Registration Dossier
Registration Dossier
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EC number: 947-974-1 | CAS number: -
Endpoint summary
Administrative data
Description of key information
Weight of evidence: Repeated dose toxicity studies are available on the components d-limonene and camphene by oral route, and on component alpha pinene by inhalation route. The absence of any d-limonene-induced renal lesions in the study with dogs provides evidence that hydrocarbon-induced nephropathy in the male rat is species- and sex-specific. Therefore, the male rat response to d-limonene, alpha pinene or camphene 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.
Thus, 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. The key value has been then selected to be the LOAEL at 1000 mg/kg bw/d. When administered orally by gavage for at least 6 months, d-limonene induces some toxicological effects at 1000 mg/kg bw/day. 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).
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:
- supporting study
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- significant methodological deficiencies
- Remarks:
- The study does not meet the main criteria of the specific testing guideline (duration, number of tested animals, haematology, clinical biochemistry, gross necropsy, etc.)
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- In this study, the effect of camphene on serum lipids and apolipoproteins (apoproteins) was investigated in male Wistar rats in order to obtain more information on the physiological role of essential oils. Camphene was mixed with the powdered commercial rat ration at the level of 1%. A control group received the commercial rat ration alone. Three or four rats were used. The rats were fasted overnight prior to blood sampling from the abdominal aorta. Serum lipids and apoproteins were determined.
- GLP compliance:
- not specified
- Limit test:
- yes
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Kyudo Co., Kumamoto
- Weight at study initiation: 248 ± 4.1 g
- Housing: The animals were kept in an air conditioned room
- Diet (e.g. ad libitum): ad libitum; NMF, Oriental Yeast Co, Tokyo.
- Water (e.g. ad libitum): ad libitum
ENVIRONMENTAL CONDITIONS
- Photoperiod: 12 hrs dark / 12 hrs light - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Camphene was mixed with the powdered commercial rat ration. - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 14 Days
- Frequency of treatment:
- Daily
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Remarks:
- Basis: 1 % of camphene mixed in diet
- No. of animals per sex per dose:
- 3-4 male rats per group
- Control animals:
- yes, concurrent no treatment
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Not specified
DETAILED CLINICAL OBSERVATIONS: Not specified
BODY WEIGHT: Yes
- Time schedule for examinations: at beginning and at the end of study
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of study.
- Animals fasted: Yes. The rats were fasted overnight prior to blood sampling.
- How many animals: 3-4
- Parameters examined: Serum lipids (cholesterol and triacylglycerol) and apoproteins (Apo A-I)
OTHER: RELATIVE LIVER WEIGHT
- Time schedule for collection of blood: At the end of study. - Sacrifice and pathology:
- GROSS PATHOLOGY: No
HISTOPATHOLOGY: No - Statistics:
- Student's t test.
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No significant differences were observed in body weight gain between treated and control animals.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No significant differences were observed in food consumption between treated and control animals.
- 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:
- no effects observed
- Description (incidence and severity):
- No significant differences in cholesterol, triacylglycerol and Apolipoprotein Apo A-I were observed between treated and control animals.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Liver weight per 100 g of body weight tended to increase by the supplementary essential oils. No significant increase was recorded for the test item.
- Gross pathological findings:
- not examined
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 500 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- clinical biochemistry
- Key result
- Critical effects observed:
- no
- Conclusions:
- After 14 days of oral exposure, the NOAEL of camphene was equal or greater than 500 mg/kg bw/day for male rats.
- Executive summary:
The effect of camphene on serum lipids and apolipoproteins (apoproteins) was investigated in male Wistar rats in order to obtain more information on the physiological role of essential oils. Camphene was mixed with the powdered commercial rat ration at the level of 1% (ca. 500 mg/kg bw/day). It was daily administered for 14 days. A control group received the commercial rat ration without test substance. Three or four rats were used. The rats were fasted overnight prior to blood sampling from the abdominal aorta. Serum lipids and apoproteins were determined. No significant differences were observed in body weight gain and/or food consumption between treated and control animals. Liver weight per 100 g of body weight tended to increase by the supplementary essential oils but not significantly. No significant differences in cholesterol, triacylglycerol and Apolipoprotein Apo A-I were observed between treated and control animals. Thus, the NOAEL of camphene was equal or greater than 500 mg/kg bw/day for male rats.
- 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:
- supporting study
- 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:
- supporting study
- 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:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 10 January 1991 - 02 May 1991
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Test method according to OECD guideline 407 and GLP.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Hoechst AG, Box reason SPF breeding
- Age at study initiation: Approximately 6 weeks
- Housing: In air-conditioned spaces, in Makrolon cages (type 4) on granulate softwood, in groups of 5 animals.
- Diet (e.g. ad libitum): Rat diet Altromin 1324 (Altromin GmbH, Lage / Lippe), ad libitum, except the time in which the animals were in metabolism cages
- Water (e.g. ad libitum): Tap water in plastic bottles, ad libitum, except the time in which the animals were in metabolism cages
- Acclimation period: At least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 20 %
- Photoperiod: 12 hrs dark / 12 hrs light
IN-LIFE DATES: From: 10.01.1991 To: 07.02.1991 - Route of administration:
- oral: gavage
- Vehicle:
- other: Sesame oil
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared daily, immediately before the administration. The test material was homogeneously dispersed using a magnetic stirrer.
Volume administered: 5 mL/kg bw
VEHICLE
Oleum Sesami, Ph. Eur III, Fa Mainland, Pharmazeutische Fabrik GmbH, Ffm. - Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- 28 Days
- Frequency of treatment:
- Daily
- Dose / conc.:
- 62.5 mg/kg bw/day (actual dose received)
- Remarks:
- (1.25 % w/v)
- Dose / conc.:
- 250 mg/kg bw/day (actual dose received)
- Remarks:
- (5 % w/v)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- (20 % w/v)
- No. of animals per sex per dose:
- 5 male and 5 female rats per group.
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Rationale for animal assignment (if not random): Randomization: Animals in cages randomisation = 946/90 and 947/90
Cages in the rack randomisation = 949/90 - Observations and examinations performed and frequency:
- In all experimental groups, the behavior and general health of animals were observed twice daily during the experiment, once a day on weekends and holidays. Every week neurological disturbances, clouding of the ocular media, adverse effects on oral mucosa and tooth development disorders were studied.
The body weight was determined at the beginning of the experiment and twice a week.
Food consumption was determined twice a week and w ater consumption, once a week.
At the end of the experiment, the bllod count was investigated without prior food deprivation in all male and female animals. The blood was taken from the retro-orbital venous plexus. The following hematological parameters were determined: erythrocyte count, hemoglobin, hematocrit, MCV, MCH, MCHC, WBC, platelet count, differential count, reticulocyte count, Heinz'sche inner body, clotting time.
The urine tests were performed on all male and female animals and took place overnight from day 26 to 27 of the experimental period. The following parameters were determined: Appearance, color, pH, hemoglobin, protein, glucose, ketone body, bilirubin, urobilinogen, density and sediment. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes - Other examinations:
- At the end of the study, animals were macroscopically examined. Alterations in organs were recorded, organs were weighed and their relative weights calculated. Histological preparations from the main organs were examined for microscopic changes. Chemical analysis were performed. The following parameters were analyzed: sodium, potassium, anorg. Phosphorus, uric acid, total and direct bilirubin, creatinine, serum glucose, urea nitrogen (BUN), calcium, chloride, AST (GOT), ALT (GPT), alkaline phosphatase (AP), gamma-glutamyl transferase (GGT), total protein, albumin.
- Statistics:
- Body weight, hematological and clinical tests, and organ weights (absolute and relative) were statistically compared with the control group.
The analysis were carried out using a program package for evaluation of toxicological tests, according to the Standard Operating Procedure (Department of Pharmaceutical Research). - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- The highest dose group (1000 mg/kg bw) showed an increased salivation. Behaviour and general health from other treated groups were not significantly different from control group.
- Mortality:
- mortality observed, treatment-related
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- The body weight was not affected by the administration of the test substance.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- The food consumption was not affected by the administration of the test substance.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- The water consumption was not affected by the administration of the test substance.
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Haematological tests revealed no evidence of compound-related toxicity.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In male animals from the highest dose group, clinical chemistry tests revealed an increase in urea nitrogen levels and a decrease in phosphorus levels.
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- The urine was normal and showed no evidence of compound-related toxicity.
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- Behaviour and general health from other treated groups were not significantly different from control group.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- In male and female animals from the highest dose group, absolute and relative liver weights were increased.
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Macroscopic examinations showed spotted kidneys in two male animals from the lowest dose group (62.5 mg / kg bw). In 3 mid-dose male animals and in all males from the highest dose group, colourless kidneys were observed.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Animals from the highest dose group showed an increased vacuolization of the hepatocytes. Female animals from dose groups of 62.5 and 250 mg / kg bw did not show toxic effects. In all dose groups of male rats deposits of the test substance in the epithelium of the proximal renal tubules associated with necrosis of single cells have been observed. These effects seem to be specific for male rats and contingent upon alpha-2 microglobinemia. The renal toxic effects found in all dose levels groups in male rats are interpreted as uniquely specific for male rats, and as having no relevance for other animal species and humans.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not examined
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Sex:
- female
- Basis for effect level:
- other: overall effects
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- < 62.5 mg/kg bw/day (actual dose received)
- Sex:
- male
- Basis for effect level:
- other: renal toxic effects
- Critical effects observed:
- not specified
- Conclusions:
- For female rats the NOEL was 250 mg / kg bw/day. For male rats, the NOEL could not be determined (it was lower than 62.5 mg/kg bw/day).
- Executive summary:
Camphene was daily administered to SPF Wistar rats (male and female) for 28 days at doses of 0, 62.5, 250 and 1000 mg / kg bw/day by gavage. Test method was according to OECD guideline 407. In all experimental groups, behaviour and general health were daily examined. The body weight and food consumption were determined twice a week, water consumption was determined once a week. Haematological and clinical tests, and urinalysis were also carried out. At the end of the study, animals were macroscopically examined. Alterations in organs were determined, organs were weighed and their relative weights calculated. Histologicalpreparations from the main organs were examined for microscopic changes. Body weight, haematological and clinical tests, and organ weights (absolute and relative) were statistically compared with the control group.
The highest dose group (1000 mg/kg bw/day) showed an increased salivation. Behaviour and general health from other treated groups were not significantly different from control group. The body weight and food- and water-consumption were not affected by the administration of the test substance.
Haematological tests revealed no evidence of compound-related toxicity. In male animals from the highest dose group, clinical chemistry tests revealed an increase in urea nitrogen levels and a decrease in phosphorus levels. The urine was normal and showed no evidence of compound-related toxicity.
In male and female animals from the highest dose group, absolute and relative liver weights were increased.Macroscopic examinations showed spotted kidneys in two male animals from the lowest dose group (62.5 mg / kg bw/day). In 3 mid-dose male animals and in all males from the highest dose group, colourless kidneys were observed. Animals from the highest dose group showed an increased vacuolization of the hepatocytes. Female animals from dose groups of 62.5 and 250 mg / kg bw/day did not show toxic effects. In all dose groups of male rats deposits of the test substance in the epithelium of the proximal renal tubules associated with necrosis of single cells have been observed. These effects seem to be specific for male rats and contingent upon alpha-2 microglobinemia. The renal toxic effects found in all dose levels groups in male rats are interpreted as uniquely specific for male rats, and as having no relevance for other animal species and humans.
Based on the results of this study, for female rats the NOEL was 250 mg / kg bw/day. For male rats, the NOEL could not be determined (it was lower than 62.5 mg/kg bw/day).
- 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.
Referenceopen allclose all
After 14 days, the NOAEL was equal or greater than 500 mg/kg bw/day for male rats.
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
For female rats the NOEL was 250 mg / kg bw/day. For male rats, the NOEL could not be determined (it was lower than 62.5 mg/kg bw/day).
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
- Species:
- dog
- Quality of whole database:
- A weight of evidence approach has been applied. Several experimental studies are available with a Klimisch score of 2.
Repeated dose toxicity: inhalation - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 28 March 2005 - 1 July 2005
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- Deviations:
- yes
- Remarks:
- no tested on preferred specie rat, no data on food consumption, no ophthalmological and clinical chemistry exams, some organ weights were not recorded (Adrenals,Brain,Ovaries,Thyroids,Uterus), animals weighed weekly and not twice weekly at the beginning.
- GLP compliance:
- yes
- Remarks:
- In compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)
- Limit test:
- no
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: NTP colony maintained at Taconic Farms, Inc. (Germantown, NY)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 22.5-23 g (male) and 19.3-19-7 g (female)
- Fasting period before study: not specified
- Housing: individually. Cages: Stainless steel, wire bottom (Lab Products, Inc., Seaford, DE); rotated weekly. Cageboard: Untreated paper cage pan liner (Shepherd Specialty Papers, Kalamazoo, MI), changed daily
- Diet (e.g. ad libitum): NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (except during exposure periods)
- Water (e.g. ad libitum): Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum
- Acclimation period: Animals were quarantined for 12 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3ºF
- Humidity (%): 50% ± 15%
- Air changes (per hr): 15 ± 2/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- whole body
- Vehicle:
- not specified
- Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Test item was held in an 8-gallon stainless-steel chemical reservoir. Test item was pumped into a heated glass column filled with glass beads that increased the surface area for vaporization. Heated nitrogen entered the column from below and assisted in vaporizing the chemical while conveying it into a short distribution manifold. Concentration in the manifold was determined by the chemical pump rate, nitrogen flow rate, and dilution air flow rate. The pressure in the distribution manifold was kept fixed to ensure constant flow through the manifold and into all chambers as the flow of vapor to each chamber was adjusted.
Metering valves at the manifold controlled flow to each chamber through individual Teflon® delivery lines that carried the vapor from the manifold to three-way exposure valves at the chamber inlets. The exposure valves diverted vapor delivery to exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. To initiate exposure, the chamber exposure valves were rotated to allow the test item vapor to flow to each exposure chamber inlet duct where it was further diluted with filtered, conditioned air to achieve the desired exposure concentration.
- Temperature, humidity, pressure in air chamber: 72 ± 3ºF; 50% ± 15%.
- Air change rate: 15 air changes per hour
- Method of particle size determination: A condensation particle detector (Model 3022A, TSI, Inc., St. Paul, MN) was used with and without animals in the exposure chambers. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) were detected.
TEST ATMOSPHERE
- Brief description of analytical method used: on-line gas chromatograph. Samples were analyzed using GC/FID to measure the stability and purity of test item in the generation and delivery system. To assess whether impurities or degradation products coeluted with test item or the solvent, a second GC/FID analysis of the samples was performed using a polar column capable of resolving compounds with similar boiling points and polarities.
