Registration Dossier
Registration Dossier
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EC number: 949-141-8 | CAS number: -
Endpoint summary
Administrative data
Description of key information
Key study: Test method OECD 422. GLP study. The NOAEL of the test substance Pine oil 50% after a repeated oral exposure of 36 days in males and approximately 63 days in females was determined to be 600 mg/kg bw/day since no adverse effects were observed at the highest dose tested.
Key study: Test method OECD 408. GLP study. In a 90-day repeated dose toxicity study conducted in Sprague Dawley Rats, the NOAEL of the test substance Pine oil 50% for both sexes was determined to be 300 mg/kg bw/day.
Supporting study: Dose range finding study according to test method OECD 407. The NOEL of the test substance Pine oil 50% after an oral exposure of 14 days was determined to be 100 mg/kg bw/day in male and female rats.
Supporting studies on some components of the test substance: Repeated dose toxicity studies are available on the components terpineol, d-limonene, cineole and camphene by oral route, and alpha pinene and analogue cumene 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 terpineol, d-limonene, alpha pinene, camphene, cineole or analogue cumene 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. Based on this reasoning the more representative study was selected to be the 180-d toxicity study by oral route in dogs (Webb, 1990). The LOAEL was established 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
- Study period:
- 16 November 2018 – 05 December 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- (The study period was only 14 days as it was conducted as a dose range finding study for a subsequent more extensive toxicity study)
- GLP compliance:
- no
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 258.22-263.41 g (range of average values of each group of males); 239.22-242.03 g (range of average values of each group of females)
- Housing: Maximum of 3 animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-23.5ºC
- Humidity (%): 42-68 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Required quantity of test item was weighed as per the dose. The weighed test item was mixed well using glass rod by adding a small quantity of vehicle and then transferred to a measuring cylinder. Again, a small quantity of vehicle was added and transferred to the measuring cylinder. The procedure was repeated until the complete transfer of the test item into the measuring cylinder. The final volume was made up with vehicle to get the desired concentration as per the dose requirement.
The freshly prepared test item formulation was used for administration.
VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not miscible with distilled water and clearly miscible with corn oil at the concentration of 100 mg/mL as per the in-house miscibility test. Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): A1712001 - Analytical verification of doses or concentrations:
- no
- Details on analytical verification of doses or concentrations:
- The stability and homogeneity of the test item in dose formulations was not established under this dose range finding study. However, freshly prepared test item formulations were administered to the animals.
- Duration of treatment / exposure:
- 14 days
- Frequency of treatment:
- Once a day
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 3
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
Doses were decided based on the US EPA (United States Environmental Protection Agency), Prevention, Pesticides and Toxic Substances (7510P), Reregistration Eligibility Decision for Pine oil (case 3113), where the LOAEL for Pine oil 80% was established in 600mg/kg/day and NOAEL was 50 mg/kg/day. - Positive control:
- None
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were included, but not be limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.
BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weight was recorded on the day of randomization, on day of treatment (Day 1) prior to treatment and weekly thereafter.
FOOD CONSUMPTION: yes
-feed consumption was measured at weekly intervals. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during acclimatization (Pre-treatment) and at week 2 of treatment.
- Dose groups that were examined: all animals
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of necropsy (day 15)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all surviving animals
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by coagulation analyzer.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy (day 15)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all surviving animals
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Triglycerides, Phosphorous, Calcium, Blood Urea Nitrogen, Globulin, Albumin/Globulin ratio. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using Prolyte Na/K/Cl analyzer (Diamond Diagnostics).
URINALYSIS: Yes
- Time schedule for collection of urine: on the day of necropsy (day 15)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
All the surviving animals and animals found dead were subjected to necropsy and detailed gross pathological examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, as well as organs and tissues of each animal with special emphasis on reproductive organs.
The following organs from all animals at the scheduled sacrifices were weighed wet as soon as possible to avoid drying: Kidneys, Adrenals, Spleen, Heart, Liver, Thymus, Brain, Lungs Testes/Ovaries, Epididymides/Uterus, Prostate along with seminal vesicles with coagulating gland. Relative organ weights were calculated against fasting body weight.
-HISTOPATHOLOGY: No - Statistics:
- After verification, the data was subjected to statistical analysis using SPSS software, version 22. Body weight, percent change in body weight with respect to day 1, hematological, clinical chemistry estimations, urinalysis parameters (whichever applicable), absolute and relative organ weights were subjected to statistical analysis. One way ANOVA followed by Dunnett’s post test was done for different treatment groups comparing with the control group data. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05).
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- There were no clinical signs of toxicity at vehicle control group and low dose group.
The mid dose group males did not reveal any clinical signs of toxicity till treatment day 3. However, one male was found with slight wet perineum on day 4, with slight wet perineum, slight nasal discharge and slight piloerection on day 5 and found recovered on day 6. The same male revealed slight nasal discharge from day 11 to 13 and slight wet perineum on day 13 which was found to be normal on day 14. Two males from mid dose group revealed slight nasal discharge from day 11 to 13 and found recovered on day 14. All the mid dose group females did not reveal any clinical signs of toxicity during the experimental period, except one female was found with slight wet perineum on day 4, recovered on day 5.
The high dose group males did not reveal any clinical signs of toxicity till day 3, one male found with slight salivation on day 5, found recovered on day 7, another male was found with slight wet perineum on days 4 and 5, found recovered on day 6. All the three males were found with slight nasal discharge from day 10 to 14 along with lethargy on day 14. All the high dose group females did not reveal any clinical signs of toxicity till day 2, all the three females were found with slight wet perineum from day 3 to 5 and recovered on day 6. Two females from this dose level revealed slight chromodacryorrhea and slight wet perineum during day 10 to 14. One female found with slight to moderate wet perineum, moderate piloerection and slight nasal discharge on days 10 and 11, found dead on day 12.
The detailed clinical examination of all the low and mid dose group animals of either sex did not reveal any treatment related changes during week 1 and 2. The detailed clinical examination of all the high dose group animals of either sex did not reveal any treatment related changes during week 1. However, all the three males and two survived females were found with rough hair coat, slight nasal discharge with reduced activities on day 15 (week 2). - Mortality:
- mortality observed, treatment-related
- Description (incidence):
- No mortality observed at vehicle control, low dose and mid dose groups. In the high dose group one female was found dead on day 12.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- There were no statistically significant changes in mean body weight and percent change in body weight with respect to day 1 at any of the tested dose group animals of either sex. However, reduction in mean body weight and percent change in body weight with respect to day 1 at mid and high dose group males and reduction in mean body weight and percent change in body weight with respect to day 1 at high dose group females were noted when compared with concurrent control group animals. These changes are considered as treatment related due to occurences of clinical signs of toxicity at both the mid and high dose groups animals, reduction in feed consumption and occurrence of motality at high dose group.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- There were no statistically significant changes in mean feed consumption at any of the tested dose group animals of either sex. However, a slight reduction in mean feed consumption during week 1 at high dose group males, during week 2 at mid and high dose group males; a slight reduction during week 1 and 2 at high dose group females was noted when compared with concurrent control group animals. These changes are considered as treatment related due to occurences of clinical signs of toxicity at both the mid and high dose groups animals, reduction in mean body weight and occurrence of motality at high dose group.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- There were no ocular changes observed during the opthalmoscopic examination carried out during week 2 (Day 14) for all control and treated dose group animals of either sex.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no treatment related changes observed in haematology at any of the tested dose group animals of either sex. However, a statistical significant increase in mean platelet volume at all the tested dose group males was noted when compared with vehicle control group males. This observed change is considered as incidental and not treatment related because of the lack of dose dependency and/or due to random biological variation.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no treatment related changes observed in clinical chemistry at any of the tested dose group animals of either sex. However, in males, a statistical significant decrease in total bilirubin (low and mid dose groups), decrease in glucose (high dose group), decrease in creatinine and calcium levels (low dose group) and increase in phosphorous (high dose group) were noted when compared with control group animals. These observed changes are considered as incidental and not treatment related due to lack of dose dependency and/or due to random biological variation and also no significant changes noted in clinical chemistry parameters at any of the tested dose group females.
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- There were no treatment related changes observed in the urinalysis parameters at any of the tested dose group animals of either sex.
- 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):
- There were no treatment related changes observed in the absolute and relative organ weights at any of the tested dose group animals of either sex. However, statistical significant increase in absolute and relative adrenals weight at low and mid dose group males, increase in absolute and relative liver weight at high dose group males, increase in relative liver weight at high dose group females, decrease in absolute lungs weight at high dose group females and increase in relative kidneys weight at high dose group males were noted when compared with concurrent control group animals.
These observed changes are considered as incidental and not treatment related due to lack of dose dependency and no gross pathological changes noted in these organs during necropsy. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no treatment gross pathological changes observed at any of the tested dose group animals of either sex during gross pathological examination. However, external gross pathological finding like, wet perineum was observed in one male at high dose group and one female which was found dead on day 12.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- LOEL
- Effect level:
- 300 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical signs
- food consumption and compound intake
- Remarks on result:
- other: Effects observed at the dose tested of 300 mg/kg bw/day.
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical signs
- food consumption and compound intake
- Key result
- Critical effects observed:
- no
- Conclusions:
- Based on the findings of this study, the NOEL of the test substance after an oral exposure of 14 days was determined to be 100 mg/kg bw/day in male and female rats.
- Executive summary:
A dose range finding study was performed for the test substance pine oil 50% in accordance with OECD guideline 407 in order to evaluate its toxic potential after repeated exposure and select the appropriate doses for a subsequent toxicity study. The test item was diluted in corn oil (vehicle) and added to Sprague-Dawley rats (3 per sex per dose) at doses of 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day for a period of 14 days. The vehicle and test item formulations were administered orally (gavage) at a dose volume of 10 mL/kg body weight. Freshly prepared test item formulations were administered to the animals as soon as possible after preparation.
All the dose group animals were observed for clinical signs of toxicity once daily, mortality and morbidity twice daily, detailed clinical examination, body weight and feed consumption weekly. Ophthalmological examination was carried out during week 2 (day 14) for all the survived animals from all the dose groups of either sex. Clinical pathology (haematology, clinical chemistry analysis and urinalysis) and gross pathological examination was carried out for all survived animals on the day of necropsy.
The low dose group did not reveal any clinical signs of toxicity and no mortality or morbidity was observed throughout the experimental period. The mid dose and high dose groups revealed treatment related clinical signs of toxicity like wet perineum, nasal discharge and lethargy and one female from high dose group was found dead on day 12 of treatment period. The detailed clinical examination carried out on during week 2 revealed treatment related changes like rough hair coat, nasal discharge and reduced activities in high dose group animals.
There were no treatment related changes noted in body weight, percent change in body weight, feed consumption, ophthalmoscopic examination, clinical pathology and gross pathological examination at low dose group animals of either sex. The mid and high dose group animals revealed treatment related reduction in body weight, percent change in body weight and feed consumption. There were no treatment related changes noted in ophthalmoscopic examination, clinical pathology and gross pathological examination at mid and high dose group animals. Some statistically significant effects were found on haematology, clinical chemistry parameters and absolute and relative organ weights in the low, mid or high dose groups, although they were considered as incidental and not treatment related because of the lack of dose dependency, due to random biological variation and/or no gross pathological changes noted in the affected organs during necropsy.
