statistically significant differences (P < 0.05, two-sided risk level) were established using least significant difference criteria, provided that Bartlett's test of homogeneity of variance was nonsignificant.
- Dose-response data were also analysed by linear regression (Dixon and Massey, 1969). - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Excretion of soft faeces; dose-related occasional mild discomfort during defaecation (presumably because of perianal contact with unabsorbed d-limonene as it passed with the faeces); sporadic episodes of emesis and diarrhoea
- Mortality:
- mortality observed, treatment-related
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- No treatment-related differences other than an increase in serum cholesterol (35%) and serum alkaline phosphatase levels (two-fold increase) at 1000 mg/kg bw/day were observed in male and female dogs.
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Linear regression analyses indicated a positive dose-related trend for absolute and relative female kidney weight and relative male kidney weight.
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 100 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: increased absolute and relative female kidney weight and relative male kidney weight at 1000 mg/kg bw/day
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- organ weights and organ / body weight ratios
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Under the test conditions, the NOAEL and LOAEL for beagle dogs were considered to be 100 and 1000 mg/kg bw/day, respectively, based on the increased absolute and relative female kidney weight and relative male kidney weight.
- Executive summary:
In a 6-month subchronic toxicity study performed similarly to OECD Guideline 409, d-limonene was administered through gavage to groups of adult beagle dogs (5/sex/dose) at dose levels of 0 (tap water), 0.12 or 1.2 mL/kg bw/day (0, 100 or 1000 mg/kg bw/day) in two divided doses for 180 days. Animals were observed daily and weighed at study initiation, weekly during the study and at the time of sacrifice. Feed consumptions were determined throughout the study and blood samples were obtained at 2 week pre-study (baseline) and then 1, 3 and 6 months during the study. At termination all animals were subjected to gross necropsy during which weights of kidneys were recorded and several tissues were processed for microscopical evaluation of haematoxylin and eosin stained sections. Mallory-Heidenhain-stained kidney sections were also prepared and evaluated for protein accumulation.
Feed consumption and body weight were unaffected by treatment. Clinical signs of toxicity noted were excretion of soft faeces, dose-related occasional mild discomfort during defaecation, sporadic episodes of emesis and diarrhoea. No treatment-related differences other than an increase in serum cholesterol (35%) and serum alkaline phosphatase levels (two-fold increase) at 1000 mg/kg bw/day were observed in male and female dogs. Linear regression analyses indicated a positive dose-related trend for absolute and relative female kidney weight and relative male kidney weight. There were no histopathological changes in the kidneys, evaluated by both haematoxylin and eosin and Mallory-Heidenhain staining that could be associated with the organ-weight changes. Furthermore, there was no evidence of hyaline droplet accumulation or any other sign of hydrocarbon-induced nephropathy typical of those seen in male rats treated with d-limonene.
Under the test conditions, the NOAEL and LOAEL for beagle dogs were considered to be 100 and 1000 mg/kg bw/day, respectively, based on the increased absolute and relative female kidney weight and relative male kidney weight.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Dose descriptor:
- NOAEL
- Based on:
- other: read across from an analogue
- Sex:
- male
- Basis for effect level:
- other: nephrotoxicity and accumulation of hyaline droplets (male rat specific)
- Remarks on result:
- not determinable
- Remarks:
- read-across from an analogue for which no NOAEL was identified.
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- As nephrotoxicity and accumulation of hyaline droplets containing α2µ-globulin were observed in male rats (mechanism known to be not relevant for humans) at all dose levels, no NOAEL could be identified in this study. Based on read across from the analogue d-limonene, a LOAEL of 75 mg/kg bw/day could be determined for alpha terpinene in a short term oral toxicity study with rats.
- Executive summary:
In a subacute toxicity study, d-limonene was administered through gavage to groups of 5 male Fisher-344 rats/dose mixed in corn oil at dose levels of 0, 75, 150 and 300 mg/kg bw/day for 1 or 4 weeks (5 days/week). Animals were weighed daily and feed consumption was recorded weekly. On Days 6 and 27, approximately 24 hours after the 5th and 20th doses, respectively, animals from the appropriate groups were subjected to gross necropsy during which weights of liver and kidneys were recorded and transverse sections were processed for histological examination. Tissues from right kidney of animals killed on Day 6 were processed for two-dimensional gel electrophoretic evaluation. Neither daily in-life observation nor examination of animals at necropsy revealed any grossly evident indication of dose-related toxicity. Weight gains and feed consumption values for treatment groups were similar to those of the vehicle control groups. Relative liver and kidney weights in 300 mg/kg bw/day group were significantly higher than the control in animals killed on Days 6 and 27. Dose-related hyaline droplet formation associated with renal accumulation of α2µ-globulin was observed in all rats killed on Day 6. Chronic nephrosis, characterised by granular casts in the outer zone of the medulla and multiple cortical changes, was observed in the kidneys of rats killed on Day 27. As nephrotoxicity and accumulation of hyaline droplets containing α2µ-globulin were observed in male rats at all dose levels, no NOAEL could be identified in this study. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known. Based on these results, the read-across approach was applied and the LOAEL for alpha terpinene could be determined to be 75 mg/kg bw/day in a short term oral toxicity study with rats.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Dose descriptor:
- NOAEL
- Basis for effect level:
- other: read-across from an analogue for which chronic nephrosis and granular cast formation were observed at all dose levels.