- Samples taken from breathing zone: yes.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Chamber and room concentrations of alpha pinene were monitored by an on-line gas chromatograph. Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period.
The average concentration measured were: 24.9 ± 1.1 ppm for the 25 ppm group, 49.8 ± 0.8 for the 50 ppm group, 99.6 ± 1.4ppm for the 100 ppm group, 200 ± 4 ppm for the 200 ppm group and 401 ± 7 ppm for the 400 ppm group. - Duration of treatment / exposure:
- 14 weeks; 6 hours plus T90 (10 minutes) per day.
- Frequency of treatment:
- Five times per week, weekdays only
- Dose / conc.:
- 0 ppm
- Dose / conc.:
- 25 ppm
- Remarks:
- (0.14 mg/L)
- Dose / conc.:
- 50 ppm
- Remarks:
- (0.28 mg/L)
- Dose / conc.:
- 100 ppm
- Remarks:
- (0.56 mg/L)
- Dose / conc.:
- 200 ppm
- Remarks:
- (1.13 mg/L)
- Dose / conc.:
- 400 ppm
- Remarks:
- (2.26 mg/L)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes
- Details on study design:
- - Dose selection rationale: doses were selected taken into account the results obtained in a previous 2-week range finding study conducted for exposure (inhalation) concentrations of 0, 100, 200, 400, 800, and 1,600 ppm.
- Positive control:
- no
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on day 8, weekly thereafter, and at the end of the studies.
BODY WEIGHT: Yes
- Time schedule for examinations: initially, on day 8, weekly thereafter, and at the end of the studies.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Not specified
- How many animals: 10 per sex per dose
- Parameters examined: Hematocrit; packed cell volume; hemoglobin; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte counts and differentials
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes. Organs weighed were heart, right kidney, liver, lung, spleen, right testis, and thymus.
HISTOPATHOLOGY: Yes. Complete histopathologic examinations were performed by the study laboratory pathologist on all chamber control and 400 ppm animals. In addition, the kidney and urinary bladder of mice were examined in the remaining groups. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin. - Other examinations:
- SPERM MOTILITY AND VAGINAL CYTOLOGY: At the end of the study, sperm samples were collected for sperm motility evaluations. Sperm heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration were evaluated. The left cauda, left epididymis, and left testis were weighed. The tail of the epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed. Test yolk was applied to slides and a small incision was made at the distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two observers. Following completion of sperm motility estimates, each left cauda epididymis was placed in buffered saline solution. Caudae were finely minced, and the tissue was incubated in the saline solution and then heat fixed at 65° C. Sperm density was then determined microscopically with the aid of a hemacytometer. To quantify spermatogenesis, the testicular spermatid head count was determined by removing the tunica albuginea and homogenizing the left testis in phosphate-buffered saline containing 10% dimethyl sulfoxide. Homogenization-resistant spermatid nuclei were counted with a hemacytometer.
Vaginal samples were collected for up to 12 consecutive days prior to the end of the study for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated. - Statistics:
- Calculation and Analysis of Lesion Incidences: Fisher exact test (Gart et al., 1979) was used to determine significance.
Analysis of Continuous Variables:
Organ and body weight data were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972),
Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).
Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the exposure-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic exposure-related trend (Dunnett’s or Dunn’s test).
Proportions of regular cycling females in each exposed group were compared to the control group using the Fisher exact test (Gart et al., 1979). Tests for extended periods of estrus, diestrus, metestrus, and proestrus, as well as skipped estrus and skipped diestrus were constructed based on a Markov chain model proposed by Girard and Sager (1987).
For each exposure group, a transition probability matrix was estimated for transitions among the proestrus, estrus, metestrus, and diestrus stages, with provision for extended stays within each stage as well as for skipping estrus or diestrus within a cycle. Equality of transition matrices among exposure groups and between the control group and each exposed group was tested using chi-square statistics. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related clinical signs.
- Mortality:
- no mortality observed
- Description (incidence):
- All mice survived until the terminal sacrifice.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Final mean body weights and body weight gains were comparable for all test animals when compared to 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:
- effects observed, non-treatment-related
- Description (incidence and severity):
- At the end of the study, there were small but statistically significant decreases in erythrocyte counts in 200 and 400 ppm females and in the hemoglobin concentration and the hematocrit value in 400 ppm females compared to concurrent controls. Decreases in erythrocyte count and hematocrit value also occurred in 400 ppm males. Leukocyte and lymphocyte counts were significantly decreased in 400 ppm males. The leukocyte changes likely represent a secondary treatment-associated stress effect. The exact mechanism for the mild decreases in the erythron are not known. Other significant changes in hematology parameters were not toxicologically relevant.
- 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:
- effects observed, treatment-related
- Description (incidence and severity):
- The absolute liver weights of 400 ppm males and females and the relative liver weights of 200 and 400 ppm males and 100, 200, and 400 ppm females were significantly greater than those of the chamber controls. The absolute and relative thymus weights of 400 ppm males were significantly less than those of the chamber controls. The absolute kidney weights of 200 and 400 ppm males were significantly less than those of the chamber controls. These organ weight changes were not accompanied by histopathologic lesions.
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- There was an increased incidence of transitional epithelium hyperplasia of the urinary bladder in males and females exposed to 100 ppm or more, the severity of which increased with increasing exposure concentration.
Transitional epithelium hyperplasia in the urinary bladder can be either reparative (e.g., regenerative or reactive) or preneoplastic, but there are no histologic features that can be used to reliably distinguish between the two etiologies.
Specific histopathologic indicators of either type of hyperplasia in male or female mice were not evident; therefore, the neoplastic potential of the transitional epithelium hyperplasia of the urinary bladder is uncertain. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- SPERM MOTILITY AND VAGINAL CYTOLOGY: There were significantly decreased numbers of sperm per mg cauda in 200 and 400 ppm males and cauda sperm in 100, 200, and 400 ppm males.
There were no changes in the proportion of regularly cycling females, estrous cycle length, or percentage of time spent in the individual stages of the estrous cycle of female mice at any exposure concentration and there were no ovarian histopathologic findings.
Thus, the test item exposure by inhalation exhibits the potential to be a reproductive toxicant in male mice, but not in female mice. - Key result
- Dose descriptor:
- LOEL
- Effect level:
- 100 ppm
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Significantly decreased sperm count per mg cauda in males treated at 200 and 400 ppm and in cauda sperm counts in 100, 200, and 400 ppm groups
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- 100 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 100 ppm
- System:
- urinary
- Organ:
- bladder
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 100 ppm
- System:
- male reproductive system
- Organ:
- cauda epididymis
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- The LOEL for alpha pinene after 14 weeks of vapour inhalation exposure to mice was determined to be 100 ppm based on the effects on transitional epithelium hyperplasia of the urinary bladder for males and females and decrease in sperm per cauda epididymis for males, both observed in the 100 ppm treated group or greater.
- Executive summary:
A 90-day repeated dose toxicity study (inhalation route) was performed on alpha pinene according to a similar method to OECD guideline 413 under GLP conditions. Groups of 10 male and 10 female mice were exposed to the test item by whole body inhalation at concentrations of 0, 25, 50, 100, 200, or 400 ppm, 6 hours per day, 5 days per week for 14 weeks. During the study, clinical observations, bodyweight, organ weight, haematology, macropathology, histopathology and sperm motility and vaginal cytology investigations were undertaken.
All exposed mice survived to the end of the studies. Some small changes in organ weights as liver or kidney, were noted but without accompanying histopathologic lesions.
The major targets for test item toxicity were the urinary system and male reproductive system.
There was an increased incidence of transitional epithelium hyperplasia of the urinary bladder in males and females exposed to 100 ppm or more (LOEL=100 ppm), the severity of which increased with increasing exposure concentration. Transitional epithelium hyperplasia in the urinary bladder can be either reparative (e.g., regenerative or reactive) or preneoplastic, but there are no histologic features that can be used to reliably distinguish between the two etiologies. Specific histopathologic indicators of either type of hyperplasia in male or female mice were not evident; therefore, the neoplastic potential of the transitional epithelium hyperplasia of the urinary bladder is uncertain.
There were also significantly decreased numbers of sperm per mg cauda in 200 and 400 ppm males and cauda sperm in 100, 200, and 400 ppm males (LOEL=100 ppm). There was an accompanying minor decrease in epididymal weights that did not reach significance. Therefore, the possibility that the change in absolute sperm per cauda was due to a decrease in epididymal weight cannot be excluded. A definitive conclusion by histopathologic investigations could not be conducted due to an artifact resulting from formalin fixation of the male reproductive tract tissues. Thus, further studies on reproductive function are warranted. However, in females there were no changes in the percentage of time spent in the individual stages of the estrous cycle or in estrous cycle length at any exposure concentration. Also, there were no ovarian histopathologic findings. Thus, no evidence of reproductive toxicity in females was found.
Based on these results, the LOEL for alpha pinene was determined to be 100 ppm.
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 28 March 2005 - 29 June 2005
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- Deviations:
- yes
- Remarks:
- no data on food consumption, no ophthalmological examination, some organ weights were not recorded (Adrenals, Brain, Ovaries, Thyroids, Uterus), animals weighed weekly and not twice weekly at the beginning.
- GLP compliance:
- yes
- Remarks:
- In compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: NTP colony maintained at Taconic Farms, Inc. (Germantown, NY)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: 96-98 g (male) and 88-89 g (female)
- Fasting period before study: not specified
- Housing: individually. Cages: Stainless steel, wire bottom (Lab Products, Inc., Seaford, DE); rotated weekly. Cageboard: Untreated paper cage pan liner (Shepherd Specialty Papers, Kalamazoo, MI), changed daily
- Diet (e.g. ad libitum): NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (except during exposure periods)
- Water (e.g. ad libitum): Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum
- Acclimation period: Animals were quarantined for 12 (male rats) or 13 (female rats) days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3ºF
- Humidity (%): 50% ± 15%
- Air changes (per hr): 15 ± 2/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- whole body
- Vehicle:
- not specified
- Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Test item was held in an 8-gallon stainless-steel chemical reservoir. Test item was pumped into a heated glass column filled with glass beads that increased the surface area for vaporization. Heated nitrogen entered the column from below and assisted in vaporizing the chemical while conveying it into a short distribution manifold. Concentration in the manifold was determined by the chemical pump rate, nitrogen flow rate, and dilution air flow rate. The pressure in the distribution manifold was kept fixed to ensure constant flow through the manifold and into all chambers as the flow of vapor to each chamber was adjusted.
Metering valves at the manifold controlled flow to each chamber through individual Teflon® delivery lines that carried the vapor from the manifold to three-way exposure valves at the chamber inlets. The exposure valves diverted vapor delivery to exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. To initiate exposure, the chamber exposure valves were rotated to allow the test item vapor to flow to each exposure chamber inlet duct where it was further diluted with filtered, conditioned air to achieve the desired exposure concentration.
- Temperature, humidity, pressure in air chamber: 72 ± 3ºF; 50% ± 15%.
- Air change rate: 15 air changes per hour
- Method of particle size determination: A condensation particle detector (Model 3022A, TSI, Inc., St. Paul, MN) was used with and without animals in the exposure chambers. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) were detected.
TEST ATMOSPHERE
- Brief description of analytical method used: on-line gas chromatograph. Samples were analyzed using GC/FID to measure the stability and purity of test item in the generation and delivery system. To assess whether impurities or degradation products coeluted with test item or the solvent, a second GC/FID analysis of the samples was performed using a polar column capable of resolving compounds with similar boiling points and polarities.
- Samples taken from breathing zone: yes.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Chamber and room concentrations of alpha pinene were monitored by an on-line gas chromatograph. Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period.
The average concentration measured were: 24.9 ± 1.1 ppm for the 25 ppm group, 49.8 ± 0.8 for the 50 ppm group, 99.6 ± 1.4ppm for the 100 ppm group, 200 ± 5 ppm for the 200 ppm group and 401 ± 6 ppm for the 400 ppm group. - Duration of treatment / exposure:
- 14 weeks; 6 hours plus T90 (10 minutes) per day.
Groups of 10 male and 10 female clinical pathology rats were exposed to the same concentrations for 23 days. - Frequency of treatment:
- Five times per week, weekdays only
- Dose / conc.:
- 0 ppm
- Dose / conc.:
- 25 ppm
- Remarks:
- (0.14 mg/L)
- Dose / conc.:
- 50 ppm
- Remarks:
- (0.28 mg/L)
- Dose / conc.:
- 100 ppm
- Remarks:
- (0.56 mg/L)
- Dose / conc.:
- 200 ppm
- Remarks:
- (1.13 mg/L)
- Dose / conc.:
- 400 ppm
- Remarks:
- (2.26 mg/L)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes
- Details on study design:
- - Dose selection rationale: doses were selected taken into account the results obtained in a previous 2-week range finding study conducted for exposure (inhalation) concentrations of 0, 100, 200, 400, 800, and 1,600 ppm.
- Positive control:
- no
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on day 7 (female), day 8 (male), weekly thereafter, and at the end of the studies.
BODY WEIGHT: Yes
- Time schedule for examinations: initially, on day 7 (female), day 8 (male), weekly thereafter, and at the end of the studies.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: on days 4 and 23 for clinical pathology rats and at the end of the studies for core study animals
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Not specified
- How many animals: 10 per sex per dose (core study animals) and 10 per sex per dose (clinical pathology rats)
- Parameters examined: Hematocrit; packed cell volume; hemoglobin; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte counts and differentials
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on days 4 and 23 for clinical pathology rats and at the end of the studies for core study animals
- Animals fasted: Not specified
- How many animals: 10 per sex per dose (core study animals) and 10 per sex per dose (clinical pathology rats)
- Parameters examined: Urea nitrogen, creatinine, total protein, albumin, globulin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile salts
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes. Organs weighed were heart, right kidney, liver, lung, spleen, right testis, and thymus.
HISTOPATHOLOGY: Yes. Complete histopathologic examinations were performed by the study laboratory pathologist on all chamber control and 400 ppm animals and 200 ppm female rats. In addition, the kidney in the remaining groups and the liver in the remaining groups of male rats were examined. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin. - Other examinations:
- SPERM MOTILITY AND VAGINAL CYTOLOGY: At the end of the study, sperm samples were collected for sperm motility evaluations. Sperm heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration were evaluated. The left cauda, left epididymis, and left testis were weighed. The tail of the epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed. Test yolk was applied to slides and a small incision was made at the distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two observers. Following completion of sperm motility estimates, each left cauda epididymis was placed in buffered saline solution. Caudae were finely minced, and the tissue was incubated in the saline solution and then heat fixed at 65° C. Sperm density was then determined microscopically with the aid of a hemacytometer. To quantify spermatogenesis, the testicular spermatid head count was determined by removing the tunica albuginea and homogenizing the left testis in phosphate-buffered saline containing 10% dimethyl sulfoxide. Homogenization-resistant spermatid nuclei were counted with a hemacytometer.