Based on these results, it can be concluded that the No Observed Effect Level (NOEL) is 100 mg/kg bw/day and Low Observed Effect Level (LOEL) is 300 mg/kg bw/day, as the dose of 100 mg/kg bw/day did not reveal any treatment related effects in either sex, the dose of 300 mg/kg bw/day revealed treatment related clinical signs of toxicity and reduction in body weight and feed consumption and the dose of 1000 mg/kg bw/day revealed treatment related clinical signs of toxicity, mortality, reduction in body weight and feed consumption.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25 January 2019 – 21 June 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to other study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Weight at study initiation: 377.18-381.03 g (range of average values of each group of males at first day of dosing); 271.24-272.21 g (range of average values of each group of females at first day of dosing)
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
Acclimatization: maximum of 3 animals of same sex per cage.
Pre mating: 2 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
Recovery animals: 3 animals of same sex per cage.
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the experimental period. Deep bore-well water passed through a Reverse Osmosis Unit was provided in plastic water bottles with stainless-steel sipper tubes.
- Acclimation period: at least 5 days. Females were screened for normal oestrous cycles (4 to 5 days) in a two weeks pre-treatment period before initiation of treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2-23.1ºC
- Humidity (%): 47-67 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed into a clean beaker and a little volume of vehicle was added into the beaker and mixed well with glass rod. The formulation was transferred into a measuring cylinder, the beaker was rinsed with some more amount of vehicle also transferred into the measuring cylinder. This procedure was repeated until the entire quantity of the test item formulation was transferred into the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.
Test item formulations were prepared daily before administration.
VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not miscible with distilled water and clearly miscible with corn oil at the concentration of 100 mg/mL as per the in-house miscibility test. Hence, corn oil was selected as vehicle for test item formulation preparations. Corn oil is universally accepted and routinely used vehicle in oral toxicity studies.
- Concentration in vehicle: 10, 30 and 60 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): A1712001 - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The stability of the test item in dose formulations was established in a preliminary study. The test item formulations were stable at room temperature for 6 hours at the concentrations of 0.5 mg/mL and 200 mg/mL in corn oil. However, freshly prepared test item formulations were administered to the animals.
Homogeneity and dose formulation analysis for dose concentration verification was done by a validated analytical method UV-Vis spectrophotometer (study nº BIO-ANM 1305). Sampling and analysis of formulations were performed during week 1 (13-02-2019) and week 5 (08-03-2019) of the treatment. Approximately 10 mL of samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%. - Duration of treatment / exposure:
- Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 36 days of treatment).
Main group females main group: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.
Recovery animals: same period as for the main group females until the first scheduled sacrifice of dams and kept without treatment for a further 14 days observation. - Frequency of treatment:
- Once a day
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- G1 - Vehicle control + G1R - Vehicle Control Recovery Group
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- G2 - Low dose
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Remarks:
- G3 - Mid dose
- Dose / conc.:
- 600 mg/kg bw/day (actual dose received)
- Remarks:
- G4 - High dose + G4R - High dose Recovery Group
- No. of animals per sex per dose:
- Main Group: 12
Recovery Group: 5 - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
Doses were decided based on the results of a dose range finding study in which the No Observed Effect Level (NOEL) was found to be 100 mg/kg bw/day and Low Observed Effect Level (LOEL) was found to be 300 mg/kg bw/day, as the dose of 100 mg/kg bw/day did not reveal any treatment related effects in either sex, the dose of 300 mg/kg bw/day revealed treatment related clinical signs of toxicity and reduction in body weight and feed consumption and the dose of 1000 mg/kg bw/day revealed treatment related clinical signs of toxicity, mortality, reduction in body weight and feed consumption. - Positive control:
- None
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the treatment on day 1 and weekly thereafter during treatment for all the animals. Signs noted were included, but not be limited to, changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.
BODY WEIGHT: Yes
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The main group females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals (both males and females) were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.
FOOD CONSUMPTION: yes
-feed consumption was measured for main group animals (both males and females) once a week during premating and weekly once for main group males during the post mating period. Feed consumption was not measured during the mating period for both males and females. Thereafter, feed consumption for main group females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13. Feed consumption was measured for recovery group animals (both males and females) once a week throughout the experimental period. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats surviving in each cage.
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once before treatment for all animals, during end of the dosing period for main group males (on day 34) and during lactation period for main group females (on lactation day 13) of vehicle control and high dose main group animals and during last week (day 64) for all recovery group animals (both males and females).
- Dose groups that were examined: vehicle control and high dose main group.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males after completion of 36 days of treatment and for females on lactation day 14).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by an "OptiClot-4 coagulation analyzer".
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males after completion of 36 days of treatment and for females on lactation day 14).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and all recovery group animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using Prolyte Na/K/Cl analyzer (Diamond Diagnostics).
URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 randomly selected males of each main group and all recovery animals.
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Microalbumin, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the dosing period for males (on day 36) and during lactation period for females (on lactation day 13). Towards the end of the recovery period (shortly prior to scheduled sacrifice on day 66) for the recovery group.
- Dose groups that were examined: all main and recovery groups.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations, Physiological Observation (Rectal temperature), Grip strength assessment and Motor activity assessment.
OTHER:
Oestrous Cyclicity: Estrous cycles were monitored during the acclimatization to evaluate its normal oestrous cyclicity (4 to 5 days). Only females with normal oestrous cyclicity were selected for the treatment. For the main group females, the vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. Oestrous cyclicity was also monitored on the day of sacrifice for main group females.
Litter Observation: The number of pups born (dead and live) in a litter, sex and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 was recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4). The number of nipples/areolae in male pups was counted on postnatal day 13 (lactation day 13). Fertility index for dams and sires, pup survival index and sex ratio at birth were calculated.
Thyroxine Hormone (T4) Level estimation: Blood samples were collected for measurement of serum T4 levels on the following schedule:
a. Two pups per litter on lactation day 4 based on the following conditions:
- Two female pups in order to retain more male pups for nipple retention on PND 13.
- No pups were eliminated when litter size dropped below 10 pups/litter.
- Only one pup was eliminated and used for blood collection in case of only one pup was available above ten.
b. All main group females (dams) at termination (lactation day 14).
c. Two pups per litter (same sex) at termination (lactation day 13).
d. All adult main group males, at termination (after completion of 36 days of treatment). - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes (see table 1)
HISTOPATHOLOGY: Yes (see table 1) - Other examinations:
- Indices calculation (see table 2)
- Statistics:
- After verification, the data was subjected to statistical analysis using SPSS software, version 22. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 3 below.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- There were no clinical signs of toxicity observed at groups G1/G1R and G2 animals of either sex throughout the experimental period.
The groups G3 and G4/G4R animals of either sex did not reveal any clinical signs of toxicity for the first three days of the treatment period. Thereafter, slight wet perineum was noted during several occasions in animals of either sex of these dose groups and found recovered later during the treatment period.
The detailed clinical examination revealed treatment related wet perineum and perinasal staining at groups G3 and G4/G4R during treatment period and found recovered later in the study.
These observed clinical signs at both the dose group animals of G3 and G4/G4R are considered as treatment related due to reduced mean body weight, percent change in mean body weight in either sex during these periods and also the observations are dose dependant when compared with control group. - Mortality:
- no mortality observed
- Description (incidence):
- There were no mortality/morbidity observed at any dose group during the experimental period.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- There were no treatment related changes noted in mean body weight and percent change in body weight with respect to day 1 at group G2 when compared with vehicle control group animals of either sex during the experimental period. However, statistically significant reduction in percent change in mean body weight with respect to day 1 at group G2 animals of either sex was noted. This change is considered as incidental due to lack of consistency and no clinical signs were noted and no effects were noted in feed consumption during this period when compared with control group.
In groups G3 and G4 animals, statistically significant reduction in percent change in body weight with respect to day 1 on days 7, 14, 21, 28 and 35 in males and days 7 and 14 in females was noted.
In group G4R animals, statistically significant reduction in percent change in body weight with respect to day 1 on days 7, 14, 28 and 35 in males, and reduction in mean body weight on day 28 and 35 and percent change in body weight with respect to day 1 during the entire experimental period in females, was noted.
The observed changes at these dose levels [300 mg/kg and 600 mg/kg] are considered as treatment related due to occurrence of treatment related clinical signs of toxicity and also the observed changes are dose dependant and consistent during the experimental period.
There were no changes observed in the gestation body weight and percent change in body weight during gestation period at G2 and G3 dose group animals. A slight reduction in mean gestation body weight was noted on GD 0, 7, 14 and 20 at group G4 animals when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to significant reduction in mean body weight at this dose group during pre-mating period and also due to occurrence of clinical signs of toxicity during treatment period.
There were no changes observed in the lactation body weight and percent change in body weight during lactation period at groups G2 and G3 when compared with control group animals. A slight reduction in mean lactation body weight was noted throughout the lactation period and the reduction is statistically significant during lactation day 1 to 4 at group G4 animals when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to significant reduction in mean body weight at this dose group during pre-mating period, gestation period and also due to occurrence of clinical signs of toxicity during treatment period. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Statistically significant reduction in mean feed consumption was noted during pre-mating period (week 1) of main group animals in either sex at all the tested dose groups when compared with vehicle control group. This occurrence is considered as incidental at group G2 and G3 of either sex as the mean feed consumption was comparable with vehicle control group during week 2. However, the reduction at group G4 can be considered as treatment related as the reduction was continued during week 2 also and this effect is correlated with reduction in percent change in body weight at this dose level.
There were no treatment related changes observed in the mean gestation feed consumption at group G2 and G3 when compared with vehicle control group. A slight reduction in mean gestation feed consumption was noted during GD 0 to 7, 7 to 14 and 14 to 20 at group G4 when compared with other dose groups and vehicle control group. This observation can be considered as treatment related due to consistency in reduction of feed consumption at this dose group during pre-mating period and also due to occurrence of clinical signs of toxicity during treatment period.
There were no treatment related changes observed in the feed consumption during lactation period at all the tested dose groups when compared with control group animals. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- There were no ocular changes observed in G1 and G4 group animals of either sex during the ophthalmological examination conducted towards end of the dosing period and no ocular changes observed in G1R and G4R group animals of either sex during the ophthalmological examination conducted at the end of recovery period.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The following statistically significant changes were noted in haematology parameters when compared with their concurrent control groups: increase in activated prothrombin time at group G4 males; decrease in haemoglobin at group G2 females; decrease in total leucocyte count, absolute lymphocytes and activated prothrombin time at group G4R males and increase in mean platelet volume at group G4R males. These changes are considered as incidental and not treatment related, due to lack of dose dependency and similar changes were not observed in other sex.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Statistically significant decrease in total bilirubin at group G3 males and statistically significant decrease in triglycerides at group G4R males were noted when compared with their concurrent control groups. These changes are considered as incidental and not treatment related, due to lack of dose dependency and similar changes were not observed in other sex.
- Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- There were no changes observed in urinalysis parameters at any of the tested dose main group males and tested dose group recovery group animals of either sex conducted at termination.
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no treatment related changes noted in neurological/functional examinations like home cage, handling, open field, sensory, neuromuscular, physiological observations and assessment of grip strength and motor activity at all the tested dose group animals of either sex from both main and recovery group animals when compared with concurrent vehicle control group animals.
However, statistically significant increase in mean number of rearing during open field examination was noted at groups G2 and G4 females when compared with vehicle control group females. This change is considered as incidental but not treatment related due to lack of dose dependency and no effects were noted in other functional observation battery parameters at these dose levels. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no treatment related changes observed in the absolute and relative organ weights at any of the tested dose group animals of either sex in both main and recovery group animals.
However, statistically significant increase in absolute and relative liver weight at group G4, statistically significant decrease in absolute seminal vesicles with coagulating glands at group G3 males; statistically significant decrease in absolute heart weight at group G4R females were noted when compared with concurrent vehicle control group. These changes are considered as incidental and not treatment related as the observations do not occur in dose dependant manner, are not found in both sexes and also no gross or histopathological changes were noted in these organs at this dose level. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- There were no gross pathological changes observed during necropsy at any of the main and recovery group animals of either sex. Also, there were no gross pathological changes noted in any of the pups sacrificed.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment related histopathological findings were noticed in this study.
Lesions considered to be spontaneous and incidental were observed in treated group and control group animals. These lesions consisted of interstitial mononuclear cell infiltrate and concretions in prostate gland, and the presence of calculi in kidney.
1 female from high dose group G4 revealed mononuclear cells infiltration at sub-urothelium of kidney. This lesion was considered spontaneous because of lack of consistency.
3 males from G4 group revealed presence of concretions in prostate gland. Inflammation and concretions of the prostate gland is common background finding in rats and mice, which increases with age (Dianne C., et.al, 2012).
Females from vehicle control group and high dose group revealed presence of placental scar characterized by brown-pigmented nodules at the uterine-mesometrial boundary. The presence of discrete pigmented nodules along the uterine-mesometrial boundary was reported. Each nodule overlies a site of placental detachment at parturition, or the site of the resorption of a feto-placental unit or decidualized implantation site. These nodules were often called placental scars and may persist for many months postpartum, perhaps even the lifespan of a rodent (Janet M., 1990).
Presences of ultimobranchial cyst or ectopic thymus in thyroid were congenital lesions and it does not have toxicological significance. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Oestrus cycle: There were no changes (irregularities) observed in the oestrus cyclicity of females at any of the tested dose group females during pre-mating treatment, mating treatment and on lactation day 14 of dams.
Litter observations: There were no treatment related changes observed in litter observation parameters at all the tested dose groups when compared with vehicle control group animals.
Serum Thyroxine (T4) Levels: There were no treatment related changes in serum Thyroxine (T4) hormone levels noted at all the tested dose group males and lactation day 13 pups. However, statistically significant decrease in T4 levels of males at group G4 was noted when compared with vehicle control group. This change is considered as incidental and not treatment related as the observations do not occur in dose dependant manner and also the values obtained are within historical control range. As no treatment related changes were noted in males and lactation day 13 pups the assessment of T4 in blood samples from the dams and day 4 pups was not performed. - Key result
- Dose descriptor:
- LOEL
- Effect level:
- 300 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
- food consumption and compound intake
- Remarks on result:
- other: Effects observed at the dose tested of 300 mg/kg bw/day.
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 100 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
- food consumption and compound intake
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 600 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- gross pathology
- histopathology: non-neoplastic
- Remarks on result:
- other: No adverse effects were found at any dose tested.
- Key result
- Critical effects observed:
- no
- No of animals showing external pathology
- No of animals showing visceral pathology
- Conclusions:
- Based on the findings of this study, the NOAEL of the test substance after a repeated oral exposure of 36 days in males and approximately 63 days in females was determined to be 600 mg/kg bw/day.
- Executive summary:
A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance Pine oil 50% by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups to detect delayed occurrence and recovery from toxic effects. Each main group (G1, G2, G3 and G4) consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle (Corn oil), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 100, 300 and 600 mg/kg body weight based on the results of a dose range finding study performed with the same species at doses of 100, 300 and 1000 mg/kg bw for a period of 14 days. The test item was administered to the main group males for a period of 36 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period), to the main group females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 and to the recovery group animals for a period of 50 days with a 14 days recovery. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. The stability of test item formulations in corn oil was established before initiation of the treatment. Dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.
All the main group and recovery group animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight, feed consumption and ophthalmological and neurological/functional examinations. The clinical pathological (haematology, clinical chemistry and urinalysis) examinations were conducted for 5 randomly selected animals from each group per sex for main group and from all the animals for recovery group at termination. The gross pathology and organ weighing were performed on the day of termination for all the main and recovery group animals. All the main group females were observed for maternal body weight and feed consumption during gestation and lactation. The main group females were evaluated for oestrus cyclicity. Each dam was observed for mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter throughout the lactation period. Histopathological examination was conducted on all the tissues collected from the main group vehicle control and high dose group animals sacrificed at termination. The pups were observed once daily for clinical signs and external examinations, weighed individually on postnatal day (PND) 1, 4, 7 and 13, measured for anogenital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13 and observed for gross pathological observations at termination. The analysis of thyroxine hormone (T4) levels in serum collected at termination was performed for main group males and PND 13 pups.
There were no clinical signs, no mortality/morbidity, no effects of test item on mean body weight, feed consumption, no changes in ophthalmological examination and neurological/functional examination, clinical pathology, organ weights and gross pathology noted at group G2 of either sex. The group G3 and G4/G4R animals of either sex revealed treatment related wet perineum and recovered later in the study. A treatment related reduction in body weight was noted at both G3 and G4/G4R groups of either sex. No treatment related changes were noted in clinical pathology and organ weights and no gross pathological changes were noted at group G3 and G4/G4R. There were no treatment related changes in oestrus cyclicity, maternal and lactation body weight and feed consumption, mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter at group G2 and G3. A treatment related reduction in mean body weight and feed consumption during gestation and lactation periods and increased gestation length was noted at group G4. There were no treatment related changes occurred in histopathological examination conducted for group G4 animals in either sex. There were no treatment related changes noted in serum thyroxine hormone (T4) levels.
It can be concluded that the No Observed Effect Level (NOEL) is 100 mg/kg bw/day based on treatment related clinical signs of toxicity and reduction in body weight found at the dose of 300 mg/kg bw/day, and the No Observed Adverse Effect Level (NOAEL) is 600 mg/kg bw/day as no adverse effects were found at any dose level tested.
- 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:
- supporting study
- 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:
- supporting study
- 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:
- supporting study
- 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:
- supporting study
- Study period:
- January 1980 - April 1980
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- GLP study performed similarly to OECD Guideline 408 but with deviations: dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- dosing 5 days/week instead of 7 days/week; food consumption, haematological and clinical biochemical test not followed
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: F344/N
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories (Portage, USA)
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: 136-153 g; females: 101-120 g
- Housing: Housed in groups of five in polycarbonate cages
- Diet (e.g. ad libitum): Purina Lab Blox (Chesapeake Feed Co., Beltsville, USA) or NIH 07 Rat and Mouse Ration (Zeigler Bros., Inc., Gardners, PA, USA), ad libitum
- Water (e.g. ad libitum): Automatic watering system (Edstrom Industries, Waterford, USA), ad libitum
- Acclimation period: 18 or 19 days
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 60-82 °F
- Humidity (%): 35-80%
- Air changes (per hour): 12-15/hour
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: Appropriate amount of test substance was weighed and mixed with corn oil by shaking in a volumetric flask.
VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - Apparatus: Periodic analysis for d-limonene in dose preparations was determined by extraction with methanol followed by gas chromatographic analysis of the extract with a 6-foot 3% OV-17 glass column, a nitrogen carrier at a flow rate of 30 mL/min, and a flame ionization detector.
- Sampling frequency: Once
- Results: 101-109% of the target concentrations - Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- Once per day; 5 days/week
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 600 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 200 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 2 400 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Doses were selected based on the mortalities observed at 3300 and 6600 mg/kg bw/day during a 16 day subacute toxicity study.
- Rationale for animal assignment (if not random): Random - Positive control:
- No
- Observations and examinations performed and frequency:
- CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: Initially and once per week thereafter - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; necropsy performed on all animals
HISTOPATHOLOGY: Yes; performed on all vehicle control and high dose animals and all female rats in the 1200 mg/kg bw/day group. Tissues examined include: adrenal glands, brain, colon, esophagus, eyes (if grossly abnormal), femur or sternebrae or vertebrae including marrow, gross lesions and tissue masses with regional lymph nodes, heart, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular or mesenteric lymph nodes, pancreas, parathyroids, pituitary gland, prostate/testes or ovaries/uterus, salivary glands, small intestine, spinal cord (if neurologic signs present), spleen, stomach, thymus, thyroid gland, trachea, and urinary bladder. Kidneys examined for all male rats. - Other examinations:
- No
- Statistics:
- No data
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- - Rough hair coats, lethargy and excessive lacrimation were observed for rats that received 1200 or 2400 mg/kg bw/day.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- - 5/10 males and 9/10 females at 2400 mg/kg bw/day died during week 1.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- - Final mean body weights of male rats in 600, 1200 or 2400 mg/kg bw/day groups were 6, 12 or 23% lower than that of the vehicle controls.
- Final body weight of the female rat that received 2400 mg/kg bw/day and lived to end of the study was 11% lower than the mean of the vehicle controls. - Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not specified
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- - Nephropathy was identified in all groups of male rats, and there was a dose-related increased severity of the lesion in dosed groups.
- Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium.
- Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOAEL
- Sex:
- male
- Basis for effect level:
- other: male-rat specific nephrotoxicity at all dose levels (considered as not relevant for humans)
- Remarks on result:
- other: no NOAEL identified
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 600 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day;
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 200 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- body weight and weight gain
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 600 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: mortality at 2400 mg/kg bw/day; occurrence of clinical signs of toxicity (rough hair coats, lethargy and excessive lacrimation) at 1200 and 2400 mg/kg bw/day
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 200 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- clinical signs
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Under the test conditions, the NOAEL for male and female rats was considered to be 600 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats could be identified in this study.