- Remarks on result:
- not determinable
- Remarks:
- read-across from an analogue for which no NOAEL was identified.
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Based on read across from the analogue d-limonene, no NOAEL for alpha terpinene can be determined based on chronic nephrosis and granular casts formation observed in male rats (mechanism known to be not relevant for humans) at all dose levels. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
- Executive summary:
In a subchronic toxicity study, d-limonene was administered through gavage to groups of male and female rats at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 91 days. Surviving animals from the appropriate groups were subjected to gross necropsy and transverse sections of right and left kidneys were processed for histological examination. Absolute mean bodyweight gain at 600, 1200 and 2400 mg/kg bw/day decreased by 12, 19 and 46%, respectively, relative to controls. Dose-related increase in the severity of chronic nephrosis and granular cast formation were observed in male rats at concentrations ranging from 150 to 1200 mg/kg bw/day. At 2400 mg/kg bw/day, the severity of chronic nephrosis was similar to that at 150 mg/kg bw/day and formation of granular cast was observed in 1/10 male. Treatment-related lesions were not observed in kidneys of female rats. Based on these results, the read-across approach was applied and no NOAEL for alpha terpinene can be determined based on chronic nephrosis and granular casts formation observed in male rats (mechanism known to be not relevant for humans) at all dose levels. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 650 mg/kg bw/day (actual dose received)
- Based on:
- other: Read across from an analogue
- Sex:
- male/female
- Basis for effect level:
- other: read-across from an analogue for which mortality at 3300 and 6600 mg/kg bw/day
- Remarks on result:
- other: read-across from an analogue for which NOAEL = 1650 mg/kg bw/day.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 3 300 mg/kg bw/day (actual dose received)
- Based on:
- other: Read across from an analogue
- Sex:
- male/female
- Basis for effect level:
- mortality
- Remarks on result:
- other: read-across from an analogue for which LOAEL = 3300 mg/kg bw/day.
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Based on read across from the analogue d-limonene, the NOAEL for alpha terpinene in mice was considered to be 1650 mg/kg bw/day, based on mortality rates. The LOAEL for male and female mice were considered to be 3300 mg/kg bw/day, based on increased mortality rates.
- Executive summary:
In a 16-day subacute toxicity study performed similarly to OECD Guideline 407 and in compliance with GLP, d-limonene was administered through gavage to groups of five B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 413, 825, 1650, 3300 and 6600 mg/kg bw/day for 16 days (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and histological examinations were performed on six mice from survivors of highest dose groups. All but one of 20 mice that received 3300 or 6600 mg/kg bw/day died within 3 days. Vehicle control mice gained little or no weight. Slight and not treatment- related bodyweight loss was observed in all treated groups. No compound-related clinical signs were observed in mice that received 1650 mg/kg bw/day and lived to the end of the studies. No treatment-related histopathologic lesions were observed. Based on these results, the read-across approach was applied and the NOAEL for alpha terpinene in mice was considered to be 1650 mg/kg bw/day, based on mortality rates. The LOAEL for male and female mice were considered to be 3300 mg/kg bw/day, based on increased mortality rates.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Details on analytical verification of doses or concentrations:
- Not applicable
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 825 mg/kg bw/day (actual dose received)
- Based on:
- other: Read across from an analogue
- Sex:
- male
- Basis for effect level:
- other: read-across from an analogue for which mortality at 3300 and 6600 mg/kg bw/day; decreased bodyweight gain at 1650 mg/kg bw/day
- Remarks on result:
- other: read-across from an analogue for which NOAEL= 825 mg/kg bw/day.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 650 mg/kg bw/day (actual dose received)
- Based on:
- other: Read across from an analogue
- Sex:
- male
- Basis for effect level:
- body weight and weight gain
- mortality
- Remarks on result:
- other: read-across from an analogue for which LOAEL= 1650 mg/kg bw/day.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 650 mg/kg bw/day (actual dose received)
- Based on:
- other: Read across from an analogue
- Sex:
- female
- Basis for effect level:
- other: read-across from an analogue for which mortalities at 3300 and 6600 mg/kg bw/day; decreased bodyweight gain at 3300 mg/kg bw/day
- Remarks on result:
- other: read-across from an analogue for which NOAEL=1650 mg/kg bw/day.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 3 300 mg/kg bw/day (actual dose received)
- Based on:
- other: Read across from an analogue
- Sex:
- female
- Basis for effect level:
- body weight and weight gain
- mortality
- Remarks on result:
- other: read-across from an analogue for which LOAEL= 3300 mg/kg bw/day.