Vaginal samples were collected for up to 12 consecutive days prior to the end of the study for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated. - Statistics:
- Calculation and Analysis of Lesion Incidences: Fisher exact test (Gart et al., 1979) was used to determine significance.
Analysis of Continuous Variables:
Organ and body weight data were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972),
Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data were analyzed using the nonparametric multiple comparison methods of Shirley (1977) (as modified by Williams, 1986) and Dunn (1964).
Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the exposure-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic exposure-related trend (Dunnett’s or Dunn’s test).
Proportions of regular cycling females in each exposed group were compared to the control group using the Fisher exact test (Gart et al., 1979). Tests for extended periods of estrus, diestrus, metestrus, and proestrus, as well as skipped estrus and skipped diestrus were constructed based on a Markov chain model proposed by Girard and Sager (1987).
For each exposure group, a transition probability matrix was estimated for transitions among the proestrus, estrus, metestrus, and diestrus stages, with provision for extended stays within each stage as well as for skipping estrus or diestrus within a cycle. Equality of transition matrices among exposure groups and between the control group and each exposed group was tested using chi-square statistics. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No signs of toxicity (e.g., abnormal breathing or behavior) were noted during clinical observations.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- In the high dose group (400 ppm), 6 females were found dead before the end of the study with no specific cause of death identified through gross examination or histopathologic analysis.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Males: the final mean body weights and mean body weight gains of exposed males were similar to those of the chamber controls.
Females: In the high dose group (400 ppm), the final mean body weights and the mean body weight gains of females exposed to 400 ppm were significantly less than those of the chamber controls. All surviving females at 400 ppm lost weight between week 12 and week 14. - 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:
- effects observed, treatment-related
- Description (incidence and severity):
- On day 4, there were mild exposure-related significant decreases in the leukocyte counts paired with mild significant decreases in the lymphocyte counts in 200 and 400 ppm male rats compared to concurrent controls. These decreases ameliorated by day 23. The leukocyte changes likely represent a secondary treatment-associated stress effect.
At week 14, there were mild significant decreases in erythrocyte counts, hemoglobin concentrations, and hematocrit values in males exposed to 100 ppm or greater.
The remaining significant differences in hematology parameters were not considered to be toxicologically relevant.
Also, no histopathological findings could be associated to these changes. Therefore, these treatment-related effects can be considered as not toxicologically significant. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Alanine aminotransferase activities were significantly decreased in males and females exposed to 50 ppm or greater at week 14. Significantly decreased alanine aminotransferase activities were also seen in 400 ppm male rats on days 4 and 23. Significant decreases in alkaline phosphatase activities were observed in 400 ppm males and 200 and 400 ppm females on day 4 and in males and females exposed to 100 ppm or greater at week 14. The reason for the decreases in these enzyme activities is not known but may be related to alterations in liver metabolism or enzyme inhibition.
The remaining significant differences in clinical chemistry parameters were not considered to be toxicologically relevant.
None of these changes in enzyme activity were related to organ weight changes or evidence of histopathology. - Urinalysis findings:
- not examined
- 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):
- Relative liver weights were significantly greater compared to controls in males at 100 ppm and greater and in all females treated groups. Absolute liver weights were significantly greater compared to controls in males at 400 ppm and in females at 50, 100 and 200 ppm.
The absolute heart weights of 100 and 200 ppm females and the relative heart weights of females exposed to 100 ppm or greater were significantly greater compared to controls.
Absolute kidney weights were increased in males at 100 ppm and greater, and relative kidney weights were increased in males at 50 ppm and greater. In females, absolute kidney weights were increased at levels of 50 and 200 ppm, relative were increased at level of 200 ppm and greater.
The absolute and relative thymus weights of 400 ppm females and the relative thymus weight of 200 ppm females were significantly less compared to controls. The absolute and relative spleen weights of 400 ppm males were significantly greater compared to controls.
The weight changes in lymphoid tissues were not accompanied by clinical chemistry or histopathologic changes indicative of immunotoxicity and, therefore, were not considered toxicologically relevant. With the exception of the male kidney, the organ weight changes in male and female rats were not accompanied by histopathologic lesions. - Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Renal tubule lesions including granular casts, hyaline droplets, and nephropathy were observed in male rats. In all male groups exposed to test item, there were significantly increased incidences of granular casts and hyaline droplet accumulation and the severities of these lesions increased with increasing exposure concentration.
Granular casts were not observed in the controls and in exposed groups the mean severity ranged from minimal in males exposed to 25 ppm to moderate in males exposed to 400 ppm.
Hyaline droplet accumulation occurred in the cytoplasm of the renal proximal convoluted tubule epithelial cells, and the severity ranged from minimal in males exposed to 25 ppm to moderate in males exposed to 400 ppm.
With the exception of one chamber control rat, nephropathy occurred in all males, with the mean severity increasing from minimal in chamber control males to moderate in males exposed to 400 ppm.
There were no exposure-related microscopic findings in females, including those that died before the end of the study.
The presence of these nonneoplastic lesions in the kidney is suggestive of α2μ-globulin nephropathy, a renal syndrome that occurs in male but not female F334/N rats and that has been linked to the development of renal tubule neoplasms (Swenberg, 1993). This syndrome has been produced by structurally diverse chemicals and is thought to be secondary to toxicity caused by accumulation of hyaline droplets within the renal tubule epithelial cells.
However, measures of α2μ-globulin were not performed in the current study. While it is possible that the observed kidney lesions are secondary to α2μ-globulin nephropathy, the increases in kidney weights in both male and female rats suggest that another independent mechanism of toxicity may have played a role in the lesion development. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- SPERM MOTILITY AND VAGINAL CYTOLOGY:
There were significantly decreased sperm count in cauda epididymis in 200 and 400 ppm males compared to control group.
Females in the 400 ppm group displayed an apparent increase in cycle length and a slight increase in the percentage of the cycle spent in metestrus, relative to the chamber control group. However, the apparent increase in cycle length may be secondary to stress, as evidenced by lower body weight and mortality in the 400 ppm group. Alternatively, the apparent changes in the 400 ppm females may have been an artifact of having too few animals available to allow for meaningful interpretation.
The minor changes in cycle length observed only in the 400 ppm group, combined with a lack of ovarian histopathology findings, did not provide sufficient evidence for female reproductive toxicity potential under the conditions of the study.
Thus, the test item exposure by inhalation exhibits the potential to be a reproductive toxicant in male rats, but not in female rats. - Key result
- Dose descriptor:
- LOEL
- Effect level:
- 25 ppm
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- histopathology: non-neoplastic
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- 200 ppm
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: Significant decrease of sperm per cauda in 200 and 400 ppm.
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 25 ppm
- System:
- urinary
- Organ:
- kidney
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 200 ppm
- System:
- male reproductive system
- Organ:
- cauda epididymis
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- In a repeated dose toxicity study with alpha pinene after 14 weeks of vapour inhalation exposure to rats, increases of incidences of kidney lesions at 25 ppm and decrease of sperm per cauda at 200 ppm in male rats were reported, thus the LOEL was determined to be 25 ppm.
- Executive summary:
A 90-day repeated dose toxicity study (inhalation route) was performed on alpha pinene according to a similar method to OECD guideline 413 under GLP conditions. Groups of 10 male and 10 female rats were exposed to the test item by whole body inhalation at concentrations of 0, 25, 50, 100, 200, or 400 ppm, 6 hours per day, 5 days per week for 14 weeks. During the study, clinical observations, bodyweight, organ weight, haematology, clinical chemistry, macropathology, histopathology and sperm motility and vaginal cytology investigations were undertaken.
All exposed male rats survived to the end of the studies, while six 400 ppm female rats died before the end of the study. The major targets for test item toxicity were the liver, urinary system, and male reproductive system.
Relative liver weights were significantly greater compared to controls in males at 100 ppm and greater and in all females treated groups (LOEL=25 ppm), however, without accompanying histopathologic changes. Increased liver weight is a common finding in toxicity studies and can be associated with induction of liver metabolizing enzymes.
Absolute kidney weights were increased in male rats exposed to 100 ppm or greater and 50 and 200 ppm female rats; in males, these increases were accompanied by histopathologic lesions including granular casts and hyaline droplet accumulation at all exposure concentrations, as well as exposure concentration-dependent increases in the severity of nephropathy (LOEL=25 ppm), which is a common spontaneous lesion observed in male rats (α2μ-globulin nephropathy). This syndrome has been produced by structurally diverse chemicals and is thought to be secondary to toxicity caused by accumulation of hyaline droplets within the renal tubule epithelial cells. However, measures of α2μ-globulin were not performed in the current study. While it is possible that the observed kidney lesions are secondary to α2μ-globulin nephropathy, the increases in kidney weights in both male and female rats suggest that another independent mechanism of toxicity may have played a role in the lesion development.
There were also significantly lower numbers of sperm per cauda compared to controls in 200 and 400 ppm male rats (LOEL=200 ppm). There was an accompanying minor decrease in epididymal weights that did not reach significance. Therefore, the possibility that the change in absolute sperm per cauda was due to a decrease in epididymal weight cannot be excluded. A definitive conclusion by histopathologic investigations could not be conducted due to an artifact resulting from formalin fixation of the male reproductive tract tissues. Thus, further studies on reproductive function are warranted.
However, in females the minor changes in cycle length observed only in the 400 ppm group, combined with a lack of ovarian histopathology findings, does not provide sufficient evidence for reproductive toxicity.
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 29 November 2004 - 16 December 2004
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Reason / purpose for cross-reference:
- reference to other study
- Remarks:
- of which it is a range-finding study
- Principles of method if other than guideline:
- - Principle of test: Mice were exposed to alpha pinene via whole body inhalation 5 days per week for 17 days
- Short description of test conditions: see below
- Parameters analysed / observed: see below - GLP compliance:
- yes
- Remarks:
- In compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)
- Limit test:
- no
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: NTP colony maintained at Taconic Farms, Inc. (Germantown, NY)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: 23.1-23.9 g (male) and 19.8-20.6 g (female)
- Fasting period before study: not specified
- Housing: individually. Cages: Stainless steel, wire bottom (Lab Products, Inc., Seaford, DE); changed weekly. Cageboard: Untreated paper cage pan liner (Shepherd Specialty Papers, Kalamazoo, MI), changed daily
- Diet (e.g. ad libitum): NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (except during exposure periods)
- Water (e.g. ad libitum): Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum
- Acclimation period: Animals were quarantined for 11 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3ºF
- Humidity (%): 50% ± 15%
- Air changes (per hr): 15 ± 2/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- whole body
- Vehicle:
- not specified
- Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Test item was held in an 8-gallon stainless-steel chemical reservoir. Test item was pumped into a heated glass column filled with glass beads that increased the surface area for vaporization. Heated nitrogen entered the column from below and assisted in vaporizing the chemical while conveying it into a short distribution manifold. Concentration in the manifold was determined by the chemical pump rate, nitrogen flow rate, and dilution air flow rate. The pressure in the distribution manifold was kept fixed to ensure constant flow through the manifold and into all chambers as the flow of vapor to each chamber was adjusted.
Metering valves at the manifold controlled flow to each chamber through individual Teflon® delivery lines that carried the vapor from the manifold to three-way exposure valves at the chamber inlets. The exposure valves diverted vapor delivery to exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. To initiate exposure, the chamber exposure valves were rotated to allow the test item vapor to flow to each exposure chamber inlet duct where it was further diluted with filtered, conditioned air to achieve the desired exposure concentration.
- Temperature, humidity, pressure in air chamber: 72 ± 3ºF; 50% ± 15%.
- Air change rate: 15 air changes per hour
- Method of particle size determination: A condensation particle detector (Model 3022A, TSI, Inc., St. Paul, MN) was used with and without animals in the exposure chambers. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) were detected.
TEST ATMOSPHERE
- Brief description of analytical method used: on-line gas chromatograph. Samples were analyzed using GC/FID to measure the stability and purity of test item in the generation and delivery system. To assess whether impurities or degradation products coeluted with test item or the solvent, a second GC/FID analysis of the samples was performed using a polar column capable of resolving compounds with similar boiling points and polarities.
- Samples taken from breathing zone: yes.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Chamber and room concentrations of alpha pinene were monitored by an on-line gas chromatograph. Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period.
The average concentration measured were: 99.8 ± 1.6 ppm for the 100 ppm group, 200 ± 1 for the 200 ppm group, 404 ± 4ppm for the 400 ppm group, 794 ± 37 ppm for the 800 ppm group and 1540 ± 129 ppm for the 1600 ppm group. - Duration of treatment / exposure:
- 17 days; 6 hours plus T90 (12 minutes) per day.
- Frequency of treatment:
- Five times per week, weekdays only
- Dose / conc.:
- 0 ppm
- Dose / conc.:
- 100 ppm
- Remarks:
- (0.56 mg/L)
- Dose / conc.:
- 200 ppm
- Remarks:
- (1.13 mg/L)
- Dose / conc.:
- 400 ppm
- Remarks:
- (2.26 mg/L)
- Dose / conc.:
- 800 ppm
- Remarks:
- (4.53 mg/L)
- Dose / conc.:
- 1 600 ppm
- Remarks:
- (9.06 mg/L)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes
- Positive control:
- no
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily on exposure days and at the end of the studies.
BODY WEIGHT: Yes
- Time schedule for examinations: initially, on days 6 and 13, and at the end of the studies.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.
HISTOPATHOLOGY: Yes. Histopathology was performed on 0, 400, 800, and 1,600 ppm rats. In addition to gross lesions and tissue masses, the lung and nose were examined. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin. - Statistics:
- Calculation and Analysis of Lesion Incidences: Fisher exact test (Gart et al., 1979) was used to determine significance.
Analysis of Continuous Variables:
Organ and body weight data were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the exposure-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic exposure-related trend (Dunnett’s or Dunn’s test). - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Lethargy and abnormal breathing were observed in three 800 ppm males and two 1,600 ppm males, and lethargy was observed in one 1,600 ppm female. Ataxia was observed in two 800 ppm males, two 1,600 ppm males, and four 800 ppm females.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- All 800 and 1,600 ppm males and females died early.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- The final mean body weights and mean body weight gains of all surviving groups of exposed mice were similar to those of the chamber 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:
- effects observed, treatment-related
- Description (incidence and severity):
- The absolute and relative liver weights of 400 ppm males and females and the relative liver weight of 200 ppm males were significantly greater (up to 20%) than those of the chamber controls. The absolute and relative kidney weights of 100 ppm females were significantly greater (18% and 15%, respectively) than those of the chamber controls as was the relative kidney weight of 400 ppm males (12%).