- Executive summary:
In a 13-week subchronic toxicity study performed similarly to OECD Guideline 408 and in compliance with GLP, d-limonene was administered through gavage to groups of 10 F344/N rats/sex/dose mixed in corn oil at dose levels of 0, 150, 300, 600, 1200 and 2400 mg/kg bw/day for 13 weeks (5 days/week). Animals were observed twice daily for clinical signs of toxicity and bodyweights were recorded weekly. Necropsy performed on all animals and microscopic examination of specified tissues was performed for all control and high dose animals scheduled to be killed at the end of the treatment period. Five of 10 males and 9/10 female rats that received 2400 mg/kg bw/day died during week 1. Final mean body weights of male rats in 600, 1200 or 2400 mg/kg bw/day groups were 6, 12 or 23% lower than that of the vehicle controls. Final body weight of the female rat that received 2400 mg/kg bw/day and lived to end of the study was 11% lower than the mean of the vehicle controls. Rough hair coats, lethargy and excessive lacrimation were observed at 1200 or 2400 mg/kg bw/day. No treatment-related histopathologic lesions were observed in female rats. Nephropathy was identified in all groups of male rats, and there was a dose-related increased severity of the lesion in dosed groups. Nephropathy was characterized by degeneration of epithelium in the convoluted tubules, granular casts within tubular lumens, primarily in the outer stripe of the outer medulla, and regeneration of the tubular epithelium. Hyaline droplets (protein reabsorption droplets) were observed in the epithelium of proximal convoluted tubules in all groups of male rats, including vehicle controls. This mechanism of nephrocarcinogenicity has been proven as being male-rat specific and not relevant for humans.
Under the test conditions, the NOAEL for female rats was considered to be 600 mg/kg bw/day. When considering the non relevance of the nephrotoxic effects for humans, the NOAEL for male rats would be 600 mg/kg bw/day, based on decrease of bodyweight gains at 1200 and 2400 mg/kg bw/day. The LOAEL for female and male rats were considered to be 1200 and 150 mg/kg bw/day, based on observation of clinical signs and nephropathy, respectively. As nephrotoxicity and accumulation of hyaline droplets were observed in male rats at all dose levels, no NOAEL for male rats had primarily been identified in this study.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- 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:
- supporting study
- 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:
- supporting study
- Study period:
- 1990
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Study conducted similarly to OECD Guideline 409 with deviations: age of animals > 9 months; no data on initial bodyweights; only two dose levels studied; ophthalmological examination not followed; individual animal data not reported
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
- Deviations:
- yes
- Remarks:
- age of animals > 9 months; no data on initial bodyweights; only two dose levels studied; ophthalmological examination not followed; individual animal data not reported
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- dog
- Strain:
- Beagle
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 10-15 months
- Housing: Housed in runs with outdoor access
- Diet (e.g. ad libitum): Meals provided for only 1 hour
- Water (e.g. ad libitum): Ad libitum - Route of administration:
- oral: gavage
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- - PREPARATION OF DOSING SOLUTIONS:
The test item was directly dosed by gavage in doses of 0.12 and 1.2 ml/kg body weight/day. - Analytical verification of doses or concentrations:
- not specified
- Details on analytical verification of doses or concentrations:
- Not applicable
- Duration of treatment / exposure:
- 180 days
- Frequency of treatment:
- Each daily dose was divided into two equal amounts, administered at approximately 9.30 am and 2.30 pm.
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- (1.2 ml tap-water/kg bw/day)
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- (0.12 ml test item/kg bw/day)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- (1.2 ml test item/kg bw/day)
- No. of animals per sex per dose:
- Five
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- - Dose selection rationale: Highest daily dose was determined in a pilot 28-day study conducted in 5 male and 5 female beagles. Kidney weights for the d-limonene-treated animals were unaffected, but absolute and relative liver weights were slightly increased. Based on these findings, the 1.2 mL/kg bw/day treatment was chosen.
- Positive control:
- No
- Observations and examinations performed and frequency:
- CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily for at least 1 hour after dosing
BODY WEIGHT: Yes
- Time schedule for examinations: At study initiation, weekly during the study and at the time of sacrifice
FOOD CONSUMPTION: Yes
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At 2 week pre-study (baseline) and then 1, 3 and 6 months during the study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- Parameters examined: White cell count, red blood cell count, haemoglobin, haematocrit, platelet count, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At 2 week pre-study (baseline) and then 1, 3 and 6 months during the study
- Animals fasted: No data
- Parameters examined: Blood urea nitrogen (BUN), BUN/creatinine, creatinine, aspartate amino transferase, alanine amino transferase, phosphorus, glucose, albumin, total protein, globulins, albumin/globulin, alkaline phosphatase, cholesterol, triglycerides, sodium, potassium, calcium and chloride
URINALYSIS: Yes
- Time schedule for collection of urine: 24-hour urine samples were collected at approximately 2 week pre-study and again at 6 months.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: Colour and appearance, specific gravity, occult blood, protein, pH, glucose, ketones, bilirubin and urobilinogen and urinary sediment - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; each animal anaesthetized with pentobarbital, killed by exsanguination and observed for gross post-mortem examinations
HISTOPATHOLOGY: Yes; samples of the following tissues were collected and fixed in 10% buffered formalin for routine histological processing, and light microscopical evaluation of haematoxylin and eosin stained sections: lungs, bronchial lymph node, heart, thoracic aorta, tongue, oesophagus, trachea, thyroid, parathyroid, submandibular lymph node, mesenteric lymph node, stomach, parotid salivary gland, palatine tonsil, liver, gall bladder, duodenum, jejunum, ileum, colon, rectum, urinary bladder, kidneys, testicles with epididymis, prostate, ovaries, uterus, vagina, cervix, adrenals, thymus, psoas muscle, spleen, pancreas, bone/marrow, skin, brain, spinal cord, sciatic nerve, pituitary gland, and eyes. Mallory-Heidenhain-stained kidney sections were also prepared and evaluated for protein accumulation. Kidney weights were determined immediately on removal from the animal. Absolute organ weights were calculated as percentages of the body weights (relative weights). - Other examinations:
- None
- Statistics:
- - Statistically significant differences (P < 0.05, two-sided risk level) were established using least significant difference criteria, provided that Bartlett's test of homogeneity of variance was nonsignificant.
- Dose-response data were also analysed by linear regression (Dixon and Massey, 1969). - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Excretion of soft faeces; dose-related occasional mild discomfort during defaecation (presumably because of perianal contact with unabsorbed d-limonene as it passed with the faeces); sporadic episodes of emesis and diarrhoea
- Mortality:
- mortality observed, treatment-related
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- No treatment-related differences other than an increase in serum cholesterol (35%) and serum alkaline phosphatase levels (two-fold increase) at 1000 mg/kg bw/day were observed in male and female dogs.
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Linear regression analyses indicated a positive dose-related trend for absolute and relative female kidney weight and relative male kidney weight.
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 100 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: increased absolute and relative female kidney weight and relative male kidney weight at 1000 mg/kg bw/day
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- organ weights and organ / body weight ratios
- Key result
- Critical effects observed:
- not specified
- Conclusions:
- Under the test conditions, the NOAEL and LOAEL for beagle dogs were considered to be 100 and 1000 mg/kg bw/day, respectively, based on the increased absolute and relative female kidney weight and relative male kidney weight.
- Executive summary:
In a 6-month subchronic toxicity study performed similarly to OECD Guideline 409, d-limonene was administered through gavage to groups of adult beagle dogs (5/sex/dose) at dose levels of 0 (tap water), 0.12 or 1.2 mL/kg bw/day (0, 100 or 1000 mg/kg bw/day) in two divided doses for 180 days. Animals were observed daily and weighed at study initiation, weekly during the study and at the time of sacrifice. Feed consumptions were determined throughout the study and blood samples were obtained at 2 week pre-study (baseline) and then 1, 3 and 6 months during the study. At termination all animals were subjected to gross necropsy during which weights of kidneys were recorded and several tissues were processed for microscopical evaluation of haematoxylin and eosin stained sections. Mallory-Heidenhain-stained kidney sections were also prepared and evaluated for protein accumulation.
Feed consumption and body weight were unaffected by treatment. Clinical signs of toxicity noted were excretion of soft faeces, dose-related occasional mild discomfort during defaecation, sporadic episodes of emesis and diarrhoea. No treatment-related differences other than an increase in serum cholesterol (35%) and serum alkaline phosphatase levels (two-fold increase) at 1000 mg/kg bw/day were observed in male and female dogs. Linear regression analyses indicated a positive dose-related trend for absolute and relative female kidney weight and relative male kidney weight. There were no histopathological changes in the kidneys, evaluated by both haematoxylin and eosin and Mallory-Heidenhain staining that could be associated with the organ-weight changes. Furthermore, there was no evidence of hyaline droplet accumulation or any other sign of hydrocarbon-induced nephropathy typical of those seen in male rats treated with d-limonene.
Under the test conditions, the NOAEL and LOAEL for beagle dogs were considered to be 100 and 1000 mg/kg bw/day, respectively, based on the increased absolute and relative female kidney weight and relative male kidney weight.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- Test method according to OECD guideline 407 with some modifications.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Deviations:
- not specified
- GLP compliance:
- not specified
- Remarks:
- No information reported
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Department of Physiology and Pharmacology of Federal University of Pernambuco (UFPE, Pernambuco, Brazil)
- Diet (e.g. ad libitum): industrialized dry food (Presence®, Purina, Brazil), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 55-65 %
- Photoperiod: 12 hrs dark / 12 hrs light - Route of administration:
- oral: gavage
- Vehicle:
- other: Tween-80 aqueous solution
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
1,8-cineole (CIN) was emulsified in a 1% Tween-80 aqueous solution before administration to the animals.
Volume administered: 10 mL/kg bw
VEHICLE
Tween-80 (CAS No: 9005-65-6) was obtained from Sigma-Aldrich® (St. Louis, MO, USA). - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 50 days
- Frequency of treatment:
- Daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- control
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
100 mg/kg dose was chosen according to previous results which showed significant gastric mucosa protection in the antiulcerogenic tests (Caldas et al., 2015). The others doses (500 and 1000 mg/kg) were extrapolated 5- and 10-fold the amount of the effective dose. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes, the animals were observed for signs of toxicity, such as piloerection, diarrhea, changes in locomotor activity or mortality.
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes, weekly
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly
HAEMATOLOGY: Yes
- Time schedule for collection of blood: 12 h
- Anaesthetic used for blood collection: Yes, thiopental (35 mg/kg, intraperitoneal route)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: red blood cell (RBC) count, hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), white blood cell (WBC) count, platelets count, mean platelet volume (MPV) and differential leukocyte count (lymphocytes, monocytes and granulocytes).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 12 h
- Anaesthetic used for blood collection: Yes, thiopental (35 mg/kg, intraperitoneal route)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked: glucose, blood urea nitrogen (BUN), creatinine, uric acid, sodium, potassium, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, triglycerides, gamma glutamyltranspeptidase (GGT), alkaline phosphatase (ALP); total, direct and indirect bilirrubin.
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.
OTHER: - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
After blood collection, all the animals were euthanized in a CO2 chamber (by inhalation) for necropsy and evaluation of vital organs. For macroscopic analysis of organs were randomly selected 10 animals (5 males and 5 females) in each group.
The organs including heart, lung, liver, kidneys, adrenal glands, spleen, stomach, intestine, pancreas, brain and reproductive organs testicles and prostate (male) or uterus and ovaries (female) were carefully removed and weighed individually. Organ weights were expressed in absolute and relative terms (g and g/100 g of body weight, respectively).