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Based on read across from the analogue d-limonene, the NOAEL for alpha terpinene for male and female rats were considered to be 825 and 1650 mg/kg bw/day, respectively. The LOAEL for male and female rats were considered to be 1650 and 3300 mg/kg bw/day, respectively, based on decreased bodyweight gains.
- Executive summary:
In a 16-day subacute toxicity study performed similarly to OECD Guideline 407 and in compliance with GLP, d-limonene was administered through gavage to groups of 5 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 413, 825, 1650, 3300 and 6600 mg/kg bw/day for 16 days (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and histological examinations were performed on seven rats from survivors of highest dose groups. All rats that received 6600 mg/kg bw/day and 5/5 males and 3/5 females that received 3300 mg/kg bw/day d-limonene died within the first 2 days. The final mean body weight of male rats that received 1650 mg/kg bw/day was 10% lower than that of the vehicle controls. The final mean body weight of female rats that received 3300 mg/kg bw/day was 8% lower than that of the vehicle controls. No treatment-related clinical signs were observed in rats that received doses of 1650 mg/kg bw/day or lower. No treatment-related lesions were observed. Based on these results, the read-across approach was applied and the NOAEL for alpha terpinene for male and female rats were considered to be 825 and 1650 mg/kg bw/day, respectively. The LOAEL for male and female rats were considered to be 1650 and 3300 mg/kg bw/day, respectively, based on decreased bodyweight gains.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 500 mg/kg bw/day (actual dose received)
- Based on:
- other: Read across from an analogue
- Sex:
- male/female
- Basis for effect level:
- other: read-across from an analogue for which mortality at 2000 mg/kg bw/day; decreased bodyweight gain and occurence of clinical signs of toxicity (rough hair coats and decreased activity) at 1000 and 2000 mg/kg bw/day.
- Remarks on result:
- other: read-across from an analogue for which NOAEL = 500 mg/kg bw/day
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- other: Read across from an analogue
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical signs
- Remarks on result:
- other: read-across from an analogue for which LOAEL = 1000 mg/kg bw/day.
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Based on read across from the analogue d-limonene, the NOAEL for alpha terpinene was considered to be 500 mg/kg bw/day. The LOAEL was considered to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.
- Executive summary:
In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene was administered through gavage to groups of 10 B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 125, 250, 500, 1000 or 2000 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all control and high dose animals scheduled to be killed at the end of the treatment period. One of 10 males and 2/10 females that received 2000 mg/kg bw/day and 1/10 females that received 500 mg/kg bw/day died before the end of the studies. Several animals in other groups died as a result of gavage error. Clinical signs of rough hair coats and decreased activity were observed at the two highest doses. Final mean bodyweights of mice that received 1000 or 2000 mg/kg bw/day were 10% lower than that of the vehicle controls for males and 2% lower for females. An alveolar cell adenoma was observed in the lung of 1/10 females that received 2000 mg/kg bw/day.
Based on these results, the read-across approach was applied and the NOAEL for alpha terpinene was considered to be 500 mg/kg bw/day. The LOAEL was considered to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Dose descriptor:
- NOAEL
- Sex:
- male
- Basis for effect level:
- other: male-rat specific nephrotoxicity at all dose levels (considered as not relevant for humans)
- Remarks on result:
- other: read-across from an analogue for which no NOAEL was identified.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 600 mg/kg bw/day (actual dose received)
- Based on:
- other: Read across from an analogue
- Sex:
- male
- Basis for effect level:
- other: read-across from an analogue for which decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day;
- Remarks on result:
- other: read-across from an analogue for which NOAEL = 600 mg/kg bw/day.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 200 mg/kg bw/day (actual dose received)
- Based on:
- other: Read across from an analogue
- Sex:
- male
- Basis for effect level:
- body weight and weight gain
- Remarks on result:
- other: read-across from an analogue for which LOAEL = 1200 mg/kg bw/day.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 600 mg/kg bw/day (actual dose received)
- Based on:
- other: Read across from an analogue
- Sex:
- female
- Basis for effect level:
- other: read-across from an analogue for which mortality at 2400 mg/kg bw/day; occurrence of clinical signs of toxicity (rough hair coats, lethargy and excessive lacrimation) at 1200 and 2400 mg/kg bw/day.
- Remarks on result:
- other: read-across from an analogue for which NOAEL = 600 mg/kg bw/day.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 200 mg/kg bw/day (actual dose received)
- Based on:
- other: Read across from an analogue
- Sex:
- female
- Basis for effect level:
- clinical signs
- Remarks on result:
- other: read-across from an analogue for which LOAEL = 1200 mg/kg bw/day.