- Gross pathological findings:
- not specified
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- In the nose, there were significantly increased incidences of minimal olfactory epithelial degeneration in 800 and 1,600 ppm males (chamber controls, 0/5; 100 ppm, 0/0; 200 ppm, 0/0; 400 ppm, 0/5; 800 ppm, 5/5; 1,600 ppm, 4/5) and females (0/5, 0/0, 0/0, 0/5, 4/5, 5/5). Degeneration was characterized by slight disorganization of the normal olfactory architecture in the Level I and II nasal sections and the epithelial cells in the mid layers of the olfactory epithelium were often pyknotic. The mucosa often had an undulating appearance, and strands of proteinaceous debris and/or mucus were present in the nasal passages.
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- Based on decreased survival and nonneoplastic lesions in the nose of 800 and 1,600 ppm males and females observed in this study, exposure concentrations of 0, 25, 50, 100, 200, and 400 ppm alpha pinene were selected for the 3-month study in mice.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 800 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- mortality
- Critical effects observed:
- not specified
- Conclusions:
- In the 2-week study with alpha-pinene, the two highest exposure concentrations (800 and 1600 ppm) were overtly toxic to mice, resulting in clinical findings of toxicity and death. Based on this experiment a maximal dose of 400 ppm was administrated in the 90-day study.
- Executive summary:
A 2-week repeated dose toxicity study (inhalation route) was performed on alpha pinene as a dose range-finding study prior to a 3 month main test. Groups of 5 male and 5 female mice were exposed to the test item by whole body inhalation at concentrations of 0, 100, 200, 400, 800 and 1600 ppm, 6 hours per day, 5 days per week for 17 days. During the study, clinical observations, bodyweight, organ weight, macropathology and histopathology investigations were undertaken. The two highest exposure concentrations (800 and 1,600 ppm) were overtly toxic to mice, resulting in clinical findings of toxicity (e.g., ataxia, tremors, abnormal breathing) and death. In the remaining exposed groups, there was evidence of a general increase in absolute and/or relative liver and kidney weights compared to chamber control mice of both sexes, but not considered to be life threatening. In the histopathologic exams only minimal olfactory epithelial degeneration of nasal tissue was found in male and female mice exposed to 800 and 1600 ppm. Therefore, 400 ppm was selected as the high concentration for the 3-month studies in mice.
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 29 November 2004 - 15 December 2004
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Reason / purpose for cross-reference:
- reference to other study
- Remarks:
- of which it is a range-finding study
- Principles of method if other than guideline:
- - Principle of test: Rats were exposed to alpha pinene via whole body inhalation 5 days per week for 16 days
- Short description of test conditions: see below
- Parameters analysed / observed: see below - GLP compliance:
- yes
- Remarks:
- In compliance with Food and Drug Administration Good Laboratory Practice Regulations (21 CFR, Part 58)
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: NTP colony maintained at Taconic Farms, Inc. (Germantown, NY)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: 100-102 g (male) and 90-92 g (female)
- Fasting period before study: not specified
- Housing: individually. Cages: Stainless steel, wire bottom (Lab Products, Inc., Seaford, DE); changed weekly. Cageboard: Untreated paper cage pan liner (Shepherd Specialty Papers, Kalamazoo, MI), changed daily
- Diet (e.g. ad libitum): NTP-2000 irradiated wafers (Zeigler Brothers, Inc., Gardners, PA), available ad libitum (except during exposure periods)
- Water (e.g. ad libitum): Tap water (Richland, WA, municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI); available ad libitum
- Acclimation period: Animals were quarantined for 11 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3ºF
- Humidity (%): 50% ± 15%
- Air changes (per hr): 15 ± 2/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- whole body
- Vehicle:
- not specified
- Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Test item was held in an 8-gallon stainless-steel chemical reservoir. Test item was pumped into a heated glass column filled with glass beads that increased the surface area for vaporization. Heated nitrogen entered the column from below and assisted in vaporizing the chemical while conveying it into a short distribution manifold. Concentration in the manifold was determined by the chemical pump rate, nitrogen flow rate, and dilution air flow rate. The pressure in the distribution manifold was kept fixed to ensure constant flow through the manifold and into all chambers as the flow of vapor to each chamber was adjusted.
Metering valves at the manifold controlled flow to each chamber through individual Teflon® delivery lines that carried the vapor from the manifold to three-way exposure valves at the chamber inlets. The exposure valves diverted vapor delivery to exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. To initiate exposure, the chamber exposure valves were rotated to allow the test item vapor to flow to each exposure chamber inlet duct where it was further diluted with filtered, conditioned air to achieve the desired exposure concentration.
- Temperature, humidity, pressure in air chamber: 72 ± 3ºF; 50% ± 15%.
- Air change rate: 15 air changes per hour
- Method of particle size determination: A condensation particle detector (Model 3022A, TSI, Inc., St. Paul, MN) was used with and without animals in the exposure chambers. No particle counts above the minimum resolvable level (approximately 200 particles/cm3) were detected.
TEST ATMOSPHERE
- Brief description of analytical method used: on-line gas chromatograph. Samples were analyzed using GC/FID to measure the stability and purity of test item in the generation and delivery system. To assess whether impurities or degradation products coeluted with test item or the solvent, a second GC/FID analysis of the samples was performed using a polar column capable of resolving compounds with similar boiling points and polarities.
- Samples taken from breathing zone: yes.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Chamber and room concentrations of alpha pinene were monitored by an on-line gas chromatograph. Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period.
The average concentration measured were: 99.6 ± 1.4 ppm for the 100 ppm group, 200 ± 1 for the 200 ppm group, 404 ± 4ppm for the 400 ppm group, 794 ± 37 ppm for the 800 ppm group and 1540 ± 130 ppm for the 1600 ppm group. - Duration of treatment / exposure:
- 16 days; 6 hours plus T90 (12 minutes) per day.
- Frequency of treatment:
- Five times per week, weekdays only
- Dose / conc.:
- 0 ppm
- Dose / conc.:
- 100 ppm
- Remarks:
- (0.56 mg/L)
- Dose / conc.:
- 200 ppm
- Remarks:
- (1.13 mg/L)
- Dose / conc.:
- 400 ppm
- Remarks:
- (2.26 mg/L)
- Dose / conc.:
- 800 ppm
- Remarks:
- (4.53 mg/L)
- Dose / conc.:
- 1 600 ppm
- Remarks:
- (9.06 mg/L)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes
- Positive control:
- no
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily on exposure days and at the end of the studies.
BODY WEIGHT: Yes
- Time schedule for examinations: initially, on days 6 and 13, and at the end of the studies.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.
HISTOPATHOLOGY: Yes. Histopathology was performed on 0, 400, 800, and 1,600 ppm rats. In addition to gross lesions and tissue masses, the lung and nose were examined. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin. - Statistics:
- Calculation and Analysis of Lesion Incidences: Fisher exact test (Gart et al., 1979) was used to determine significance.
Analysis of Continuous Variables:
Organ and body weight data were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).
Jonckheere’s test (Jonckheere, 1954) was used to assess the significance of the exposure-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic exposure-related trend (Dunnett’s or Dunn’s test). - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Abnormal breathing, ataxia, lethargy, and nasal/eye discharge occurred in one 1,600 ppm male. In rats exposed to 800 ppm, nasal/eye discharge was observed in two males and five females, ataxia was observed in two males and two females, and tremors and abnormal breathing were observed in one male. Nasal/eye discharge and tremors were observed in three females exposed to 400 ppm.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- All rats exposed to 1600 ppm were found dead by day 2. All rats exposed to 800 ppm were found dead by day 16.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Final mean body weights of all exposed rats that survived to the end of the study were similar to those of the chamber controls; the mean body weight gain of 400 ppm females was significantly less than that of the chamber control group.
- 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:
- effects observed, treatment-related
- Description (incidence and severity):
- The absolute liver weights of 400 ppm males and 200 ppm females were significantly greater than those of the chamber controls. The relative liver weights of 400 ppm males and all surviving groups of exposed females were significantly greater than those of the chamber controls. The absolute kidney weight of 200 ppm females was significantly greater than that of the chamber controls, and the relative kidney weights of all surviving groups of exposed males and 200 and 400 ppm females were significantly greater than those of the chamber controls. In females, the absolute lung weights of the 100 and 200 ppm groups and the relative lung weight of the 100 ppm group were significantly greater than those of the chamber controls.
There were no exposure-related microscopic findings. - Gross pathological findings:
- not specified
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- There were no exposure-related microscopic findings
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- Based on the mortality of 800 and 1600 ppm rats and a lack of significant histopathologic findings in rats exposed to 400 ppm or less, exposure concentrations of 0, 25, 50, 100, 200, and 400 ppm alpha pinene were selected for the 3-month rat study.
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 800 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- mortality
- Critical effects observed:
- not specified
- Conclusions:
- In the 2-week study with alpha-pinene, the two highest exposure concentrations (800 and 1600 ppm) were overtly toxic to rats, resulting in clinical findings of toxicity and death. Based on this experiment a maximal dose of 400 ppm was administrated in the 90-day study.
- Executive summary:
A 2-week repeated dose toxicity study (inhalation route) was performed on alpha pinene as a dose range-finding study prior to a 3 month main test. Groups of 5 male and 5 female rats were exposed to the test item by whole body inhalation at concentrations of 0, 100, 200, 400, 800 and 1600 ppm, 6 hours per day, 5 days per week for 16 days. During the study, clinical observations, bodyweight, organ weight, macropathology and histopathology investigations were undertaken. The two highest exposure concentrations (800 and 1,600 ppm) were overtly toxic to rats, resulting in clinical findings of toxicity (e.g., ataxia, tremors, abnormal breathing) and death. In the remaining exposed groups, there was evidence of a general increase in absolute and/or relative liver and kidney weights compared to chamber control rats of both sexes, but not considered to be life threatening. There were no significant histopathologic findings in rats exposed to 400 ppm or less. Therefore, 400 ppm was selected as the high concentration for the 3-month studies in rats.
Referenceopen allclose all
Table 1: Survival and Body Weights
|
Concentration (ppm) |
Survivalb |
Initial Body Weight (g) |
Final Body Weight (g) |
Change in Body Weight (g) |
Final Weight Relative to Controls (%) |
Male
|
0 |
10/10 |
22.9 ± 0.2 |
37.1 ± 0.6 |
14.3 ± 0.6 |
|
25 |
10/10 |
23.0 ± 0.3 |
36.9 ± 0.7 |
13.9 ± 0.8 |
99 |
|
50 |
10/10 |
22.7 ± 0.3 |
38.3 ± 0.9 |
15.6 ± 0.8 |
103 |
|
100 |
10/10 |
22.5 ± 0.2 |
35.9 ± 0.7 |
13.4 ± 0.7 |
97 |
|
200 |
10/10 |
22.8 ± 0.3 |
35.5 ± 1.0 |
12.7 ± 0.9 |
96 |
|
400 |
10/10 |
22.8 ± 0.2 |
36.2 ± 0.5 |
13.5 ± 0.4 |
98 |
|
Female |
0 |
10/10 |
19.5 ± 0.4 |
31.5 ± 0.6 |
12.0 ± 0.5 |
|
25 |
10/10 |
19.6 ± 0.4 |
30.3 ± 0.6 |
10.8 ± 0.7 |
96 |
|
50 |
10/10 |
19.7 ± 0.3 |
32.7 ± 0.7 |
12.9 ± 0.7 |
104 |
|
100 |
10/10 |
19.7 ± 0.4 |
31.5 ± 1.1 |
11.8 ± 0.9 |
100 |
|
200 |
10/10 |
19.3 ± 0.3 |
30.7 ± 0.6 |
11.4 ± 0.6 |
97 |
|
400 |
10/10 |
19.4 ± 0.3 |
30.6 ± 0.5 |
11.2 ± 0.4 |
97 |
a Weights and weight changes are given as mean ± standard error.
b Number of animals surviving at 14 weeks/number initially in group
Table 2: Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios
|
Chamber control |
25 ppm |
50 ppm |
100 ppm |
200 ppm |
400 ppm |
Male |
||||||
n |
10 |
10 |
10 |
10 |
10 |
10 |
Necropsy body wt |
37.1 ± 0.6 |
36.9 ± 0.7 |
38.3 ± 0.9 |
35.9 ± 0.7 |
35.5 ± 1.0 |
36.2 ± 0.5 |
R Kidney absolute |
0.330 ± 0.006 |
0.318 ± 0.009 |
0.336 ± 0.010 |
0.309 ± 0.008 |
0.295 ± 0.006* |
0.307 ± 0.007* |
R kidney relative |
8.903 ± 0.167 |
8.629 ± 0.208 |
8.793 ± 0.267 |
8.617 ± 0.205 |
8.348 ± 0.145 |
8.469 ± 0.155 |
Liver absolute |
1.617 ± 0.022 |
1.589 ± 0.028 |
1.702 ± 0.040 |
1.637 ± 0.024 |
1.660 ± 0.043 |
1.957 ± 0.057** |
Liver relative |
43.671 ± 0.880 |
43.123 ± 0.458 |
44.487 ± 0.806 |
45.651 ± 0.678 |
46.903 ± 0.750* |
54.009 ± 1.465** |
Thymus absolute |
0.066 ± 0.004 |
0.063 ± 0.004 |
0.067 ± 0.003 |
0.057 ± 0.001 |
0.062 ± 0.004 |
0.051 ± 0.003** |
Thymus relative |
1.777 ± 0.081 |
1.699 ± 0.090 |
1.742 ± 0.063 |
1.591 ± 0.052 |
1.739 ± 0.115 |
1.397 ± 0.081** |
Female |
||||||
n |
10 |
10 |
10 |
10 |
10 |
10 |
Necropsy body wt |
31.5 ± 0.6 |
30.3 ± 0.6 |
32.7 ± 0.7 |
31.5 ± 1.1 |
30.7 ± 0.6 |
30.6 ± 0.5 |
Liver absolute |
1.466 ± 0.041 |
1.475 ± 0.053 |
1.442 ± 0.036 |
1.548 ± 0.053 |
1.587 ± 0.037 |
1.730 ± 0.032** |
Liver relative |
46.542 ± 0.988 |
48.567 ± 1.239 |
44.214 ± 0.880 |
49.280 ± 0.672* |
51.728 ± 0.795** |
56.511 ± 0.705** |
* Significantly different (P≤0.05) from the chamber control group by Williams’ test
** Significantly different (P≤0.01) from the chamber control group by Williams’ or Dunnett’s test
a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean ± standard error).