HISTOPATHOLOGY: Yes
The remaining animals in each group (10 animals, 5 males and 5 females) were perfused with saline (to remove blood), and then, the organs previously described were removed and fixed "in totum" in 10% buffered formalin for 48h at room temperature. After fixing, each sample was washed with water and immersed in 70% ethyl alcohol for 3 to 4 days, then were embedded in paraffin. Paraffin sections of 5 μm were obtained and stained with hematoxylin/eosin (HE). Histological analyses of organs were made using an automatic microscopy system MICRO DIP® (Kacil Inc.). - Statistics:
- Values were expressed as mean ± standard error of mean (S.E.M.) and the differences are analyzed by variance analysis (ANOVA) followed by Dunnett’s test or Student’s T test for unpaired samples. The level of significance for rejection of the null hypothesis was set at 5% (p < 0.05). Statistical analyses were performed using GraphPad Prism 5.0®.
- Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No signs of toxicity (such as piloerection or alteration in locomotor activity) were recorded during the 50 consecutive days of treatment by oral route at doses of 100, 500 or 1000 mg/kg. However, animals treated at doses of 500 and 1000 mg/kg presented diarrhea during the first week of treatment although this ceased from the second week onwards.
- Mortality:
- no mortality observed
- Description (incidence):
- No deaths occurred during the study.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Although it was observed a decrease in body weight gain in animals treated with test susbstance at doses of 500 and 1000 mg/kg during the first week, this reduction did not affect the total weight gain of animals, indicating that this response may be associated with the occurrence of diarrhea in rats treated during this period.
- Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Changes were observed in the consumption of food during all the treatment in animals of both sexes treated with all doses of test substance.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Changes were observed in the consumption of water during all the treatment in animals of both sexes treated with all doses of test substance.
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- For male rats, there was a significant increase of 6.93% in MCV (1000 mg/kg) and of 43.54 and 38.98% in the platelet count (500 and 1000 mg/kg, respectively) and a decrease of 6.74 and 6.67 % in MCHC (500 and 1000 mg/kg) and MPV of 10.40, 10.60 and 15.73% (100, 500 and 1000 mg/kg, respectively), when compared to the control group. In the female groups, no statistically significant clinical findings were recorded for any of the parameters examined.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- No significant differences were observed in the serum levels of glucose, creatinine, uric acid, sodium, potassium, AST, ALT, total cholesterol, triglycerides, gamma glutamyltranspeptidase (GGT), total bilirubin, or direct and indirect bilirubin. However, a decrease was observed in the level of alkaline phosphatase (30.00%) in male rats treated with 100 mg/kg and an increase of 29.27% and 25.86% in the level of urea (BUN) in the female groups treated with 500 and 1000 mg/kg, respectively, in relation to the control group.
- 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):
- There was a decrease in absolute weight of the lungs (15.10%) and spleen (20.25%) in males rats at doses of 500 and 1000 mg/kg, respectively, as well as an increase in absolute (32.55%) and relative (39.33%) weight of the liver in females at a dose of 1000 mg/kg compared to control group.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- The macroscopic analysis of the target organs of the animals treated with 1,8-cineole did not show significant changes in the color or texture when compared with the control group.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Microscopic examination of the organs showed the presence of eosinophilic and lymphocytic infiltrate in the lungs of males and females and into the uterus of females at all doses and lymphocytic infiltrate in the liver of males treated with all doses and females (500 and 1000 mg/kg). It was also observed increase of the glomerular space in the kidneys, weak for males (1000 mg/kg), and moderate for females (500 and 1000 mg/kg), which was more evident in females. The others organs of male and female animals in the treated and control groups exhibited no changes, with histological findings all within normal limits.
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: water consumption and mean platelet volume and alkaline phosphatase
- Key result
- Critical effects observed:
- no
- Conclusions:
- In a short term repeated dose toxicity study, oral routed performed with 1,8-cineole using Wistar rats for a exposure time of 50 days, occasional alterations in rats of both sexes were observed, as evidenced in hematological and biochemical parameters and histopathological analysis. However, these changes showed low clinically relevant, since they occurred in a non-generalized manner between animals of the treated groups. Furthermore, a NOAEL was not established since effects were found at the lowest dose tested (100 mg/kg bw/day).
- Executive summary:
1,8-cineole was daily administered to Wistar rats (male and female) for 50 days at doses of 0, 100, 500 and 1000 mg / kg bw/day by gavage. Test method was according to OECD guideline 407. In all experimental groups, animals were observed for signs of toxicity, such as piloerection, diarrhea, changes in locomotor activity or mortality. Body weight, food and water consumption were determined once a week. Haematological and clinical tests 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. Histological preparations 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.
No deaths or significant clinical signs of toxicity were reported during the study. Although it was observed a decrease in body weight gain in animals at doses of 500 and 1000 mg/kg during the first week, this reduction did not affect the total weight gain of animals, indicating that this response may be associated with the occurrence of diarrhea in rats treated during this period. Furthermore, oscillations in the consumption of water and food observed did not prevent an increase of the body weight from the second week of treatment in animals of both sexes with all doses.
Haematological tests revealed an increase in MCV and platelet count and a decrease in MCHC and MPV in male animals treated with 500 and 1000 mg/kg. Biochemical parameters showed a decreased level of alkaline phosphatase in male rats treated with 100 mg/kg and a slight increase in the level of urea in the group of females treated with 500 and 1000 mg/kg in relation to the control group. However, these values lie within the physiological limits described for the species (Harkness and Wagner, 2010).
There was a decrease in the absolute mass of the spleen and lungs in males (500 and 1000 mg/kg, respectively) and an increase in the absolute and relative mass of the liver in the females (1000 mg/kg). Although no change in the color or texture of organs has been found in rats of both sexes, the microscopic analysis showed eosinophilic and/or lymphocytic infiltrate in the lungs and liver of animals of both sexes and uterus of females treated with all doses. Moreover, an increase of the glomerular space in the kidneys of males females treated with highest doses was observed.
In conclusion, occasional alterations in rats of both sexes were observed, as evidenced in hematological and biochemical parameters and histopathological analysis. However, these changes showed low clinically relevant, since they occurred in a non-generalized manner between animals of the treated groups.
Since the lower dose (100 mg/kg) induced reduction of water consumption and mean platelet volume and alkaline phosphatase, it was not possible to establish the NOAEL.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- GLP compliance:
- not specified
- Remarks:
- (The experiment was carried out following the Regulations of Animal Experimentation of College of Veterinary Medicine, Sichuan Agricultural University, which is based on the Guidelines of the International Committee on Laboratory Animals)
- Limit test:
- no
- Species:
- mouse
- Strain:
- other: Kunming (a closed strain coming from Kunming, Yun nan province, P.R. China)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Chengdu Dossy Experimental Animals Co. Ltd. (License No. SCXK (Sichuan) 2008-24)
- Weight at study initiation: 18-22 g
- Housing: no data
- Diet (e.g. ad libitum): ad libitum, Laboratory animals-mice and rats formula feeds
- Water (e.g. ad libitum): ad libitum
- Acclimation period: The test animals were quarantined for 13 days before experiment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22ºC
- Humidity (%): 40-70%
- phothoperiod: 12 h light/dark cycle - Route of administration:
- oral: gavage
- Vehicle:
- other: 2% Tween 80
- Details on oral exposure:
- VEHICLE
- Amount of vehicle (if gavage): 10 ml total dose/kg/day - Analytical verification of doses or concentrations:
- no
- Duration of treatment / exposure:
- 30 days
- Frequency of treatment:
- Daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Group I
- Dose / conc.:
- 21.38 mg/kg bw/day (nominal)
- Remarks:
- Group II
- Dose / conc.:
- 64.15 mg/kg bw/day (nominal)
- Remarks:
- Group III
- Dose / conc.:
- 192.45 mg/kg bw/day (nominal)
- Remarks:
- Group IV
- No. of animals per sex per dose:
- 10 animals per dose either sex
- Control animals:
- yes, concurrent vehicle
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes, animal behavior and mortality.
BODY WEIGHT: Yes
- Time schedule for examinations: weekly - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
The animals were sacrificed on day 31. After fasted overnight, all the mice were euthanized. The organs/tissues were carefully examined macroscopically and gross lesions were recorded. The relative weight (organ to body weight ratios) of liver, spleen and double kidneys was immediately calculated.
HISTOPATHOLOGY: Yes
Heart, liver, spleen, lungs, kidney, testis and ovary of treatment groups and control group were excised and then fixed in 10% neutral buffered formalin. Following dehydration and embedding, they were sectioned at 5 μm, stained with Hematoxylin and Eosin (H and E) and examined microscopically.
Ultrastructural observation:
The organs were excised and prefixed in 2.5% glutaraldehyde solution, diced into 1 mm3, followed by three 15 min rinses with 0.1 M phosphate buffer (pH 7.4). Post-fixation was in cold 1% aqueous osmium tetroxide for 1 h. After rinsing with phosphate buffer again, the specimens were dehydrated in a graded ethanol series of 50~100% and then embedded in Epon 812. Ultra-thin sections were sliced with glass knives on a LKB-V ultramicrotome (Nova, Sweden), and stained with uranyl acetate and lead citrate. The sections were examined under a Hitachi H-600 transmission electron microscope. - Statistics:
- The statistical significance of differences between means was calculated using One-way Analysis of Variance (ANOVA) followed by Dunnett’s test for multiple comparisons with the control group.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- The animals in all groups were in good condition.
- Mortality:
- no mortality observed
- Description (incidence):
- No deaths occurred during the study.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- The body weights of the mice was increasing weekly in 1,8-cineole treated groups and the control group, and there was no significant difference among all the groups (P<0.05).
- 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 specified
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- There were no significant differences in organ weights and relative organ weights between the 1,8-cineole treated groups and the control group after administration for 30 days.
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- After 30 days of treatment the obvious pathological changes appeared in the liver and kidney of mice. There were no distinct histopathological changes in the other organs examined in this test.
Liver: Central venous congestion of liver lobule and granular degeneration of hepatocytes were appeared in Group II and III, while in Group IV, more serious pathological changes involving central venous congestion, granular degeneration, vacuolar degeneration and hepatic necrosis appeared.
Kidney: Normal appearances of kidney were appeared in Group I and II, in which glomerulus and renal tubule structure was clearly conserved. Group III and Group IV showed pathological changes: capillary of glomerulus and interstitial angiectasis hyperemia, renal tubular epithelial cells swelling, granular degeneration and partially separating from basement membrane; amount of eosinophilic protein exudation existing in tubular lumen. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- Ultrastructural observation:
Electron microscope has revealed specific ultrastructural changes in hepatic cells and renal tubular epithelial cells under experimental conditions.
Hepatic cells: After 30 days of administrated with 1,8-cineole (192.45 mg/kg), the endoplasmic reticulum was distorted and fractured. The ribosomal fell off from the rough endoplasmic reticulum membrane and scattered into the cytoplasm. The mitochondria were found to be swollen and the cristae within the mitochondria were disorganized.