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Based on read across from the analogue d-limonene, the NOAEL for alpha terpinene was considered to be 600 mg/kg bw/day for male and female rats. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats could be identified in this study.
- Executive summary:
In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene was administered through gavage to groups of 10 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all control and high dose animals scheduled to be killed at the end of the treatment period. Five of 10 males and 9/10 female rats that received 2400 mg/kg bw/day died during week 1. Final mean body weights of male rats in 600, 1200 or 2400 mg/kg bw/day groups were 6, 12 or 23% lower than that of the vehicle controls. Final body weight of the female rat that received 2400 mg/kg bw/day and lived to end of the study was 11% lower than the mean of the vehicle controls. Rough hair coats, lethargy and excessive lacrimation were observed at 1200 or 2400 mg/kg bw/day. No treatment-related histopathologic lesions were observed in female rats. Nephropathy was identified in all groups of male rats, and there was a dose-related increased severity of the lesion in dosed groups. Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium. Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. This mechanism of nephrocarcinogenicity has been proven as being male-rat specific and not relevant for humans.
Under the test conditions, the NOAEL for female rats was considered to be 600 mg/kg bw/day. When considering the non relevance of the nephrotoxic effects for humans, the NOAEL for male rats would be 600 mg/kg bw/day, based on decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats had primarily been identified in this study.
Based on these results, the read-across approach was applied and the NOAEL for alpha terpinene was considered to be 600 mg/kg bw/day for male and female rats.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- other: Read across from an analogue
- Sex:
- male
- Basis for effect level:
- other: read-across from an analogue for which decreased body weight and protein casts observed in the renal tubule at 3000 mg/lg bw/d.
- Remarks on result:
- other: read-across from an analogue for which NOAEL=1000 mg/kg bw/day.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 3 000 mg/kg bw/day (nominal)
- Based on:
- other: Read across from an analogue
- Sex:
- male
- Basis for effect level:
- other: read-across from an analogue for which decreased body weight and protein casts observed in the renal tubule at 3000 mg/lg bw/d.
- Remarks on result:
- other: read-across from an analogue for which LOAEL=3000 mg/kg bw/day.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 340 mg/kg bw/day (nominal)
- Based on:
- other: Read across from an analogue
- Sex:
- female
- Basis for effect level:
- other: read-across from an analogue for which decreased body weight and protein casts observed in the renal tubule at 1000 mg/lg bw/d.
- Remarks on result:
- other: read-across from an analogue for which NOAEL=340 mg/kg bw/day.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 000 mg/kg bw/day (nominal)
- Based on:
- other: Read across from an analogue
- Sex:
- female
- Basis for effect level:
- other: read-across from an analogue for which decreased body weight and protein casts observed in the renal tubule at 1000 mg/lg bw/d.
- Remarks on result:
- other: read-across from an analogue for which LOAEL=1000 mg/kg bw/day.
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Based on read across from the analogue d-limonene, the NOAEL for alpha terpinene was considered to be 1000 mg/kg bw/day in males and 340 mg/kg bw/day in females on the basis of decreased body weight and protein casts observed in the renal tubule at the next dose level.
- Executive summary:
Three dogs per sex and per dose group were administered d-limonene by gavage once per day for 6 months at the dose level of 0, 0.4, 1.2 or 3.6 mL/kg bw/day. All 6 animals (males and females) from the high dose group except one female and all females in the intermediate dose group lost weight between the first and the last day of study. Food consumption only decreased in the intermediate dose group females. Urinalysis and hematology were not affected by treatment. The glucose and total cholesterol levels in blood decreased in the high dose group males and females when compared to the pre-treatment levels; the total cholesterol level recovered the pre-test level by the end of the 6 -month treatment period. The relative to body weight kidney and liver weights were slightly higher in the high dose group males than in other groups. A dose-related increased incidence of protein casts were observed in the renal tubule: all males in the high dose group and all females in the intermediate and high dose groups showed this effect. Based on these results, the read-across approach was applied and the NOAEL for alpha terpinene was considered to be 1000 mg/kg bw/day in males and 340 mg/kg bw/day in females.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 5 mg/kg bw/day (nominal)
- Based on:
- other: Read across from an analogue
- Sex:
- male
- Basis for effect level:
- other: read-across from an analogue for which chronic nephrosis and a dose-related trend in the increased relative weights of the kidney and liver were observed at 30 and 75 mg/kg bw/day.
- Remarks on result:
- other: read-across from an analogue for which NOAEL = 5 mg/kg bw/day.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 30 mg/kg bw/day (nominal)
- Based on:
- other: Read across from an analogue
- Sex:
- male
- Basis for effect level:
- other: read-across from an analogue for which chronic nephrosis and a dose-related trend in the increased relative weights of the kidney and liver were observed at 30 and 75 mg/kg bw/day.