Table 3: Hematology data
Treatment concentration (ppm) |
0 (control group) |
25 |
50 |
100 |
200 |
400 |
Male |
|
|
|
|
|
|
Hematocrit (spun) (%) |
51.3 ± 0.3 |
50.5 ± 0.4 |
50.1 ± 0.3 |
51.1 ± 0.3 |
50.9 ± 0.4 |
49.8 ± 0.3* |
Hemoglobin (g/dL) |
16.0 ± 0.1 |
16.0 ± 0.1 |
15.7 ± 0.1 |
16.0 ± 0.0 |
16.1 ± 0.1 |
15.7 ± 0.1 |
Erythrocytes (106/µL) |
10.51 ± 0.06 |
10.47 ± 0.06 |
10.23 ± 0.09 |
10.52 ± 0.04 |
10.55 ± 0.08 |
10.10 ± 0.07** |
Reticulocytes(103/µL) |
223.7 ± 19.4 |
200.3 ± 14.9 |
193.9 ± 16.4 |
205.2 ± 13.0 |
214.3 ± 16.4 |
202.2 ± 15.9 |
Nucleated erythrocytes/100 leukocytes |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.00 ± 0.00 |
Mean cell volume (fL) |
49.3 ± 0.3 |
49.2 ± 0.2 |
49.6 ± 0.2 |
49.4 ± 0.2 |
49.6 ± 0.2 |
50.6 ± 0.2** |
Mean cell hemoglobin (pg) |
15.3 ± 0.1 |
15.2 ± 0.1 |
15.4 ± 0.1 |
15.2 ± 0.0 |
15.2 ± 0.0 |
15.6 ± 0.1* |
Mean cell hemoglobin concentration (g/dL) |
31.0 ± 0.2 |
31.0 ± 0.1 |
31.0 ± 0.1 |
30.8 ± 0.2 |
30.7 ± 0.1 |
30.8 ± 0.1 |
Leukocytes (103/µL) |
3.10 ± 0.40 |
2.94 ± 0.43 |
2.03 ± 0.26 |
2.47 ± 0.15 |
2.31 ± 0.26 |
1.87 ± 0.17* |
Lymphocytes (103/µL) |
2.60 ± 0.34 |
2.41 ± 0.39 |
1.73 ± 0.23 |
2.03 ± 0.13 |
1.93 ± 0.22 |
1.48 ± 0.14* |
Female |
|
|
|
|
|
|
Hematocrit (spun) (%) |
49.6 ± 0.3 |
50.1 ± 0.4 |
49.4 ± 0.5 |
50.1 ± 0.3 |
48.9 ± 0.3 |
48.3 ± 0.3* |
Hemoglobin (g/dL) |
15.8 ± 0.1 |
16.0 ± 0.1 |
15.7 ± 0.2 |
15.9 ± 0.1 |
15.5 ± 0.1 |
15.5 ± 0.1* |
Erythrocytes (106/µL) |
10.21 ± 0.05 |
10.26 ± 0.06 |
10.10 ± 0.11 |
10.14 ± 0.06 |
9.96 ± 0.09* |
9.85 ± 0.08** |
Reticulocytes(103/µL) |
269.5 ± 15.4 |
248.9 ± 14.9 |
251.9 ± 16.5 |
282.5 ± 18.3 |
240.1 ± 20.8 |
251.2 ± 15.3 |
Nucleated erythrocytes/100 leukocytes |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.00 ± 0.00 |
0.00 ± 0.00 |
Mean cell volume (fL) |
49.3 ± 0.2 |
49.7 ± 0.2 |
49.5 ± 0.3 |
50.1 ± 0.2* |
49.7 ± 0.2 |
49.7 ± 0.2 |
Mean cell hemoglobin (pg) |
15.5 ± 0.1 |
15.6 ± 0.1 |
15.5 ± 0.1 |
15.6 ± 0.1 |
15.6 ± 0.1 |
15.7 ± 0.1 |
Mean cell hemoglobin concentration (g/dL) |
31.5 ± 0.2 |
31.4 ± 0.1 |
31.4 ± 0.2 |
31.1 ± 0.1 |
31.4 ± 0.1 |
31.6 ± 0.1 |
Leukocytes (103/µL) |
3.65 ± 0.35 |
3.10 ± 0.27 |
3.34 ± 0.32 |
2.80 ± 0.29 |
3.11 ± 0.32 |
3.16 ± 0.34 |
Lymphocytes (103/µL) |
3.09 ± 0.30 |
2.65 ± 0.23 |
2.78 ± 0.30 |
2.39 ± 0.25 |
2.60 ± 0.26 |
2.66 ± 0.27 |
* Significantly different (P≤0.05) from the chamber control group by Dunn’s or Shirley’s test
** P≤0.01
a Data are presented as mean ± standard error.Statistical tests were performed on unrounded data.
Table 4: Incidence of Non neoplastic lesions of the urinary bladder
Sex |
Treatment concentration (ppm) |
0 (control group) |
25 |
50 |
100 |
200 |
400 |
Male |
Number Examined Microscopically |
10 |
10 |
10 |
10 |
10 |
10 |
Male |
Transitional Epithelium, Hyperplasiaa |
0 |
0 |
0 |
7** (1.0)b
|
10** (2.0)b
|
10** (2.5)b
|
Female |
Number Examined Microscopically |
10 |
10 |
10 |
10 |
10 |
10 |
Female |
Transitional Epithelium, Hyperplasiaa |
0 |
0 |
0 |
6** (1.0)b
|
10** (1.6)b
|
10** (2.2)b
|
** Significantly different (P≤0.01) from the chamber control group by the Fisher exact test
a Number of animals with lesion
b Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked
Table 5: Epididymal Spermatozoal Measurements for Male Mice
|
Chamber control |
100 ppm |
200 ppm |
400 ppm |
n |
10 |
10 |
10 |
10 |
Epididymal spermatozoal measurements |
||||
Sperm motility (%) |
90.25 ± 0.34 |
88.31 ± 0.86 |
89.74 ± 0.80 |
87.95 ± 1.08 |
Sperm (103/mg cauda epididymis) |
704.8 ± 64.9 |
690.7 ± 55.9 |
537.5 ± 27.0* |
445.8 ± 13.5** |
Sperm (106/cauda epididymis) |
24.45 ± 0.95 |
18.40 ± 0.41** |
16.48 ± 0.72** |
14.64 ± 0.25** |
* Significantly different (P≤0.05) from the chamber control group by Shirley’s test.
** P≤0.01
a Data are presented as mean ± standard error.
Table 1: Survival and Body Weights
|
Concentration (ppm) |
Survivalb |
Initial BodyWeight (g) |
Final BodyWeight (g) |
Change in BodyWeight (g) |
Final Weight Relativeto Controls(%) |
Male |
0 |
10/10 |
98 ± 3 |
335 ± 6 |
238 ± 5 |
|
25 |
10/10 |
98 ± 2 |
329 ± 11 |
231 ± 9 |
98 |
|
50 |
10/10 |
98 ± 2 |
333 ± 6 |
235 ± 5 |
99 |
|
100 |
10/10 |
98 ± 2 |
334 ± 7 |
236 ± 5 |
100 |
|
200 |
10/10 |
96 ± 2 |
330 ± 4 |
234 ± 4 |
98 |
|
400 |
10/10 |
97 ± 2 |
322 ± 6 |
225 ± 7 |
96 |
|
Female |
0 |
10/10 |
89 ± 2 |
194 ± 3 |
105 ± 3 |
|
25 |
10/10 |
89 ± 2 |
199 ± 4 |
110 ± 4 |
102 |
|
50 |
10/10 |
89 ± 2 |
206 ± 4 |
117 ± 4 |
106 |
|
100 |
10/10 |
88 ± 2 |
199 ± 3 |
112 ± 2 |
103 |
|
200 |
10/10 |
88 ± 2 |
201 ± 3 |
113 ± 2 |
104 |
|
400 |
4/10c |
89 ± 2 |
159 ± 5** |
72 ± 5** |
82 |
** Significantly different (P≤0.01) from the chamber control group by unnett’s test
a Weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of the study.
b Number of animals surviving at 14 weeks/number initially in group
c Weeks of death: 6, 6, 6, 6, 8, 13
Table 2: Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios
|
Chamber control |
25 ppm |
50 ppm |
100 ppm |
200 ppm |
400 ppm |
Male |
||||||
n |
10 |
10 |
10 |
10 |
10 |
10 |
Necropsy body wt |
335 ± 6 |
329 ± 11 |
333 ± 6 |
334 ± 7 |
330 ± 4 |
322 ± 6 |
R Kidney absolute |
1.025 ± 0.019 |
1.012 ± 0.037 |
1.061 ± 0.026 |
1.137 ± 0.027** |
1.209 ± 0.020** |
1.286 ± 0.039** |
R kidney relative |
3.058 ± 0.038 |
3.073 ± 0.037 |
3.186 ± 0.042* |
3.405 ± 0.036** |
3.660 ± 0.040** |
3.991 ± 0.056** |
Liver absolute |
10.54 ± 0.27 |
10.31 ± 0.40 |
10.44 ± 0.32 |
11.08 ± 0.36 |
11.37 ± 0.26 |
11.87 ± 0.45* |
Liver relative |
31.402 ± 0.375 |
31.270 ± 0.317 |
31.298 ± 0.490 |
33.152 ± 0.569* |
34.393 ± 0.531** |
36.807 ± 0.864** |
Spleen absolute |
0.628 ± 0.012 |
0.630 ± 0.013 |
0.663 ± 0.014 |
0.659 ± 0.009 |
0.655 ± 0.010 |
0.677 ± 0.023* |
Spleen relative |
1.874 ± 0.028 |
1.925 ± 0.045 |
1.997 ± 0.058 |
1.978 ± 0.030 |
1.983 ± 0.022 |
2.103 ± 0.057** |
Female |
||||||
n |
10 |
10 |
10 |
10 |
10 |
4 |
Necropsy body wt |
194 ± 3 |
199 ± 4 |
206 ± 4 |
199 ± 3 |
201 ± 3 |
159 ± 5** |
Heart absolute |
0.584 ± 0.010 |
0.612 ± 0.012 |
0.618 ± 0.010 |
0.629 ± 0.012* |
0.638 ± 0.011** |
0.530 ± 0.006* |
Heart relative |
3.010 ± 0.039 |
3.081 ± 0.054 |
3.002 ± 0.041 |
3.156 ± 0.034* |
3.175 ± 0.049* |
3.349 ± 0.084** |
R Kidney absolute |
0.618 ± 0.011 |
0.641 ± 0.009 |
0.680 ± 0.013** |
0.659 ± 0.015 |
0.679 ± 0.014** |
0.595 ± 0.021 |
R kidney relative |
3.185 ± 0.040 |
3.230 ± 0.062 |
3.301 ± 0.041 |
3.307 ± 0.058 |
3.376 ± 0.050* |
3.757 ± 0.138** |
Liver absolute |
5.486 ± 0.179 |
5.990 ± 0.121 |
6.270 ± 0.115** |
6.269 ± 0.151** |
6.424 ± 0.144** |
4.840 ± 0.247 |
Liver relative |
28.216 ± 0.637 |
30.152 ± 0.550** |
30.438 ± 0.319** |
31.459 ± 0.586** |
31.916 ± 0.317** |
30.470 ± 0.715** |
Thymus absolute |
0.347 ± 0.012 |
0.349 ± 0.010 |
0.352 ± 0.010 |
0.346 ± 0.010 |
0.330 ± 0.014 |
0.204 ± 0.010** |
Thymus relative |
1.785 ± 0.054 |
1.751 ± 0.029 |
1.707 ± 0.041 |
1.739 ± 0.048 |
1.638 ± 0.058* |
1.286 ± 0.035** |
* Significantly different (P≤0.05) from the chamber control group by Williams’ or Dunnett’s test
** P≤0.01
a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean ± standard error).