Kidney cells: After 30 days of administrated with 1,8-cineole (192.45 mg/kg), some proximal convoluted tubule epithelial cells was interrupted and escape from the cytoplasm to the lumen of the tubule. The usually elongated mitochondria in the basilar portion of the distal convoluted tubule epithelial cells became more spherical, with marked disorganization and swelling of the cristae. However, the basement membrane of mitochondria was intact. No significant alteration was observed in the glomeruli. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 64.15 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- no
- Lowest effective dose / conc.:
- 192.45 mg/kg bw/day (nominal)
- System:
- urinary
- Organ:
- kidney
- Key result
- Critical effects observed:
- no
- Lowest effective dose / conc.:
- 192.45 mg/kg bw/day (nominal)
- System:
- hepatobiliary
- Organ:
- liver
- Conclusions:
- The NOAEL of 1,8-cineole after an oral exposure of 30 days was 64.15 mg/kg bw/day in mice.
- Executive summary:
A short term repeated dose toxicity study (oral route) was performed on 1,8-cineole according to OECD guideline 407. The substance was emulsified in 2% Tween 80 and administered daily to mice by oral gavage in doses of 0 (control, only 2% Tween 80), 21.38, 64.15 and 192.45mg/kg bw/day during 30 days.Animal behavior, body weight and mortality were recordedduring the study. After being sacrificed on day 31,organs/tissues were examined macroscopically and gross lesions were recorded. Relative weight of liver, spleen and double kidneys was calculated.Heart, liver, spleen, lungs, kidney, testis and ovary of treatment groups and control group were examined microscopically. Also, ultrastructural observation by electron microscopy was performed on those organ cells with effects. There were no significant differences in body weight and relative organ weight between the control group and treatment groups. The histopathological examinations showed that granular degeneration and vacuolar degeneration appeared in liver and kidney tissue after administration of high dose (192.45 mg/kg bw/day). Under electron microscopy, a series of ultrastructural changes were observed. At the subcellular level the influence of test substance was found on the mitochondria, endoplasmic reticulum and other membrane type structure of liver and kidney. Based on these results, the NOAEL of the test substance was determined to be 64.15 mg/kg bw/day.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- The study although predating current guidelines was performed similarly to OECD Guideline 408.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- (no data on details of test animals and environmental conditions)
- GLP compliance:
- no
- Limit test:
- no
- Species:
- rat
- Strain:
- Osborne-Mendel
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Housing: The animals were housed individually in wire cages
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- - DIET PREPARATION
- Rate of preparation of diet (frequency): weekly - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- 7% loss of test compound from lab animal diet during a 7-day period.
- Duration of treatment / exposure:
- 20 weeks
- Frequency of treatment:
- Fresh diets were made and distributed weekly.
- Dose / conc.:
- 10 000 ppm
- Remarks:
- Equivalent to 400 mg/kg bw/ day (based on food consumption of 40 g/kg bw/day included in the Guidance on information requirements and chemical safety assessment R8, table R 8 -17)
- Dose / conc.:
- 2 500 ppm
- Remarks:
- Equivalent to 100 mg/kg bw/ day (based on food consumption of 40 g/kg bw/day included in the Guidance on information requirements and chemical safety assessment R8, table R 8 -17)
- Dose / conc.:
- 1 000 ppm
- Remarks:
- Equivalent to 40 mg/kg bw/ day (based on food consumption of 40 g/kg bw/day included in the Guidance on information requirements and chemical safety assessment R8, table R 8 -17)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent no treatment
- Observations and examinations performed and frequency:
- CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes, weekly - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes; At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. Tissues from rats dying during the experiment were examined for gross changes and were preserved if autolysis was not advanced. Organs were not weighed but abnormalities and the suspected reason for death were noted.
HISTOPATHOLOGY: Yes; Organs weighed at termination, the remaining abdominal and thoracic viscera, and one hind leg, for bone, bone marrow, and muscle, were preserved in 10% buffered formalin-saline solution for histopathological examination. For routine histopathology, sections were embedded in paraffin wax and stained with haematoxylin and eosin. Detailed microscopic examinations were done on 6 or 8 rats, evenly divided by sex, from the high dose group and the control group. If changes attributable to the test compound were found in the high dose group, additional animals on lower dosage levels were examined as indicated. - Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- 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:
- 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
- Description (incidence and severity):
- No macroscopic change in the tissues in the 10000, 2500 and 1000 ppm exposure groups.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- No microscopic change in the tissues in the 10000 ppm exposure group.
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- > 10 000 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical signs
- food consumption and compound intake
- gross pathology
- haematology
- histopathology: non-neoplastic
- Remarks on result:
- other: Equivalent to NOAEL > 400 mg/kg bw/day.
- Key result
- Critical effects observed:
- no
- Conclusions:
- No effects were observed in a 20-weeks repeated dose toxicity study in rats at the tested concentrations (up to 10000 ppm).
- Executive summary:
In a 20 -weeks oral exposure study, Osborne-Mendel rats (10/dose/sex) were administered alpha-Terpinyl Acetate via diet intake at concentrations of 0 (control), 10000, 2500 and 1000 ppm. Animals were then observed for mortality, weight, food intake and general condition. Haematological examinations were made at termination. At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. Detailed microscopic examinations was done on the animals from the high dose group and the control group. No effect on growth or haematology, and no macroscopic or microscopic change in the tissues in the 10000 ppm exposure group were observed. No effect on growth or haematology, and no macroscopic change in the tissues in the 2500 and 1000 ppm exposure groups. No microscopic examination was performed on rats exposed to 2500 and 1000 ppm. According to the Guidance on information requirements and chemical safety assessment R8 table R 8 -17, rats eat 40 to 50 g/kg bw/day. 10000 ppm is equivalent to 1% in the diet. Consequently, rats exposed to 10000 ppm in the diet consumed between 400 and 500 mg Terpinyl-Acetate-Alpha per kg bodyweight per day. Therefore, the NOAEL was calculated to be higher than 400 mg/kg bw/day.
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 18 September 2012 – 28 March 2013
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- Test method according to OECD guideline 422 and GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- yes
- Remarks:
- (mating females were only administered test chemical up to 4 days of lactation instead of 13 days)
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River. Atsugi Breeding Center.
- Age at study initiation: 10 weeks
- Weight at study initiation: 350-420 g (378 g average in males); 215-257 g (231 g average in females)
- Housing: bracket-type metal net cage (W 254 × D 350 × H 170 mm). During mating (1 male: 1 female) and from 17th day of pregnancy to 4th day of lactation a plastic cage (W340 × D400 × H185 mm) was used.
- Diet (e.g. ad libitum): NMF solid form (radiation sterilization, Oriental Yeast Co., Ltd.), ad libitum.
- Water (e.g. ad libitum): Tap water (metal cage) and from bottle (during time of lactation in plastic cage), ad libitum.
- Acclimation period: 20 days (including 3 days of quarantine)
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24ºC
- Humidity (%): 39-65 %
- Photoperiod: 12 hrs dark / 12 hrs light - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Preparation of the test solutions was conducted by taking a required amount of the test substance with a glass syringe for each concentration and accurately weighing it in a beaker. The weighed test substance was transferred to a measuring cylinder. The beaker was washed several times with a small amount of solvent, and the liquid was also added to the measuring cylinder. The mixture was shaken by hand and mixed, and it was visually confirmed that it was completely dissolved. Next, the solvent was added to the measuring cylinder to the required amount of test material.
VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- A sample of 10 mL of each formulation prepared for administration in the first and last week of the dosing period was analysed for achieved concentration of the test substance. The mean concentrations of terpineol in test formulations analysed for the study were within applied limits (99.5 to 103.0% of nominal concentrations), confirming accurate formulation.
The determination of the concentrations was performed by HPLC. - Duration of treatment / exposure:
- Males: From 14th day before mating up to 30 days after mating (total 44 days).
Females: From 14th day before mating up to 4 days of lactation (total 41-51 days).
Non-mating female group: 44 days - Frequency of treatment:
- Once a day, 7 days a week
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- Mating male group: 12 per sex per dose (including 5 in the control and high dose group for 14-day observation after treatment)
Mating female group: 12 per sex per dose
Non-mating female group: 10 (control group, including 5 for 14-day observation after treatment) and 10 (high dose group, including 3 for 14-day observation after treatment) - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
A preliminary range finding study was conducted with doses of 250, 500 and 1000 mg/kg bw/ day. One male in the 1000 mg/kg bw/day dose group showed a decrease in body weight and food intake, a decrease in feces volume, a decrease in locomotor activity and a decrease in respiratory rate, and died on the 12 th day of administration. A high value of liver and kidney weight and a high value of urea nitrogen and total protein were observed in autopsy of surviving males of high dose group. In each female of 500 and 1000 mg/kg bw/day group, a significant reduction in walking and muscle tone and a licking gait were observed only on 1 day of administration. Therefore, in this study, 1000 mg/kg bw/day at which obvious signs of toxicity is expected was set as high dose, and 300 and 100 mg/kg bw/day were set as medium and low dose, respectively, divided by the common ratio of about 3. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily during the quarantine period, three times daily for the administration period (before administration, immediately after administration and 1 to 3 hours after administration), twice daily during the recovery period (morning, afternoon)
- Cage side observations: The presence or absence of abnormalities in general conditions such as life and death, appearance of the body, posture, behavior and emissions (urine, feces) were observed. Regular observation accompanied by handling of animals after 2 weeks of administration was also carried out as observation of detailed general condition.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed general condition observation was performed once for all individuals, before administration. Also, males and non-mating females were observed once a week during the administration period and during the recovery period. Females in the mating group were observed once a week during the pre-mating period, during the mating period, during the pregnancy period and during the lactation period.
BODY WEIGHT: Yes
- Time schedule for examinations: For all individuals, body weight was measured on acclimated days 1, 3, 8, 13 and 20 and on the day of administration 1, 8, 15, 22, 29, 36, 42, 44 and on the day of necropsy. For the recovered animals further recovered the body weight was measured on days 1, 8 and 14 and on the day of necropsy. Body weights of female mating group were measured on day of administration 1, 8, 15, 22, on day of gestation 0, 7, 14, and on day 0 and 4 of lactation.
FOOD CONSUMPTION: yes
-Food consumption was determined daily by weighing the residue. Measurements were not made during the mating period.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: The day before necropsy after administration period and after 14-day observation period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 animals per sex and per group
- Parameters checked: Erythrocyte count (RBC), Haemoglobin concentration (HGB), Haematocrit (HCT), Mean cell volume (MCV), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Reticulocyte count (Retic), Platelet count (PLT), White blood cell count (WBC), differential leucocyte count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M) and Large unstained cells (LUC)), Prothrombin time (PT), Activated partial thromboplastin time (APTT) and Fibrinogen (FIB).
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The day before necropsy after administration period and after 14-day observation period.