- Remarks on result:
- other: read-across from an analogue for which LOAEL = 30 mg/kg bw/day.
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Based on read across from the analogue d-limonene, the NOAEL and LOAEL for alpha terpinene were considered to be 5 and 30 mg/kg bw/day, respectively, based on observation of chronic nephrosis. Mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
- Executive summary:
In a subchronic toxicity study, d-limonene was administered through gavage to groups of 5 or 10 male Fisher-344 rats/dose mixed in corn oil at dose levels of 0, 2, 5, 10, 30 and 75 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed and weighed daily, and feed consumption was recorded weekly. Rats from selected dose groups received interim necropsies from Days 8-29, while all groups were necropsied at the end of the study. In the preliminary acute toxicity study, d-limonene (200 mg/kg bw; 200 µCi/kg bw in corn oil) was administered to a group of male and female Fischer 344 rats by oral gavage. After 24 hours, an increase in the incidence and severity of hyaline droplets containing alpha-2µ-globulin was observed in the kidneys of males only. In the main study, incidence and type of gross pathological lesions observed at necropsy, the cumulative body-weight gain, feed consumption and feed efficiency for treated males did not differ significantly from those of the control males. Linear regression analyses indicated a dose-related trend in the increased relative weights of the kidney and liver at 30 and 75 mg/kg bw/day. Histological examination of kidney tissue confirmed induction of chronic nephrosis characterized by hyaline droplets, granular casts at the corticomedullary junction and multiple cortical changes. At the earliest necropsy, 8 days after the start of the treatment, it was evident that d-limonene exacerbated the hyaline droplets at the 10 mg/kg body weight dose.
Based on these results, the read-across approach was applied and the NOAEL and LOAEL for alpha terpinene were considered to be 5 and 30 mg/kg bw/day, respectively, but mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance (R)-p-mentha-1,8-diene (d-limonene) which shares the same functional groups with the substance p-mentha-1,3-diene (alpha terpinene) also has comparable values for the relevant molecular properties.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 100 mg/kg bw/day (actual dose received)
- Based on:
- other: read across from an analogue
- Sex:
- male/female
- Basis for effect level:
- other: read-across from an analogue for which increased absolute and relative female kidney weight and relative male kidney weight at 1000 mg/kg bw/day were observed.
- Remarks on result:
- other: read-across from an analogue for which NOAEL= 100 mg/kg bw/day.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- other: Read across from an analogue
- Sex:
- male/female
- Basis for effect level:
- organ weights and organ / body weight ratios
- Remarks on result:
- other: read-across from an analogue for which LOAEL= 1000 mg/kg bw/day.
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Based on read across from the analogue d-limonene, the NOAEL and LOAEL for alpha terpinene were considered to be 100 and 1000 mg/kg bw/day, respectively, based on the increased absolute and relative female kidney weight and relative male kidney weight in beagle dogs.
- Executive summary:
In a 6-month subchronic toxicity study performed similarly to OECD Guideline 409, d-limonene was administered through gavage to groups of adult beagle dogs (5/sex/dose) at dose levels of 0 (tap water), 0.12 or 1.2 mL/kg bw/day (0, 100 or 1000 mg/kg bw/day) in two divided doses for 180 days. Animals were observed daily and weighed at study initiation, weekly during the study and at the time of sacrifice. Feed consumptions were determined throughout the study and blood samples were obtained at 2 week pre-study (baseline) and then 1, 3 and 6 months during the study. At termination all animals were subjected to gross necropsy during which weights of kidneys were recorded and several tissues were processed for microscopical evaluation of haematoxylin and eosin stained sections. Mallory-Heidenhain-stained kidney sections were also prepared and evaluated for protein accumulation.
Feed consumption and body weight were unaffected by treatment. Clinical signs of toxicity noted were excretion of soft faeces, dose-related occasional mild discomfort during defaecation, sporadic episodes of emesis and diarrhoea. No treatment-related differences other than an increase in serum cholesterol (35%) and serum alkaline phosphatase levels (two-fold increase) at 1000 mg/kg bw/day were observed in male and female dogs. Linear regression analyses indicated a positive dose-related trend for absolute and relative female kidney weight and relative male kidney weight. There were no histopathological changes in the kidneys, evaluated by both haematoxylin and eosin and Mallory-Heidenhain staining that could be associated with the organ-weight changes. Furthermore, there was no evidence of hyaline droplet accumulation or any other sign of hydrocarbon-induced nephropathy typical of those seen in male rats treated with d-limonene.
Based on these results, the read-across approach was applied and the NOAEL and LOAEL for alpha terpinene were considered to be 100 and 1000 mg/kg bw/day, respectively, based on the increased absolute and relative female kidney weight and relative male kidney weight in beagle dogs.