Table 3: Hematology data
Treatment dose (ppm) |
0 |
25 |
50 |
100 |
200 |
400 |
Male |
||||||
Hematocrit (spun) (%) |
|
|
|
|
|
|
Day 4 |
45.6 ± 0.3 |
45.1 ± 0.6 |
46.5 ± 0.6 |
45.5 ± 0.4 |
44.8 ± 0.5 |
44.4 ± 0.4 |
Day 23 |
47.9 ± 0.5 |
48.0 ± 0.6 |
47.4 ± 0.4 |
47.8 ± 0.4 |
47.7 ± 0.3 |
48.6 ± 0.5 |
Week 14 |
49.5 ± 0.5 |
48.3 ± 0.4 |
49.0 ± 0.3 |
48.3 ± 0.7* |
47.6 ± 0.3** |
47.7 ± 0.4** |
Packed cell volume (mL/dL) |
|
|
|
|
|
|
Day 4 |
44.6 ± 0.4 |
44.1 ± 0.6 |
45.0 ± 0.6 |
44.2 ± 0.4 |
43.1 ± 0.3* |
43.3 ± 0.4* |
Day 23 |
47.4 ± 0.4 |
47.1 ± 0.5 |
46.5 ± 0.5 |
47.2 ± 0.4 |
46.9 ± 0.4 |
48.1 ± 0.4 |
Week 14 |
49.9 ± 0.5 |
48.9 ± 0.4 |
49.5 ± 0.3 |
48.3 ± 0.3* |
48.0 ± 0.3** |
47.8 ± 0.6** |
Hemoglobin (g/dL) |
|
|
|
|
|
|
Day 4 |
13.5 ± 0.1 |
13.4 ± 0.2 |
13.7 ± 0.1 |
13.6 ± 0.2 |
13.3 ± 0.1 |
13.2 ± 0.1 |
Day 23 |
14.9 ± 0.1 |
14.9 ± 0.1 |
14.6 ± 0.1 |
15.0 ± 0.1 |
14.8 ± 0.1 |
15.1 ± 0.1 |
Week 14 |
15.7 ± 0.1 |
15.5 ± 0.1 |
15.5 ± 0.1 |
15.3 ± 0.1* |
15.0 ± 0.1** |
15.1 ± 0.2** |
Erythrocytes (106/µL) |
|
|
|
|
|
|
Day 4 |
7.15 ± 0.09 |
7.12 ± 0.11 |
7.27 ± 0.07 |
7.26 ± 0.08 |
7.09 ± 0.07 |
7.06 ± 0.07 |
Day 23 |
8.06 ± 0.06 |
7.95 ± 0.07 |
7.84 ± 0.09 |
8.04 ± 0.08 |
7.92 ± 0.07 |
8.10 ± 0.06 |
Week 14 |
9.35 ± 0.07 |
9.09 ± 0.05 |
9.25 ± 0.06 |
8.94 ± 0.05** |
8.92 ± 0.08** |
8.86 ± 0.10** |
Mean cell volume (fL) |
|
|
|
|
|
|
Day 4 |
62.3 ± 0.3 |
62.0 ± 0.4 |
61.8 ± 0.3 |
60.9 ± 0.5 |
60.9 ± 0.4* |
61.3 ± 0.4 |
Day 23 |
58.8 ± 0.2 |
59.2 ± 0.4 |
59.4 ± 0.2 |
58.8 ± 0.4 |
59.3 ± 0.4 |
59.4 ± 0.3 |
Week 14 |
53.4 ± 0.2 |
53.9 ± 0.4 |
53.5 ± 0.3 |
54.0 ± 0.4 |
53.9 ± 0.3 |
53.9 ± 0.3 |
Mean cell hemoglobin (pg) |
|
|
|
|
|
|
Day 4 |
18.8 ± 0.1 |
18.8 ± 0.1 |
18.9 ± 0.0 |
18.8 ± 0.1 |
18.8 ± 0.1 |
18.7 ± 0.0 |
Day 23 |
18.5 ± 0.1 |
18.7 ± 0.1 |
18.7 ± 0.1 |
18.6 ± 0.1 |
18.6 ± 0.1 |
18.6 ± 0.1 |
Week 14 |
16.9 ± 0.1 |
17.0 ± 0.1 |
16.8 ± 0.1 |
17.1 ± 0.1 |
16.9 ± 0.1 |
17.1 ± 0.1 |
Mean cell hemoglobin concentration (g/dL) |
||||||
Day 4 |
30.2 ± 0.1 |
30.4 ± 0.2 |
30.5 ± 0.1 |
30.9 ± 0.2* |
30.9 ± 0.1** |
30.5 ± 0.1 |
Day 23 |
31.5 ± 0.2 |
31.7 ± 0.2 |
31.4 ± 0.2 |
31.7 ± 0.2 |
31.5 ± 0.1 |
31.4 ± 0.2 |
Week 14 |
31.6 ± 0.1 |
31.6 ± 0.1 |
31.4 ± 0.1 |
31.5 ± 0.1 |
31.3 ± 0.1 |
31.7 ± 0.2 |
Leukocytes (103/µL) |
|
|
|
|
|
|
Day 4 |
9.08 ± 0.45 |
8.94 ± 0.32 |
9.75 ± 0.44 |
9.04 ± 0.72 |
7.57 ± 0.36* |
6.95 ± 0.25** |
Day 23 |
6.29 ± 0.23 |
7.10 ± 0.25 |
7.44 ± 0.29 |
8.12 ± 0.42** |
7.52 ± 0.64 |
7.20 ± 0.31 |
Week 14 |
7.01 ± 0.36 |
6.99 ± 0.39 |
7.86 ± 0.25 |
7.34 ± 0.37 |
6.71 ± 0.40 |
6.69 ± 0.47 |
Lymphocytes (103/µL) |
|
|
|
|
|
|
Day 4 |
7.99 ± 0.40 |
7.82 ± 0.30 |
8.39 ± 0.38 |
7.75 ± 0.61 |
6.44 ± 0.33** |
5.88 ± 0.24** |
Day 23 |
5.25 ± 0.23 |
5.91 ± 0.25 |
6.38 ± 0.22d |
6.89 ± 0.40* |
6.10 ± 0.57 |
6.06 ± 0.27 |
Week 14 |
5.36 ± 0.35 |
5.31 ± 0.39 |
6.18 ± 0.25 |
5.66 ± 0.40 |
5.12 ± 0.41 |
4.93 ± 0.38 |
Treatment dose (ppm) |
0 |
25 |
50 |
100 |
200 |
400 |
female |
||||||
Hematocrit (spun) (%) |
|
|
|
|
|
|
Day 4 |
47.7 ± 0.4 |
47.0 ± 0.2 |
47.0 ± 0.2 |
47.5 ± 0.3 |
47.1 ± 0.6 |
46.2 ± 0.4 |
Day 23 |
48.7 ± 0.4 |
49.2 ± 0.5 |
49.1 ± 0.4 |
49.2 ± 0.3 |
48.9 ± 0.5 |
49.7 ± 0.6 |
Week 14 |
48.9 ± 0.4 |
47.2 ± 0.4* |
47.8 ± 0.2 |
48.3 ± 0.4 |
48.7 ± 0.4 |
50.9 ± 0.8 |
Packed cell volume (mL/dL) |
|
|
|
|
|
|
Day 4 |
46.7 ± 0.5 |
46.1 ± 0.3 |
46.0 ± 0.3 |
46.8 ± 0.4 |
46.1 ± 0.5 |
45.3 ± 0.5 |
Day 23 |
48.5 ± 0.4 |
49.0 ± 0.4 |
49.3 ± 0.3 |
49.2 ± 0.3 |
48.8 ± 0.3 |
50.0 ± 0.6* |
Week 14 |
49.1 ± 0.3 |
48.6 ± 0.3 |
48.7 ± 0. |
49.0 ± 0.5 |
49.7 ± 0.4 |
52.7 ± 0.4 |
Hemoglobin (g/dL) |
|
|
|
|
|
|
Day 4 |
14.3 ± 0.1 |
14.2 ± 0.1 |
14.2 ± 0.1 |
14.5 ± 0.1 |
14.3 ± 0.1 |
14.1 ± 0.1 |
Day 23 |
15.3 ± 0.1 |
15.5 ± 0.1 |
15.5 ± 0.1 |
15.5 ± 0.1 |
15.3 ± 0.1 |
15.7 ± 0.1 |
Week 14 |
15.7 ± 0.1 |
15.4 ± 0.1 |
15.5 ± 0.1 |
15.6 ± 0.1 |
15.8 ± 0.1 |
16.7 ± 0.1 |
Erythrocytes (106/µL) |
|
|
|
|
|
|
Day 4 |
7.64 ± 0.07 |
7.56 ± 0.05 |
7.56 ± 0.05 |
7.74 ± 0.07 |
7.67 ± 0.10 |
7.55 ± 0.08 |
Day 23 |
8.13 ± 0.08 |
8.13 ± 0.08 |
8.20 ± 0.07 |
8.24 ± 0.05 |
8.13 ± 0.06 |
8.31 ± 0.09 |
Week 14 |
8.67 ± 0.06 |
8.53 ± 0.05 |
8.54 ± 0.06 |
8.62 ± 0.09 |
8.71 ± 0.06 |
9.23 ± 0.09 |
Mean cell volume (fL) |
|
|
|
|
|
|
Day 4 |
61.1 ± 0.3 |
60.9 ± 0.3 |
60.9 ± 0.2 |
60.5 ± 0.3 |
60.2 ± 0.5 |
60.0 ± 0.3* |
Day 23 |
59.6 ± 0.3 |
60.3 ± 0.3 |
60.1 ± 0.3 |
59.7 ± 0.3 |
60.1 ± 0.3 |
60.2 ± 0.2 |
Week 14 |
56.6 ± 0.2 |
56.9 ± 0.1 |
57.0 ± 0.1 |
56.9 ± 0.1 |
57.0 ± 0.2 |
57.1 ± 0.3 |
Mean cell hemoglobin (pg) |
|
|
|
|
|
|
Day 4 |
18.7 ± 0.1 |
18.8 ± 0.1 |
18.8 ± 0.1 |
18.7 ± 0.1 |
18.7 ± 0.1 |
18.7 ± 0.1 |
Day 23 |
18.8 ± 0.1 |
19.0 ± 0.1 |
18.9 ± 0.1 |
18.8 ± 0.1 |
18.8 ± 0.1 |
18.9 ± 0.1 |
Week 14 |
18.1 ± 0.0 |
18.1 ± 0.1 |
18.2 ± 0.1 |
18.1 ± 0.1 |
18.1 ± 0.0 |
18.1 ± 0.0 |
Mean cell hemoglobin concentration (g/dL) |
||||||
Day 4 |
30.6 ± 0.2 |
30.8 ± 0.1 |
30.8 ± 0.1 |
31.0 ± 0.2 |
31.1 ± 0.2 |
31.2 ± 0.2 |
Day 23 |
31.6 ± 0.2 |
31.5 ± 0.1 |
31.4 ± 0.1 |
31.6 ± 0.1 |
31.4 ± 0.1 |
31.4 ± 0.1 |
Week 14
|
32.1 ± 0.1 |
31.8 ± 0.1 |
31.9 ± 0.1 |
31.9 ± 0.1 |
31.8 ± 0.1 |
31.7 ± 0.2 |
Leukocytes (103/µL) |
|
|
|
|
|
|
Day 4 |
10.52 ± 0.52 |
10.89 ± 0.26 |
10.25 ± 0.34 |
11.26 ± 0.61 |
10.39 ± 0.63 |
8.52 ± 0.68 |
Day 23 |
7.96 ± 0.36 |
8.01 ± 0.24 |
7.87 ± 0.43 |
8.04 ± 0.39 |
7.78 ± 0.56 |
6.84 ± 0.48 |
Week 14 |
5.86 ± 0.27 |
5.70 ± 0.24 |
6.05 ± 0.29 |
5.60 ± 0.29 |
5.22 ± 0.33 |
6.08 ± 0.58 |
Lymphocytes (103/µL) |
|
|
|
|
|
|
Day 4 |
9.34 ± 0.52 |
9.58 ± 0.25 |
8.90 ± 0.30 |
9.76 ± 0.59 |
9.19 ± 0.51d |
7.34 ± 0.62 |
Day 23 |
6.79 ± 0.35 |
6.86 ± 0.20 |
6.83 ± 0.41 |
6.96 ± 0.38 |
6.62 ± 0.54 |
5.83 ± 0.42 |
Week 14 |
4.67 ± 0.24 |
4.44 ± 0.25 |
4.67 ± 0.23 |
4.36 ± 0.28 |
4.17 ± 0.29 |
4.93 ± 0.53 |
* Significantly different (P≤0.05) from the chamber control group by Dunn’s or Shirley’s test
** P≤0.01
Table 4: clinical chemistry data
Male |
|||||||||
Dose treatment (ppm) |
0 (control group) |
25
|
50 |
100 |
200 |
400 |
|||
Urea nitrogen (mg/dL) |
|
|
|
|
|
|
|||
Day 4 |
7.5 ± 0.4 |
7.8 ± 0.4 |
7.5 ± 0.3 |
7.4 ± 0.3 |
7.0 ± 0.4 |
7.5 ± 0.4 |
|||
Day 23 |
9.9 ± 0.5 |
8.9 ± 0.4 |
9.1 ± 0.2 |
9.5 ± 0.3 |
9.8 ± 0.4 |
11.4 ± 0.6 |
|||
Week 14 |
12.3 ± 0.3 |
13.7 ± 0.3* |
12.8 ± 0.3 |
13.3 ± 0.2 |
13.3 ± 0.3 |
13.6 ± 0.4* |
|||
Creatinine (mg/dL) |
|
|
|
|
|
|
|||
Day 4 |
0.29 ± 0.01 |
0.26 ± 0.02 |
0.23 ± 0.02* |
0.25 ± 0.02 |
0.25 ± 0.02 |
0.24 ± 0.02 |
|||
Day 23 |
0.30 ± 0.00 |
0.32 ± 0.01 |
0.32 ± 0.03 |
0.31 ± 0.01 |
0.36 ± 0.02** |
0.38 ± 0.01** |
|||
Week 14 |
0.37 ± 0.02 |
0.37 ± 0.02 |
0.37 ± 0.03 |
0.39 ± 0.02 |
0.39 ± 0.01 |
0.40 ± 0.03 |
|||
Glucose (mg/dL) Day |
|||||||||
Day 4 |
137 ± 3 |
134 ± 1 |
133 ± 5 |
137 ± 3 |
139 ± 6 |
130 ± 2 |
|||
Day 23 |
145 ± 12 |
126 ± 7 |
134 ± 9 |
127 ± 5 |
117 ± 4 |
116 ± 5 |
|||
Week 14 |
127 ± 2 |
130 ± 3 |
124 ± 2 |
129 ± 3 |
136 ± 6 |
128 ± 3 |
|||
Total protein (g/dL) |
|
|
|
|
|
|
|||
Day 4 |
6.0 ± 0.0 |
6.0 ± 0.1 |
6.1 ± 0.1 |
6.0 ± 0.0 |
6.1 ± 0.1 |
6.1 ± 0.0 |
|||
Day 23 |
6.5 ± 0.1 |
6.5 ± 0.1 |
6.5 ± 0.1 |
6.5 ± 0.1 |
6.8 ± 0.1** |
6.8 ± 0.1** |
|||
Week 14 |
7.4 ± 0.1 |
7.4 ± 0.1 |
7.5 ± 0.1 |
7.4 ± 0.1 |
7.5 ± 0.1 |
7.5 ± 0.0 |
|||