- Animals fasted: Yes
- How many animals: 5 animals per sex and per group
- Parameters checked: Serum was analysed for: Alkaline phosphatase (ALP), Total bile acid (TBA), Total cholesterol (T-CHO), Triglyceride (TG), Phospholipids (PL), Total bilirubin (T-BIL), Glucose (GLU), Urea nitrogen (BUN), Creatinine (CRNN), Sodium (Na), Potassium (K), Chlorine (Cl), Canoesium (Ca), Inorganic phosphorus (P), Total protein (TP) and Anolebumin (ALB). Heparinized blood was examined for Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Lactate dehydrogenase (LDH) and Gamma-glutamyl transpeptidase (GTP).
URINALYSIS: Yes
- Time schedule for collection of urine: During the last week of the administration period and during the recovery period urine was collected for 4 and 20 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: For the first 4 hours urine collected pH and urine volume were taken. Osmotic pressure, electrolytes (sodium, potassium and chlorine) and urine volume were measured for the 20 hours urine obtained. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy, which included a visual examination of the external tissues and organs of the head, thorax and abdomen.
HISTOPATHOLOGY: Yes
- ORGAN WEIGHTS:
The absolute weight and relative weight of the following organs were measured: Brain, pituitary, thyroid (including parathyroid gland), adrenal gland, spleen, thymus, tongue, heart, liver, Kidney, testis, epididymis, prostate, seminal vesicle (including coagulated gland), ovary and uterus.
-HISTOPATHOLOGY:
For all individuals, the following organs / tissues were fixed for histopathology examination: cerebrum, cerebellum, sciatic nerve, spinal cord (chest), the eyeball, optic nerve, pituitary, thyroid, parathyroid, adrenal gland, spleen, thymus, submandibular lymph node, mesenteric lymph node, heart, aortic aorta, trachea, lung (including bronchus), tongue, larynx, oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, kidney, testis, ovary, epididymis, uterus, prostate, seminal vesicle (coagulating glands mate), skin (inguinal region), mammary gland (inguinal region), sternum, femur (including bone marrow) and femoral skeletal muscle. Samples of any abnormal tissues were also retained and processed for examination. Testis and epididymis were fixed with Bouin's fixative. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- CAGE SIDE OBSERVATIONS: There was no abnormality in any animal.
SIGNS AND ARENA OBSERVATIONS: Wiggling was observed on the first day of pregnancy in one female in the mating group of 1000 mg/kg/day administration group.
In addition, two animals were observed gait abnormality and jumping respectively but they were temporary findings and it was judged that there was no relation with the administration of the test substance because it was not related to the dose. Furthermore, facial cleansing behavior was observed in another animal, which is a normal behavior observed when the animal calms down, and is not related to administration of the test substance.
General condition of surviving animals: In the 1000 mg/kg/day dose group, 4 males had nursing behaviour and 1 male a decrease in feces volume, and in females, a decrease in feces volume was observed. In 3 females of the non - mating group, decrease in fecal volume, scaling, decreased motor activity, wiggling gait or decrease in respiration rate were observed.
These symptoms were not seen during the recovery period, thus it was judged as no treatment related.
Gripping strength: There was no significant difference between the control group and each test substance administration group.
Motor activity: There was no significant difference between the control group and each test substance administration group. Meanwhile, in the females of the non-mating group, a significant low value was observed in the measurement values of 0 - 10 and 10 - 20 intervals in the 1000 mg/kg/day dose group compared with the control group, but there was no significant difference in the total measurement value (0-60 interval) and it was equivalent to the measurement value of the control group at the end of the recovery period week. Thus it was judged to be a change within the variation range. - Mortality:
- mortality observed, treatment-related
- Description (incidence):
- 6 females in the 1000 mg/kg/day dose group were found dead or euthanized. These animals showed worsening status before moribund and death, suppression and reduction in body weight gain, low food consumption, decreased fecal volume, scraping, wiggling, decreased motor activity, decreased respiratory rate, abdominal drainage and decrease in body temperature. In addition, it was mainly characterized by malnutrition, reduction of spleen and thymus, downsizing of kidney (renal tubular dilation, vacuolization of proximal renal tubule, vacuolation of distal tubule / collecting duct, papillary necrosis, single cell necrosis of teat canal, regeneration of the papillary collecting duct, papillary cell infiltration), single cell necrosis of the transitional epithelial cell, hypertrophy / hyperplasia of the transitional epithelial cell, adrenal gland cell vacuolation, eosinophilic droplets and vacuolation of hepatocytes in the liver. Other secondary changes accompanying state deterioration include atrophy of various lymphoid tissues, atrophy of colonic mucosa, land mucosa and uterine atrophy or reduction of zymogen granules of pancreas, mesenterium lymph nodes, submandibular lymph nodes and thymus.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In males, suppression of body weight gain was observed in the 1000 mg/kg/day dose group, and a significant lower value was observed in body weight gain during the administration period. In the group administered with 300 mg/kg/day or less, the body weight transition was similar to that of the control group, and no significant difference was observed.
In the mating group females, body weight was lower than that of the control group through premating, pregnancy and lactation period in the 1000 mg/kg/day dose group, but no significant difference was observed. A significant low value was observed on the 0th day of lactation compared to the control group in the 300 mg/kg/day administration group, but it was a temporary change, and the weight gain increase in the subsequent lactation period was found to be a significant high value. It was judged that it was not related to administration of the test substance. In the 100 mg/kg/day administration group, no significant difference was observed between the control group and the control group.
In the non-mating group females, mean body weight decreased due to the moribund / death cases on day 4 of administration in the 1000 mg/kg/day dose group, and a significant lower value was observed than in the control group. However, it is a transient fluctuation due to the moribund / death cases, not an influence on the body weight by administration of the test substance.
In the 1000 mg/kg/day dose group, both sexes showed weight gain above the control group during the recovery period, but no significant difference was found between the treated group and the control group. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- In males, a significant high value was observed on the 30th and 44th days of administration in the 1000 mg/kg/day dose group, but it was judged as a change within the variation range because it was temporary. In the group administered with 300 mg/kg/day or less, the food consumption was equivalent to that of the control group, and no significant difference was observed. In the mating group females, there was no significant difference between the control groups and the test substance administration group in the pre-mating, pregnancy and lactation periods. In the non-mating group females, a significant low value was observed on the 8th day of administration compared with the control group in the 1000 mg/kg/day dose group, but it was judged that it was a slight temporary change and was not related to the administration of the test substance. In the 1000 mg/kg/day dose group, males showed food intake over the control group during the recovery period, and a significant difference was observed on administration 1 and 14 days. In females there was no significant difference between the control group and the 1000 mg/kg/day dose group.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- During administration and recovery period, a significant high value of water intake was observed in the 1000 mg/kg/day dose group of males and non-mating group females as compared with the control group.
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In males, a significant lower value of hemoglobin was observed in the 1000 mg/kg/day dose group compared to the control group. This difference was not seen during the recovery period, thus it was judged as no treatment related.
In the mating group females, no change was observed by administration of the test substance. In addition, compared with the control group, the leukocyte count, the basophil count and the large unstained cell count were significantly lower in the 100 mg/kg/day administration group. Significantly high levels of white blood cell count, eosinophil count and monocyte count were observed in the 300 mg/kg/day administration group, but there was no significant difference in the leukocyte percentage and there was no relation with the dose and the females of the non-mating group. It is considered that it is temporary fluctuation accompanying increase / decrease of mild leukocyte counts at the beginning of lactation after childbirth because there is no such change, and it was judged to be within the physiological variation range.
Moreover, a significant shortening of the prothrombin time was observed in the group administered with 300 mg/kg/day, but it was judged within the physiological variation range because it was slight shortening and there was no toxicological significance.
Significantly low levels of hemoglobin and hematocrit and significant high values of mean red blood cell hemoglobin concentration and significant high levels of fibrinogen were observed in non-mating group females as compared with the control group. In addition, although a significant low value of eosinophil ratio was observed in the 1000 mg/kg/day dose group compared with the control group, it was a slight change and there was no significant difference in the eosinophil count, so it was judged as an accidental change.
During recovery period, significantly low platelet count was observed in the 1000 mg/kg/day dose group compared with the control group, and the lymphocyte ratio was significantly lower in the 1000 mg/kg/day dose group in the non-mating group female, but since it was not recognized during administration period and it was a slight change in the background data, it was judged that it was not related to the administration of the test substance. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In male, a significant high value was observed in GTP in the 1000 mg/kg/day dose group compared to the control group. This difference was not seen during the recovery period, thus it was judged as no treatment related.
In the mating group females, a significant high value was found in GTP in the group administered with 300 mg/kg/day compared to the control group, but it was a slight change and it was not related to the dose, so it was judged within the physiological variation range.
In the non-mating group females, significant high values of calcium and inorganic phosphorus were found in the background data of the test facility, but this change was equivalent to the control group of the mating group female, thus it was judged that there was no relation with the administration of the test substance.
At the end of recovery period, significant low value for total bilirubin and significant high value for calcium in the male 1000 mg/kg/day dose group, and significant high value of total cholesterol in the 1000 mg/kg/day dose group of non-mating group females were observed. These were minor changes compared with background data and results at the end of the administration period, thus it was judged that there was no relation with the administration of the test substance. - Urinalysis findings:
- effects observed, treatment-related
- Description (incidence and severity):
- During administration period: Regarding qualitative items, neither sex was abnormal in any animals. Regarding quantitative items, a significant high value of urine volume accompanied by a significant high value of water intake and a significant low value of urinary osmotic pressure were observed in the 1000 mg/kg/day dose group of males and non-mating group females as compared with the control group.
During the recovery period: Regarding the qualitative items, neither sex was abnormal in any animals. For quantitative items, high or significant urine volume with a significant high value of water intake and significant low value of urinary osmotic pressure were observed in the 1000 mg/kg/day dose group of males and non-mating females compared with the control group. Sodium, potassium and chlorine emissions were high or significantly high. - Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- During dosing period, neither animal had any abnormality in the auditory reaction, approach reaction, contact reaction, pain sense reaction, pupillary reflex, and airborne forward reflection.
In the landing leg width, there was no effect of administration of the test substance. In the non-mating group females, a significant high value was observed at 6 weeks of administration in the 1000 mg/kg/day administration group compared with the control group, but since it was equivalent to the measurement value of the control group at the end of the recovery period, it was judged as no treatment related.
During recovery period, neither animal had any abnormality in the auditory reaction, approach reaction, contact reaction, pain sense reaction, pupillary reflex, and airborne forward reflection.
There was no significant difference between the control group and the 1000 mg/kg/day administration group in the landing leg open width. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- In males, significant high values for liver and kidney and significant low values for testes and epididymis in absolute and relative weights were observed in the 1000 mg/kg/day dose group.
In addition, a significant high value was observed in the relative weight of the pituitary, but since there was no change in the absolute weight and there was no histological abnormality, it was judged as a change within the physiological variation range. In addition, a significant high value was found in the absolute and relative weights of the prostate in the 300 mg/kg/day administration group compared to the control group, but it was not related to the dose.