Referenceopen allclose all
Table 1: Survival and mean body weights of mice in the 16-day gavage studies of d-limonene
Dose (mg/kg bw/day) |
Survival (a) |
Mean body weights (grams) |
Final weight relative to vehicle controls (%) |
|||
Initial (b) |
Final |
Change (c) |
||||
Male |
0 |
5/5 |
25.4 ± 0.4 |
26.0 ± 0.9 |
0.6 ± 0.9 |
- |
413 |
5/5 |
25.2 ± 0.5 |
23.0 ± 0.9 |
-2.2 ± 0.8 |
88.5 |
|
825 |
5/5 |
24.6 ± 0.2 |
24.2 ± 0.7 |
-0.4 ± 0.6 |
93.1 |
|
1650 |
(d) 4/5 |
25.6 ± 0.6 |
25.3 ± 1.3 |
-0.5 ± 1.7 |
97.3 |
|
3300 |
(e) 1/5 |
24.8 ± 0.4 |
19 |
-5 |
73.1 |
|
6600 |
(f) 0/5 |
24.6 ± 0.2 |
(g) |
(g) |
(g) |
|
Female |
0 |
5/5 |
20.2 ± 0.2 |
21.8 ± 1.1 |
1.6 ± 1.1 |
- |
413 |
5/5 |
21.4 ± 0.5 |
20.8 ± 0.5 |
-0.6 ± 0.5 |
95.4 |
|
825 |
5/5 |
20.0 ± 0.3 |
19.8 ± 0.6 |
-0.2 ± 0.4 |
90.8 |
|
1650 |
(h) 4/5 |
21.2 ± 0.4 |
22.3 ± 0.6 |
1.0 ± 1.0 |
102.3 |
|
3300 |
(i) 0/5 |
20.4 ± 0.4 |
(g) |
(g) |
(g) |
|
6600 |
(j) 0/5 |
19.8 ± 0.2 |
(g) |
(g) |
(g) |
(a) Number surviving/number initially in group
(b) Initial group mean body weight ± standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.
(c) Mean body weight change of the survivors ± standard error of the mean
(d) Day of death: 2
(e) Day of death: 2, 2, 3, 3
(f) Day of death: all 1
(g) No data are reported due to the 100% mortality in this group.
(h) Death due to gavage error
(i) Day of death: 1, 2, 2, 2, 2
(j) Day of death: 1, 1, 1, 2, 2
Table 1: Survival and mean body weights of rats in the 16-day gavage studies of d-limonene
Dose (mg/kg bw/day) |
Survival (a) |
Mean body weights (grams) |
Final weight relative |
|||
Initial (b) |
Final |
Change (c) |
||||
Male |
0 |
5/5 |
115 ± 2 |
173 ± 3 |
58 ± 3 |
- |
413 |
5/5 |
113 ± 2 |
171 ± 4 |
58 ± 3 |
99 |
|
825 |
5/5 |
113 ± 3 |
173 ± 4 |
60 ± 5 |
100 |
|
1650 |
5/5 |
113 ± 3 |
156 ± 4 |
43 ± 3 |
90 |
|
3300 |
(d) 0/5 |
114 ± 2 |
(e) |
(e) |
(e) |
|
6600 |
(f) 0/5 |
111 ± 2 |
(e) |
(e) |
(e) |
|
Female |
0 |
5/5 |
98 ± 1 |
123 ± 1 |
25 ± 1 |
- |
413 |
5/5 |
101 ± 2 |
(g) 139 ± 2 |
38 ± 3 |
113 |
|
825 |
5/5 |
100 ± 2 |
131 ± 3 |
31 ± 4 |
107 |
|
1650 |
5/5 |
101 ± 2 |
127 ± 1 |
27 ± 2 |
103 |
|
3300 |
(f) 2/5 |
102 ± 2 |
113 ± 8 |
10 ± 5 |
92 |
|
6600 |
(f) 0/5 |
103 ± 4 |
(e) |
(e) |
(e) |
(a) Number surviving/number initially in the group
(b) Initial group mean body weight ± standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.
(c) Mean body weight change of the survivors ± standard error of the mean
(d)Day of death: 1, 1, 1, 1, 2
(e) No data are reported due to the 100% mortality in this group.
(f) Day of death all 1
(g) One final body weight not recorded; weight change based on remaining four animals.