Albumin (g/dL) |
|
|
|
|
|
|
|||
Day 4 |
4.3 ± 0.0 |
4.3 ± 0.0 |
4.3 ± 0.0 |
4.3 ± 0.0 |
4.4 ± 0.0 |
4.4 ± 0.0 |
|||
Day 23 |
4.6 ± 0.0 |
4.6 ± 0.0 |
4.5 ± 0.0 |
4.5 ± 0.1 |
4.7 ± 0.0 |
4.7 ± 0.1 |
|||
Week 14 |
4.9 ± 0.1 |
4.9 ± 0.0 |
4.9 ± 0.1 |
4.8 ± 0.0 |
4.9 ± 0.0 |
4.9 ± 0.0 |
|||
Globulin (g/dL) |
|
|
|
|
|
|
|||
Day 4 |
1.7 ± 0.0 |
1.7 ± 0.0 |
1.8 ± 0.0 |
1.7 ± 0.0 |
1.8 ± 0.0 |
1.8 ± 0.0 |
|||
Day 23 |
1.9 ± 0.0 |
2.0 ± 0.0 |
2.0 ± 0.0 |
2.0 ± 0.0 |
2.1 ± 0.0** |
2.1 ± 0.0** |
|||
Week 14 |
2.6 ± 0.0 |
2.5 ± 0.0 |
2.6 ± 0.0 |
2.5 ± 0.0 |
2.6 ± 0.0 |
2.6 ± 0.0 |
|||
A/G ratio |
|||||||||
Day 4 |
2.5 ± 0.0 |
2.5 ± 0.0 |
2.5 ± 0.0 |
2.5 ± 0.0 |
2.5 ± 0.0 |
2.4 ± 0.0 |
|||
Day 23 |
2.4 ± 0.0 |
2.4 ± 0.0 |
2.3 ± 0.1 |
2.3 ± 0.0 |
2.3 ± 0.0 |
2.2 ± 0.0** |
|||
Week 14 |
1.9 ± 0.0 |
1.9 ± 0.0 |
1.9 ± 0.0 |
1.9 ± 0.0 |
1.9 ± 0.0 |
2.0 ± 0.0 |
|||
Alanine aminotransferase (IU/L) |
|
|
|
|
|
|
|||
Day 4 |
57 ± 1 |
57 ± 1 |
55 ± 1 |
53 ± 1 |
55 ± 1 |
52 ± 1** |
|||
Day 23 |
41 ± 1 |
41 ± 1 |
41 ± 1 |
39 ± 2 |
38 ± 1 |
35 ± 0** |
|||
Week 14 |
85 ± 3 |
83 ± 3 |
70 ± 3** |
60 ± 2** |
56 ± 2** |
51 ± 2** |
|||
Alkaline phosphatase (IU/L) |
|
|
|
|
|
|
|||
Day 4 |
575 ± 7 |
578 ± 10 |
566 ± 10 |
566 ± 11 |
554 ± 7 |
546 ± 11* |
|||
Day 23 |
406 ± 6 |
423 ± 11 |
433 ± 9 |
407 ± 11 |
420 ± 8 |
404 ± 12 |
|||
Week 14 |
223 ± 5 |
227 ± 7 |
211 ± 4 |
200 ± 3** |
204 ± 4** |
199 ± 6** |
|||
Creatine kinase (IU/L) |
|
|
|
|
|
|
|||
Day 4 |
545 ± 121 |
507 ± 42 |
430 ± 52 |
449 ± 56 |
515 ± 54 |
434 ± 44 |
|||
Day 23 |
404 ± 37 |
390 ± 40 |
409 ± 66 |
393 ± 37 |
354 ± 30 |
413 ± 45 |
|||
Week 14 |
171 ± 8 |
186 ± 18 |
144 ± 14 |
155 ± 13 |
150 ± 14 |
183 ± 15 |
|||
Sorbitol dehydrogenase (IU/L) |
|
|
|
|
|
|
|||
Day 4 |
13 ± 1 |
14 ± 0 |
13 ± 0 |
12 ± 0* |
14 ± 1 |
13 ± 0 |
|||
Day 23 |
14 ± 1 |
14 ± 1 |
16 ± 1 |
15 ± 1 |
18 ± 1** |
15 ± 1 |
|||
Week 14 |
24 ± 1 |
24 ± 1 |
22 ± 1 |
22 ± 1 |
21 ± 1* |
20 ± 1** |
|||
Bile acids (µmol/L) |
|
|
|
|
|
|
|||
Day 4 |
4.7 ± 0.4 |
4.7 ± 0.5 |
5.6 ± 0.8 |
4.6 ± 0.4 |
7.2 ± 1.3 |
4.6 ± 0.7 |
|||
Day 23 |
5.7 ± 0.9 |
3.3 ± 0.2** |
4.9 ± 0.6* |
3.6 ± 0.3** |
3.8 ± 0.3** |
3.6 ± 0.7** |
|||
Week 14 |
3.3 ± 0.1 |
3.5 ± 0.4 |
3.4 ± 0.3 |
3.2 ± 0.1 |
3.8 ± 0.6 |
3.0 ± 0.1 |
|||
Female |
|
||||||||
Dose treatment (ppm) |
0 (control group) |
25 |
50 |
100 |
200 |
400 |
|
||
Urea nitrogen (mg/dL) |
|
|
|
|
|
|
|
||
Day 4 |
7.8 ± 0.4 |
8.5 ± 0.3 |
8.1 ± 0.3 |
8.3 ± 0.4 |
9.1 ± 0.3* |
8.6 ± 0.3 |
|
||
Day 23 |
11.5 ± 0.3 |
11.8 ± 0.4 |
11.5 ± 0.4 |
10.6 ± 0.4 |
10.8 ± 0.3 |
9.1 ± 0.4** |
|
||
Week 14 |
14.1 ± 0.4 |
14.4 ± 0.4 |
13.0 ± 0.5 |
13.6 ± 0.5 |
13.4 ± 0.5 |
11.3 ± 0.5* |
|
||
Creatinine (mg/dL) |
|
|
|
|
|
|
|
||
Day 4 |
0.29 ± 0.01 |
0.28 ± 0.01 |
0.29 ± 0.02 |
0.26 ± 0.02 |
0.28 ± 0.01 |
0.26 ± 0.02 |
|
||
Day 23 |
0.31 ± 0.01 |
0.30 ± 0.00 |
0.28 ± 0.01 |
0.30 ± 0.00 |
0.30 ± 0.00 |
0.31 ± 0.01 |
|
||
Week 14 |
0.37 ± 0.02 |
0.35 ± 0.02 |
0.36 ± 0.02 |
0.38 ± 0.01 |
0.34 ± 0.02 |
0.35 ± 0.03 |
|
||
Glucose (mg/dL) |
|
|
|
|
|
|
|
||
Day 4 |
138 ± 2 |
135 ± 2 |
136 ± 4 |
136 ± 2 |
139 ± 5 |
130 ± 2 |
|
||
Day 23 |
127 ± 3 |
123 ± 6 |
133 ± 5 |
123 ± 3 |
122 ± 3 |
122 ± 5 |
|
||
Week 14 |
141 ± 8 |
131 ± 5 |
123 ± 2 |
133 ± 3 |
131 ± 4 |
114 ± 12 |
|
||
Total protein (g/dL) |
|
|
|
|
|
|
|
||
Day 4 |
5.9 ± 0.0 |
6.0 ± 0.1 |
6.1 ± 0.0 |
6.0 ± 0.0 |
6.1 ± 0.1* |
6.1 ± 0.0 |
|
||
Day 23 |
6.3 ± 0.1 |
6.4 ± 0.1 |
6.4 ± 0.1 |
6.5 ± 0.1 |
6.5 ± 0.1 |
6.6 ± 0.1 |
|
||
Week 14 |
7.5 ± 0.1 |
7.4 ± 0.1 |
7.5 ± 0.1 |
7.6 ± 0.1 |
7.5 ± 0.1 |
7.2 ± 0.1 |
|
||
Albumin (g/dL) |
|
|
|
|
|
|
|
||
Day 4 |
4.3 ± 0.0 |
4.4 ± 0.0 |
4.4 ± 0.0 |
4.4 ± 0.0 |
4.4 ± 0.0 |
4.4 ± 0.0 |
|
||
Day 23 |
4.5 ± 0.0 |
4.6 ± 0.0 |
4.6 ± 0.0 |
4.6 ± 0.1 |
4.6 ± 0.0 |
4.7 ± 0.1 |
|
||
Week 14 |
5.2 ± 0.1 |
5.2 ± 0.1 |
5.2 ± 0.1 |
5.3 ± 0.0 |
5.2 ± 0.0 |
5.0 ± 0.1 |
|
||
Globulin (g/dL) |
|
|
|
|
|
|
|
||
Day 4 |
1.6 ± 0.0 |
1.6 ± 0.0 |
1.6 ± 0.0 |
1.7 ± 0.0 |
1.7 ± 0.0* |
1.6 ± 0.0 |
|
||
Day 23 |
1.8 ± 0.0 |
1.8 ± 0.0 |
1.8 ± 0.0 |
1.9 ± 0.0* |
1.9 ± 0.0* |
2.0 ± 0.0** |
|
||
Week 14 |
2.3 ± 0.1 |
2.2 ± 0.0 |
2.3 ± 0.0 |
2.3 ± 0.0 |
2.4 ± 0.0 |
2.2 ± 0.1 |
|
||
A/G ratio |
|
|
|
|
|
|
|
||
Day 4 |
2.8 ± 0.0 |
2.7 ± 0.1 |
2.7 ± 0.0 |
2.7 ± 0.1 |
2.6 ± 0.1 |
2.7 ± 0.0 |
|
||
Day 23 |
2.6 ± 0.0 |
2.5 ± 0.0 |
2.5 ± 0.0 |
2.4 ± 0.0 |
2.5 ± 0.0 |
2.4 ± 0.0** |
|
||
Week 14 |
2.3 ± 0.1 |
2.4 ± 0.0 |
2.3 ± 0.0 |
2.3 ± 0.0 |
2.2 ± 0.0 |
2.4 ± 0.1 |
|
||
Alanine aminotransferase (IU/L) |
|
||||||||
Day 4 |
47 ± 1 |
49 ± 1 |
49 ± 1 |
46 ± 1 |
45 ± 1 |
44 ± 2 |
|
||
Day 23 |
35 ± 1 |
36 ± 1 |
35 ± 1 |
36 ± 1 |
34 ± 1 |
31 ± 1 |
|
||
Week 14 |
69 ± 4 |
65 ± 5 |
55 ± 3** |
56 ± 4* |
47 ± 2** |
49 ± 5** |
|
||
Alkaline phosphatase (IU/L) |
|
||||||||
Day 4 |
487 ± 8 |
493 ± 10 |
475 ± 6 |
468 ± 7 |
454 ± 5** |
457 ± 8** |
|||
Day 23 |
305 ± 5 |
311 ± 8 |
304 ± 5 |
302 ± 8 |
289 ± 8 |
289 ± 7 |
|||
Week 14 |
197 ± 6 |
182 ± 4 |
182 ± 8 |
177 ± 8** |
181 ± 5* |
164 ± 13* |
|||
Creatine kinase (UI/L) |
|
|
|
|
|
|
|||
Day 4 |
364 ± 20b |
332 ± 27 |
388 ± 29b |
443 ± 74 |
460 ± 39b |
375 ± 48 |
|||
Day 23 |
299 ± 30 |
305 ± 27 |
292 ± 41 |
369 ± 43 |
338 ± 26 |
250 ± 16 |
|||
Week 14 |
162 ± 16 |
165 ± 38 |
172 ± 22 |
139 ± 14 |
170 ± 22 |
145 ± 26 |
|||
Sorbitol dehydrogenase (IU/L |
|
|
|
|
|
|
|||
Day 4 |
13 ± 1 |
13 ± 0 |
14 ± 0 |
12 ± 0* |
11 ± 1* |
12 ± 0* |
|||
Day 23 |
14 ± 0 |
15 ± 1 |
15 ± 1 |
15 ± 1 |
14 ± 1 |
16 ± 0 |
|||
Week 14 |
21 ± 1 |
20 ± 1 |
18 ± 1 |
17 ± 1 |
17 ± 1 |
18 ± 1 |
|||
Bile acids (µmol/L) |
|
|
|
|
|
|
|||
Day 4 |
5.3 ± 0.5 |
5.0 ± 0.5 |
6.5 ± 1.1 |
5.8 ± 0.6 |
6.8 ± 1.3 |
4.9 ± 0.5 |
|||
Day 23 |
4.0 ± 0.3 |
4.7 ± 0.4 |
5.4 ± 0.7 |
4.5 ± 0.4 |
3.9 ± 0.4 |
4.0 ± 0.7 |
|||
Week 14 |
9.1 ± 2.3 |
4.9 ± 0.5** |
4.7 ± 0.4** |
4.3 ± 0.3** |
5.1 ± 1.1** |
16.9 ± 4.7* |
|||
* Significantly different (P≤0.05) from the chamber control group by Dunn’s or Shirley’s test
** P≤0.01
Table 5: Incidences of Non neoplastic Lesions of the Kidney in Male Rats
|
Chamber control |
25 ppm |
50 ppm |
100 ppm |
200 ppm |
400 ppm |
Number Examined Microscopically |
10 |
10 |
10 |
10 |
10 |
10 |
Casts, Granulara |
0 |
9** (1.0)b |
10** (1.2) |
10** (1.5) |
10** (2.5) |
10** (3.0) |
Accumulation, Hyaline Droplet |
1 (2.0) |
10** (1.1) |
10** (1.8) |
10** (2.0) |
10** (2.7) |
10** (3.0) |
Nephropathy |
9 (1.1) |
10 (1.6) |
10 (2.0) |
10 (2.0) |
10 (2.5) |
10 (3.0) |
** Significantly different (P≤0.01) from the chamber control group by the Fisher exact test
a Number of animals with lesion
b Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked
Table 6: Epididymal Spermatozoal Measurements for Male Rats
|
Chamber control |
100 ppm |
200 ppm |
400 ppm |
n |
10 |
10 |
9 |
10 |
Epididymal spermatozoal measurements |
||||
Sperm motility (%) |
91.73 ± 1.26 |
91.40 ± 0.93 |
91.24 ± 0.80 |
90.93 ± 0.89 |
Sperm (103/mg cauda epididymis) |
615.0 ± 34.3 |
596.5 ± 31.8 |
526.3 ± 19.0 |
547.4 ± 14.0 |
Sperm (106/cauda epididymis) |
120.89 ± 6.79 |
113.16 ± 3.11 |
97.52 ± 3.51** |
98.40 ± 3.02** |
** Significantly different (P≤0.01) from the chamber control group by Shirley’s test.
a Data are presented as mean ± standard error.