Table 1. Survival and mean body weights of mice in the 13-week gavage studies of d-limonene
Dose (mg/kg bw/day) |
Survival (a) |
Mean body weights (grams) |
Final weight relative to vehicle controls (%) |
|||
Initial (b) |
Final |
Change (c) |
||||
Male |
0 |
10/10 |
26.6 ± 1.0 |
37.1 ± 1.0 |
+10.5 ± 1.3 |
- |
125 |
10/10 |
28.8 ± 0.7 |
37.9 ± 1.1 |
+9.1 ± 0.7 |
102.2 |
|
250 |
(d) 9/10 |
26.5 ± 0.8 |
33.9 ± 0.8 |
+7.6 ± 0.8 |
91.4 |
|
500 |
(d) 7/10 |
24.7 ± 0.9 |
34.4 ± 0.9 |
+9.7± 1.1 |
92.7 |
|
1000 |
(d) 9/10 |
28.2 ± 0.9 |
33.3 ± 0.8 |
+5.1 ± 1.1 |
89.8 |
|
2000 |
(e) 9/10 |
27.7±0.7 |
33.0 ± 0.8 |
+5.6 ± 0.8 |
88.9 |
|
Female |
0 |
10/10 |
21.3 ± 0.2 |
24.7±0.5 |
+3.4 ±0.4 |
- |
125 |
(d) 9/10 |
20.6 ± 0.3 |
25.9± 0.5 |
+5.2 ± 0.4 |
104.9 |
|
250 |
10/10 |
20.7 ± 0.3 |
25.4 ± 0.6 |
+4.7 ± 0.4 |
102.8 |
|
500 |
(f) 9/10 |
20.9 ± 0.2 |
24.9 ±0.5 |
+4.1 ± 0.4 |
100.8 |
|
1000 |
10/10 |
20.4 ±0.2 |
24.1 ± 0.7 |
+3.7 ±0.7 |
97.6 |
|
2000 |
(g) 8/10 |
21.0 ± 0.3 |
24.1 ± 0.4 |
+3.4 ± 0.3 |
97.6 |
(a) Number surviving/number initially in group
(b) Initial group mean body weight f standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.
(c) Mean body weight change of the survivors ± standard error of the mean
(d) Death due to gavage error
(e) Week of death: 1
(f) Week of death: 5
(g) Week of death: 3,4
Table 1. Survival and mean body weights of rats in the 13-week gavage studies of d-limonene
Dose (mg/kg bw/day) |
Survival (a) |
Mean body weights (g) |
Final weight relative to vehicle controls (%) |
|||
Initial (b) |
Final |
Change (c) |
||||
Male |
0 |
10/10 |
144 ± 2 |
333 ± 6 |
+189 ± 5 |
- |
150 |
10/10 |
145 ± 3 |
332 ± 4 |
+187 ± 3 |
100 |
|
300 |
10/10 |
149 ±2 |
330 ± 3 |
+181 ± 4 |
99 |
|
600 |
10/10 |
148 ± 2 |
314± 5 |
+166 ± 5 |
94 |
|
1200 |
10/10 |
139 ± 3 |
292 ± 5 |
+153 ± 6 |
88 |
|
2400 |
(d) 5/10 |
150 ±3 |
255 ± 10 |
+103 ± 10 |
77 |
|
Female |
0 |
10/10 |
118 ± 2 |
185 ± 2 |
+67± 4 |
- |
150 |
10/10 |
115 ± 1 |
186± 2 |
+71± 2 |
101 |
|
300 |
10/10 |
105 ± 4 |
181 ± 2 |
+76± 4 |
98 |
|
600 |
10/10 |
114 ± 1 |
184± 2 |
+70± 1 |
99 |
|
1200 |
10/10 |
116 ± 2 |
182 ± 3 |
+66± 3 |
98 |
|
2400 |
(d) 1/10 |
113 ± 1 |
164 |
+ 56 |
89 |
(a) Number surviving/number initially in group
(b) Initial group mean body weight ± standard error of the mean. Subsequent calculations are based on animals surviving to the end of the study.
(c) Mean body weight change of the survivors ± standard error of the mean
(d) Week of death: all 1
Table 2. Severity of kidney lesions in male rats in the 13-week gavage study of d-limonene (a)
Lesion |
Dose (mg/kg) |
|||||
Vehicle Control |
150 |
300 |
600 |
1200 |
2400 |
|
Regeneration |
(b) 0.8 |
2.4 |
2.5 |
2.5 |
3.7 |
0.9 |
Granular casts |
0 |
1.6 |
2.4 |
2.7 |
3.5 |
0.3 |
(a) Severity grades: 1 = minimal; 2 = mild; 3 = moderate; 4 = marked
(b) Average severity grade for all rats in the group
Table 1: Protein casts observed in the renal tubule
Male | Female | |||||||
Control | 0.4 | 1.2 | 3.6 | Control | 0.4 | 1.2 | 3.6 | |
No. animals examined | 3 | 3 | 3 | 3 | 3 | 2 | 3 | 3 |
Protein casts | 1 | 1 | 2 | 3 | 1 | 2 | 3 | 3 |
Preliminary acute toxicity study:
- An increase in the incidence and severity of hyaline droplets was observed in the kidneys of males only. This histological change was accompanied by a treatment-related increase in alpha 2µ-globulin in males only and a greater accumulation of radioactivity in renal cortex of the male rat compared with that in the females dosed with [14C]d-limonene.
Table 1. Organ and bodyweight data for dogs after 6 months daily administration of d-limonene
|
Dose (mg/kg bw/day) |
||
|
Control |
100 |
1000 |
Male |
|||
Final body weight (kg) |
11.381 ± 0.828 |
10.889 ± 0.595 |
11.008 ± 0.688 |
Kidney weight (g) |
57.09 ± 6.02 |
64.56 ± 6.60 |
73.33 ± 8.91 |
Kidney/body weight (%) |
0.498 ± 0.023 |
0.588 ± 0.035 |
0.661 ± 0.61* |
Female |
|||
Final body weight (kg) |
9.158 ± 0.789 |
9.513 ± 0.315 |
9.176 ± 0.823 |
Kidney weight (g) |
42.18 ± 3.48 |
45.61 ± 2.29 |
55.36 ±2.58* |
Kidney/body weight (%) |
0.461 ± 0.006 |
0.479 ± 0.016 |
0.614 ± 0.032* |
* Statistical significance at P < 0.05,
Values are means ± SEM for 5 dogs.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- LOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subchronic
- Experimental exposure time per week (hours/week):
- 168
- Species:
- dog
- Quality of whole database:
- Weight of evidence and read-across from the analogue substance d-limonene for which there are subacute and subchronic studies in rats, mice and dogs.
- System:
- urinary
- Organ:
- kidney
Repeated dose toxicity: inhalation - systemic effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: inhalation - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - systemic effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Summary of results of the experimental studies on analogue substance d-limonene:
Subacute (27 days, rats, oral):
In a subacute toxicity study (Kanerva, 1987), d-limonene was administered through gavage to groups of 5 male Fisher-344 rats/dose mixed in corn oil at dose levels of 0, 75, 150 and 300 mg/kg bw/day for 1 or 4 weeks (5 days/week). As nephrotoxicity and accumulation of hyaline droplets containing α2µ-globulin were observed in male rats at all dose levels, no NOAEL could be identified in this study. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
Subchronic (90 days, rats, oral):
In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP (NTP, 1990), d-limonene was administered through gavage to groups of 10 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 13 weeks (5 days/week). The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium. Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. This mechanism of nephrocarcinogenicity has been proven as being male-rat specific and not relevant for humans.
In another subchronic toxicity study (Webb, 1989), d-limonene was administered through gavage to groups of 5 or 10 male Fisher-344 rats/dose mixed in corn oil at dose levels of 0, 2, 5, 10, 30 and 75 mg/kg bw/day for 13 weeks (5 days/week). The NOAEL and LOAEL were considered to be 5 and 30 mg/kg bw/day, respectively, but mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
In a third subchronic toxicity study (Kanerva, 1987), d-limonene was administered through gavage to groups of male and female rats at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 91 days. As chronic nephrosis and granular casts formation were observed in male rats at all dose levels, no NOAEL could be identified in this study. However, mechanisms and specificity of toxicity of d-limonene on kidney of male rats and its non relevance for humans are well known.
Subchronic (90 days, mouse, oral):
In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP (NTP, 1990), d-limonene was administered through gavage to groups of 10 B6C3F1 mice/sex/dose mixed in corn oil at dose levels of 0, 125, 250, 500, 1000 or 2000 mg/kg bw/day for 13 weeks (5 days/week). The LOAEL was considered to be 1000 mg/kg bw/day for both female and male mice, based on observation of clinical signs in both sexes and decreased bodyweights in males.
Subchronic (180 days, dogs, oral):
In a 6-month subchronic toxicity study performed similarly to OECD Guideline 409 (Webb, 1990), d-limonene was administered through gavage to groups of adult beagle dogs (5/sex/dose) at dose levels of 0 (tap water), 0.12 or 1.2 mL/kg bw/day (0, 100 or 1000 mg/kg bw/day) in two divided doses for 180 days. The NOAEL and LOAEL for beagle dogs were considered to be 100 and 1000 mg/kg bw/day, respectively, based on the increased absolute and relative female kidney weight and relative male kidney weight.
In another 6-month subchronic toxicity study (Tsuji, 1975), three dogs per sex and per dose group were administered d-limonene by gavage once per day for 6 months at the dose level of 0, 0.4, 1.2 or 3.6 mL/kg bw/day. The NOAEL in this study is 1.2 mL/kg bw/day (equivalent to 1000 mg/kg bw/day) in males and 0.4 mL/kg bw/day (equivalent to 340 mg/kg bw/day) in females on the basis of decreased body weight and protein casts observed in the renal tubule at the next dose level. Food consumption was also decreased in the intermediate dose females.
Justification for classification or non-classification
Based on the available data, the substance is not classified for specific target organ toxicity by repeated exposure (STOT-RE) according to CLP Regulation (EC) no 1272/2008.
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