Table 1: Survival and Body Weights
|
Concentration (ppm) |
Survivalb |
Initial Body Weight (g) |
Final Body Weight (g) |
Change in Body Weight (g) |
Final Weight Relative to Controls (%) |
Male
|
0 |
5/5 |
23.7 ± 0.4 |
27.9 ± 0.7 |
4.1 ± 0.4 |
|
100 |
5 /5 |
23.4 ± 0.7 |
26.7 ± 0.7 |
3.4 ± 0.3 |
96 |
|
200 |
5/5 |
23.1 ± 0.8 |
26.6 ± 0.9 |
3.5 ± 0.4 |
95 |
|
400 |
5/5 |
23.9 ± 0.5 |
27.0 ± 0.6 |
3.1 ± 0.2 |
97 |
|
800 |
0/5 c |
23.5 ± 05 |
- |
- |
- |
|
1600 |
0/5 d |
23.5 ± 0.6 |
- |
- |
- |
|
Female |
0 |
5/5 |
20.2 ± 0.4 |
23.0 ± 0.4 |
2.8 ± 0.3 |
|
100 |
5/5 |
20.6 ± 0.5 |
23.6 ± 0.5 |
3.0± 0.3 |
103 |
|
200 |
5/5 |
20.2 ± 0.5 |
23.2 ± 0.7 |
3.0 ± 0.7 |
101 |
|
400 |
5/5 |
19.9 ± 0.5 |
22.6 ± 0.5 |
2.7 ± 0.3 |
99 |
|
800 |
0/5 e |
19.8 ± 0.3 |
- |
- |
- |
|
1600 |
0/5 f |
19.8 ± 0.2 |
- |
- |
- |
a Weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of the study.
b Number of animals surviving at day 18/number initially in group
c Days of death: 2, 2, 3, 4, 16
d Days of death: 1, 1, 1, 1, 2
e Day of deaths: 2
f Day of deaths: 1
Table 2: Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios
|
|
|
Chamber Control |
100 ppm |
200 ppm |
400 ppm |
|
|
n |
5 |
5 |
5 |
5 |
Male |
|
Necropsy body wt |
27.9 ± 0.7 |
26.7 ± 0.7 |
26.6 ± 0.9 |
27.0 ± 0.6 |
Heart |
Absolute |
0.136 ± 0.006 |
0.124 ± 0.004 |
0.126 ± 0.005 |
0.136 ± 0.008 |
|
Relative |
4.872 ± 0.140 |
4.640 ± 0.127 |
4.743 ± 0.182 |
5.028 ± 0.223 |
||
R. Kidney |
Absolute |
0.234 ± 0.011 |
0.246 ± 0.009 |
0.236 ± 0.015 |
0.254 ± 0.012 |
|
Relative |
8.377 ± 0.226 |
9.194 ± 0.191 |
8.832 ± 0.297 |
9.401 ± 0.337* |
||
Liver |
Absolute |
1.436 ± 0.066 |
1.394 ± 0.044 |
1.476 ± 0.069 |
1.672 ± 0.056* |
|
Relative |
51.415 ± 1.352 |
52.117 ± 0.648 |
55.355 ± 0.721* |
61.935 ± 1.281** |
||
Lung |
Absolute |
0.184 ± 0.011 |
0.186 ± 0.007 |
0.200 ± 0.017 |
0.184 ± 0.006 |
|
Relative |
6.579 ± 0.260 |
6.953 ± 0.154 |
7.498 ± 0.510 |
6.814 ± 0.120 |
||
R. Testis |
Absolute |
0.099 ± 0.002 |
0.095 ± 0.004 |
0.089 ± 0.010 |
0.094 ± 0.002 |
|
Relative |
3.539 ± 0.048 |
3.536 ± 0.109 |
3.301 ± 0.287 |
3.478 ± 0.074 |
||
Thymus |
Absolute |
0.057 ± 0.007 |
0.048 ± 0.004 |
0.050 ± 0.003 |
0.049 ± 0.007 |
|
Relative |
2.030 ± 0.192 |
1.801 ± 0.131 |
1.882 ± 0.105 |
1.831 ± 0.256 |
||
Female
|
|
Necropsy body wt |
23.0 ± 0.4 |
23.6 ± 0.5 |
23.2 ± 0.7 |
22.6 ± 0.5 |
Heart |
Absolute |
0.118 ± 0.004 |
0.122 ± 0.004 |
0.120 ± 0.003 |
0.118 ± 0.002 |
|
Relative |
5.135 ± 0.090 |
5.163 ± 0.137 |
5.165 ± 0.034 |
5.221 ± 0.137 |
||
R. Kidney |
Absolute |
0.166 ± 0.005 |
0.196 ± 0.007* |
0.184 ± 0.008 |
0.180 ± 0.006 |
|
Relative |
7.225 ± 0.137 |
8.283 ± 0.132** |
7.919 ± 0.273 |
7.952 ± 0.234 |
||
Liver |
Absolute |
1.230 ± 0.029 |
1.300 ± 0.033 |
1.320 ± 0.065 |
1.426 ± 0.036* |
|
Relative |
53.557 ± 0.672 |
54.996 ± 0.772 |
56.718 ± 1.676 |
63.001 ± 0.995** |
||
Lung |
Absolute |
0.182 ± 0.006 |
0.190 ± 0.005 |
0.188 ± 0.004 |
0.198 ± 0.015 |
|
Relative |
7.932 ± 0.261 |
8.033 ± 0.085 |
8.114 ± 0.273 |
8.756 ± 0.661 |
||
Thymus |
Absolute |
0.074 ± 0.003 |
0.073 ± 0.005 |
0.068 ± 0.003 |
0.060 ± 0.004 |
|
Relative |
3.220 ± 0.122 |
3.059 ± 0.157 |
2.913 ± 0.123 |
2.654 ± 0.114* |
* Significantly different (P≤0.05) from the chamber control group by Williams’ or Dunnett’s test
** P≤0.01
a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean ± standard error).
No data were available for the 800 and 1,600 ppm groups due to 100% mortality.
Table 1: Survival and Body Weights
Concentration (ppm) |
Survivalb |
Initial BodyWeight (g) |
Final BodyWeight (g) |
Change in Body Weight (g) |
Final Weight Relative to Controls (%) |
|
Male
|
0 |
5/5 |
101± 3 |
172 ± 3 |
74 ± 4 |
|
100 |
5/5 |
101± 3 |
171 ± 2 |
70 ± 1 |
99 |
|
200 |
5/5 |
102 ± 3 |
173 ± 7 |
71 ± 5 |
100 |
|
400 |
5/5 |
100 ± 3 |
176 ± 4 |
75 ± 2 |
102 |
|
800 |
0/5 c |
101± 3 |
- |
- |
- |
|
1600 |
0/5 d |
102 ± 4 |
- |
- |
- |
|
Female |
0 |
5/5 |
91± 2 |
125 ± 3 |
34 ± 2 |
|
100 |
5/5 |
90 ± 2 |
130 ± 3 |
40 ± 2 |
104 |
|
200 |
5/5 |
91± 2 |
129 ± 2 |
38 ± 2 |
103 |
|
400 |
5/5 |
92 ± 1 |
118 ± 2 |
26 ± 2* |
94 |
|
800 |
0/5 e |
92 ± 2 |
- |
- |
- |
|
1600 |
0/5 f |
91 ± 1 |
- |
- |
- |
* Significantly different (P≤0.05) from the chamber control group by Dunnett’s test
a Weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of the study.
b Number of animals surviving at day 17/number initially in group
c Days of death: 8, 8, 8, 8, 16
d Days of death: 1, 1, 1, 1, 2
e Day of deaths: 8
f Day of deaths: 1
Table 2: Selected Organ Weights and Organ-Weight-to-Body-Weight Ratios
|
|
Chamber control |
100 ppm |
200 ppm |
400 ppm |
|
Male |
|
n |
5 |
5 |
5 |
5 |
|
Necropsy body wt |
172 ± 3 |
171 ± 2 |
173 ± 7 |
176 ± 4 |
|
Heart |
Absolute |
0.620 ± 0.008 |
0.614 ± 0.006 |
0.592 ± 0.028 |
0.620 ± 0.018
|
|
Relative |
3.602 ± 0.098 |
3.584 ± 0.033 |
3.418 ± 0.039 |
3.534 ± 0.059 |
||
R. Kidney |
Absolute |
0.714 ± 0.014 |
0.758 ± 0.020 |
0.790 ± 0.044 |
0.796 ± 0.026 |
|
Relative |
4.142 ± 0.075 |
4.425 ± 0.117* |
4.556 ± 0.098** |
4.535 ± 0.069** |
||
Liver |
Absolute |
7.988 ± 0.154 |
8.064 ± 0.196 |
8.284 ± 0.410 |
9.668 ± 0.422** |
|
Relative |
46.331 ± 0.589 |
47.091 ± 1.287 |
47.824 ± 0.791 |
55.069 ± 1.780** |
||
Lung |
Absolute |
1.294 ± 0.119 |
1.216 ± 0.067 |
1.228 ± 0.060 |
1.566 ± 0.075 |
|
Relative |
7.504 ± 0.660 |
7.081 ± 0.309 |
7.100 ± 0.245 |
8.971 ± 0.568 |
||
R. Testis |
Absolute |
0.994 ± 0.015 |
0.984 ± 0.016 |
0.981 ± 0.034 |
0.991 ± 0.016 |
|
Relative |
5.770 ± 0.130 |
5.744 ± 0.064 |
5.681 ± 0.157 |
5.654 ± 0.085 |
||
Thymus |
Absolute |
0.403 ± 0.012 |
0.439 ± 0.016 |
0.427 ± 0.016 |
0.426 ± 0.007 |
|
Relative |
2.344 ± 0.095 |
2.565 ± 0.106 |
2.477 ± 0.096 |
2.431 ± 0.037 |
||
Female |
|
Necropsy body wt wtwwt |
125 ± 3 |
130 ± 3 |
129 ± 2 |
118 ± 2 |
Heart
|
Absolute |
0.466 ± 0.012 |
0.484 ± 0.022 |
0.480 ± 0.008 |
0.444 ± 0.017 |
|
Relative |
3.728 ± 0.089 |
3.727 ± 0.080 |
3.736 ± 0.073 |
3.752 ± 0.091 |
||
R. Kidney |
Absolute |
0.530 ± 0.021 b |
0.586 ± 0.016 |
0.602 ± 0.007* |
0.578 ± 0.017 |
|
Relative |
4.220 ± 0.058 b |
4.521 ± 0.038 |
4.686 ± 0.071** |
4.896 ± 0.166** |
||
Liver |
Absolute |
4.854 ± 0.194 |
5.404 ± 0.260 |
5.764 ± 0.051** |
5.244 ± 0.138 |
|
Relative |
38.750 ± 0.728 |
41.602 ± 1.031* |
44.888 ± 0.926** |
44.379 ± 1.012** |
||
Lung |
Absolute |
0.850 ± 0.022 |
1.066 ± 0.041** |
1.042 ± 0.046* |
0.862 ± 0.060 |
|
Relative |
6.808 ± 0.233 |
8.221 ± 0.223* |
8.114 ± 0.371 |
7.315 ± 0.572 |
||
Thymus |
Absolute |
0.317 ± 0.003 |
0.335 ± 0.006 |
0.361 ± 0.011* |
0.285 ± 0.017 |
|
Relative |
2.535 ± 0.052 |
2.590 ± 0.059 |
2.807 ± 0.062 |
2.413 ± 0.149 |
* Significantly different (P≤0.05) from the chamber control group by Williams’ or Dunnett’s test
** P≤0.01
a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean ± standard error).
No data were available for the 800 and 1,600 ppm groups due to 100% mortality.
b n=4
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
Weight of evidence from experimental results with individual components:
Camphene (28 days, rats, oral):
The test item was daily administered to SPF Wistar rats (male and female) for 28 days at doses of 0, 62.5, 250 and 1000 mg / kg bw/day by gavage. Test method was according to OECD guideline 407. Based on the results of this study, for female rats the NOEL was 250 mg / kg bw/day. For male rats, the NOEL could not be determined (it was lower than 62.5 mg/kg bw/day). The renal toxic effects found in all dose levels groups in male rats are interpreted as uniquely specific for male rats, and as having no relevance for other animal species and humans.
d-limonene (90 days, rats, oral):
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). 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 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). 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 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. 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.
d-limonene (28 days, rats, oral):
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). 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.
d-limonene (90 days, mouse, oral):
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). 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.
d-limonene (180 days, dogs, oral):
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. 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 a 6-month subchronic toxicity study, 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.
alpha pinene (90 days, rats, inhalation):
A 90-day repeated dose toxicity study (inhalation route) was performed on alpha pinene according to a similar method to OECD guideline 413 under GLP conditions. Groups of 10 male and 10 female rats were exposed to the test item by whole body inhalation at concentrations of 0, 25, 50, 100, 200, or 400 ppm, 6 hours per day, 5 days per week for 14 weeks. Relative liver weights were significantly greater compared to controls in males at 100 ppm and greater and in all females treated groups (LOEL=25 ppm), however, without accompanying histopathologic changes. Increased liver weight is a common finding in toxicity studies and can be associated with induction of liver metabolizing enzymes. Absolute kidney weights were increased in male rats exposed to 100 ppm or greater and 50 and 200 ppm female rats; in males, these increases were accompanied by histopathologic lesions including granular casts and hyaline droplet accumulation at all exposure concentrations, as well as exposure concentration-dependent increases in the severity of nephropathy (LOEL=25 ppm), which is a common spontaneous lesion observed in male rats (α2μ-globulin nephropathy). There were also significantly lower numbers of sperm per cauda compared to controls in 200 and 400 ppm male rats (LOEL=200 ppm). There was an accompanying minor decrease in epididymal weights that did not reach significance. Therefore, the possibility that the change in absolute sperm per cauda was due to a decrease in epididymal weight cannot be excluded. A definitive conclusion by histopathologic investigations could not be conducted due to an artifact resulting from formalin fixation of the male reproductive tract tissues. Thus, further studies on reproductive function are warranted. In females the minor changes in cycle length observed only in the 400 ppm group, combined with a lack of ovarian histopathology findings, does not provide sufficient evidence for reproductive toxicity.
alpha pinene (90 days, mouse, inhalation):
A 90-day repeated dose toxicity study (inhalation route) was performed on alpha pinene according to a similar method to OECD guideline 413 under GLP conditions. Groups of 10 male and 10 female mice were exposed to the test item by whole body inhalation at concentrations of 0, 25, 50, 100, 200, or 400 ppm, 6 hours per day, 5 days per week for 14 weeks. There was an increased incidence of transitional epithelium hyperplasia of the urinary bladder in males and females exposed to 100 ppm or more (LOEL=100 ppm), the severity of which increased with increasing exposure concentration. Transitional epithelium hyperplasia in the urinary bladder can be either reparative (e.g., regenerative or reactive) or preneoplastic, but there are no histologic features that can be used to reliably distinguish between the two etiologies. Specific histopathologic indicators of either type of hyperplasia in male or female mice were not evident; therefore, the neoplastic potential of the transitional epithelium hyperplasia of the urinary bladder is uncertain. There were also significantly decreased numbers of sperm per mg cauda in 200 and 400 ppm males and cauda sperm in 100, 200, and 400 ppm males (LOEL=100 ppm). There was an accompanying minor decrease in epididymal weights that did not reach significance. Therefore, the possibility that the change in absolute sperm per cauda was due to a decrease in epididymal weight cannot be excluded. A definitive conclusion by histopathologic investigations could not be conducted due to an artifact resulting from formalin fixation of the male reproductive tract tissues. Thus, further studies on reproductive function are warranted. However, in females there were no changes in the percentage of time spent in the individual stages of the estrous cycle or in estrous cycle length at any exposure concentration. Also, there were no ovarian histopathologic findings. Thus, no evidence of reproductive toxicity in females was found.
Weight of evidence: conclusion:
The absence of any d-limonene-induced renal lesions in the study with dogs provides evidence that hydrocarbon-induced nephropathy in the male rat is species- and sex-specific. Therefore, the male rat response to d-limonene, alpha pinene or camphene 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.
Thus, 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. The key value has been then selected to be the LOAEL at 1000 mg/kg bw/d.
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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