In the mating group females, significant high values for liver in absolute and relative weights were observed in the 300 mg/kg/day dose group.
In addition, in the group administered with 300 mg/kg/day, there was a significant low value in the absolute weight of the brain and a significant high value in the relative weight of the heart, but there was no change in the same direction as the absolute or relative weight, and it was histologically judged as a change within the physiological variation range because there was no abnormality.
In non-mating group females, significant high values for liver, kidney and adrenal glands in absolute and relative weights were observed in the 1000 mg/kg/day dose group.
In males at the end of the recovery period, significant high values for liver and kidney and significant low values for testes and epididymis in absolute and relative weights were observed in the 1000 mg/kg/day dose group.
In addition, a significant high value was found in the absolute and relative weights of the thymus and the adrenal glands, but these were changes not seen at the end of the administration period and thus, they were histologically judged as changes within the physiological variation range because there were no abnormalities.
In non-mating group females at the end of the recovery period, significant high values for liver in relative weight were observed in the 1000 mg/kg/day dose group. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- In male, 2/7 cases of white kidney and 7/7 cases of small testes were observed in the 1000 mg/kg/day dose group.
In females of non-mating group, 1/5 cases of discoloured, pale kidney were observed in the 1000 mg/kg/day dose group.
No abnormality was observed by administration of the test substance in the mating group female (necropsy on 5th of lactation).
However, in the 1000 mg/kg/day dose group of the mating group female, 9/12 cases (including 1 untreated female) were infertile, 2/12 cases were pregnant and 1/12 cases died on 3rd pregnancy. The cause of female infertility was the miniaturization of the testes (histologically moderate seminiferous tube atrophy, the appearance of mild to moderate multinucleated giant cells, minor or mild seminiferous tube vacuolation).
In males at the end of the recovery period, 5/5 cases of small testes were observed in the 1000 mg/kg/day dose group. There was no abnormality in females of non-mating group. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- drenal gland: The vacuolation (minor or mild) of cortical cells was found in the group administered 300 mg/kg/day or more in the mating group female and in the 1000 mg/kg/day dose group of the non-mating group female.
Testis: Atrophy (moderate) of seminiferous tubules, multinucleated giant cells (minor to moderate) and vacuolization of seminiferous tubules (minor, mild) were found in the1000 mg/kg/day dose group. The testicular disorder caused by the administration of the test substance was considered to be the cause of female infertility.
At the end of the recovery period, atrophy of seminiferous tubules (mild to moderate), multinucleated giant cells (minor) and vacuolization of seminiferous tubules (minor, mild) were found in the1000 mg/kg/day dose group. Mature spermatozoa were not observed, but the length of seminal epithelium was higher than the examination at the end of administration and recovery was noted.
Epididymis: Reduction of sperm (mild to severe) and cell debris content of the lumen (mild, moderate) were observed in the 1000 mg/kg/day dose group, reflecting the influence on the testes by administration of the test substance.
At the end of the recovery period, reduction of sperm (moderate) and cell debris content of the lumen (mild, moderate) were seen in the 1000 mg/kg/day dose group. Although it was thought that it was an image reflecting the change of the testes, it was thought that it will disappear with the recovery of the testes in the future.
Kidney: The vacuolization (minor) of the proximal tubule was observed in the 300 mg/kg/day dose group in the mating group female, the vacuolization (minor, mild) in the distal tubule / collecting duct was in the male, the mating group female and the non-mating group female in the 1000 mg/kg/day dose group, the regeneration of the cortical renal tubule (minor, mild) was in the group administered with 300 mg/kg/day or more in males and in the 1000 mg/kg/day dose group of non-mating females, renal tubule dilation (minor, mild), single cell necrosis of papillary duct (minor) and cortical cell infiltration (minor) were observed in the 1000 mg/kg/day dose group of males and non-mating group females, regeneration (minor) of papillary collecting duct was in the 1000 mg/kg/day male group. Papillary necrosis (minor, mild) and papillary cell infiltration (minor) were found in the 1000 mg/kg/day dose group of mating female and non-mating females. In addition, an increase in the frequency of eosinophilic corpuscles in renal tubules was observed in the male group receiving 300 mg/kg/ day or more. Eosinophilic bodies of renal tubular epithelial cells were positive for α2μ globulin immunohistochemistry and were negative for PAS staining and were considered to be derived from α2μ globulin.
At the end of the recovery period, papillary necrosis (minor) was observed in the 1000 mg/kg/day dose group of male and non-mating females, regeneration (mild) of cortical renal tubules was observed 1000 mg/kg/day dose group of males. Although renal papillary necrosis was not seen at the end of male administration, similar changes were observed in mating and non-mating females, and even in male, regeneration of papillary collecting ducts and single-cell necrosis of papilla were seen, thus it was thought that the change at the end of the administration remained, but the degree of finding was minor. In females, animals with strong papillary changes were deceased or euthanized, and it seemed that they recovered for those with minor changes. Regeneration of cortical renal tubules decreased frequency and showed recovery.
Liver: centrilobular, hepatocytic hypertrophy (minor) was found in the 1000 mg/kg/day dose group of male; hepatocyte vacuolation (minor, mild) was found in the 1000 mg/kg/day dose group of mating female and the non-mating female.
Urinary bladder: Atrophy of umbrella cells (minor, mild) were observed in the 1000 mg/kg/day dose group of males, mating females and non-mating females. Vacuolation of umbrella cells (minor to moderate) were observed in the 300 mg/kg/day dose group of males and mating females. Hypertrophy / hyperplasia of epithelial cells (minor, mild) was observed in the male, mating group female and non - mating group females administered with 1000 mg/kg/day.
Pancreas: An increase in the frequency of decreased zymogen granules (minor to moderate) was observed in the 300 mg/kg/day dose group of mating females and in the 1000 mg/kg/day dose group of non-mating females.
In two female animals all littermate died on 2nd day of lactation in the 300 and 1000 mg/kg/day dose group, respectively. In both cases the death of all littermate was considered to be a possibility of deterioration of the condition of the mother animal. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 100 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Remarks on result:
- other: Effects observed at the dose tested of 300 mg/kg bw/day.
- Key result
- Critical effects observed:
- no
- Conclusions:
- Based on the findings of this study, the NOEL of terpineol after an oral exposure of 44 days was determined to be 100 mg/kg bw/day in male and female rats.
- Executive summary:
A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of terpineol (CAS 8000-41-7) by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. Terpineol was diluted with corn oil and added to Sprague-Dawley SPF rats (12 per sex per dose) at doses of 0, 100, 300 and 1000 mg/kg bw/day, from day 14 before mating up to 30 days after mating for males (total 44 days) and up to 4 days of lactation for females (total 41-51 days). A non-mating female group (10) was exposed at doses of 0 and 1000 mg/kg bw/day for 44 days. Also, after administration period some animals from the control and high dose groups (5 males of mating group, 5 females of control and 3 females of high dose of non-mating group) were kept for further 14 days without treatment to detect delayed occurrence and recovery from toxic effects. During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, haematology, blood chemistry and urinalysis. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults.
After administration and recovery periods no treatment-related effects were identified on clinical sings and general conditions of animals, body weight gain, haematology and blood chemistry parameters.
In the 1000 mg/kg/day dose group, 6 females were found dead or had to be euthanized. Significant low value of urinary osmotic pressure was observed in the 1000 mg/kg/day dose group of males and non-mating females compared with the control group. At the end of the recovery period significant high values for liver and kidney and significant low values for testes and epididymis in absolute and relative weights of males and significant high relative weights for liver of non-mating females were observed in the 1000 mg/kg/day dose group. In the mating group females, significant high values for liver in absolute and relative weights were observed in the 300 mg/kg/day dose group. In the 1000 mg/kg/day dose group of mating females, 9/12 cases were infertile, 2/12 cases were pregnant, and 1/12 cases died on day 3 pregnancy. Female infertility was considered to be caused by the effects found in histopathology of testis at the end of recovery period: Atrophy (mild to moderate) of seminiferous tubules, multinucleated giant cells (minor) and vacuolization of seminiferous tubules (minor, mild). For epididymis, reduction of sperm (moderate) and cell debris content of the lumen (mild, moderate) were observed at the end of recovery period, reflecting the influence on the testes by administration of the test substance. The vacuolation (minor or mild) of cortical cells in the adrenal gland was found in the group administered 300 mg/kg/day or more in the mating group female and in the 1000 mg/kg/day dose group of the non-mating group female. After histopathological examination of kidney, vacuolization (minor) of the proximal tubule, vacuolization (minor, mild) in the distal tubule / collecting duct, regeneration of the cortical renal tubule (minor, mild), renal tubule dilation (minor, mild), single cell necrosis of papillary duct (minor), cortical cell infiltration (minor), regeneration (minor) of papillary collecting duct, papillary necrosis (minor, mild) and papillary cell infiltration (minor) were found in the 1000 mg/kg/day dose group and/or 300 mg/kg/day dose group. After histopathological examination of liver, centrilobular, hepatocytic hypertrophy (minor) and hepatocyte vacuolation (minor, mild) was found in the 1000 mg/kg/day dose group. After histopathological examination of urinary bladder, atrophy of umbrella cells (minor, mild) and hypertrophy / hyperplasia of epithelial cells (minor, mild) were observed in the 1000 mg/kg/day dose group, and vacuolation of umbrella cells (minor to moderate) were observed in the 300 mg/kg/day dose group. After histopathological examination of pancreas, an increase in the frequency of decreased zymogen granules (minor to moderate) was observed in the 300 mg/kg/day dose group of mating females and in the 1000 mg/kg/day dose group of non-mating females.
Based on these findings, the NOEL for repeated exposure was determined to be 100 mg/kg bw/day in male and female rats.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 06-04-2022 to 19-09-2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on species / strain selection:
- Rat is one of the recommended species by regulatory agencies for conducting toxicity assessment studies among rodents.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: In-house bred animals
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 to 8 weeks
- Housing: Two animals of same sex were housed in a standard standard polysulphonate cage (Size: L 430 X B 280 X H 210 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized corn cob was provided as bedding material.
- Diet (e.g. ad libitum): ad libitum. Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): ad libitum. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: The animals were acclimatized for a period of six days to experimental room conditions prior to dosing and were observed for clinical signs once daily. Veterinary examination and body weight recording of all the animals were performed on the day of receipt and on the day of grouping. Ophthalmoscopic examination of all the animals was performed on the day of grouping.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6ºC to 22.9 ºC
- Humidity (%): 45%-66%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle
IN-LIFE DATES: From: To: 08 April 2022 to 10 August 2022 - Route of administration:
- oral: gavage
- Details on route of administration:
- The test formulations and vehicle was administered through oral gavage using oral intubation cannula attached to disposable syringe.
- Vehicle:
- corn oil
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item formulations were prepared daily before dose administration. Required quantity of test item was weighed into a clean glass beaker and there by adding little volume of vehicle in to beaker, mixed well with clean glass rod and transferred to a measuring cylinder. This procedure was repeated until to ensure entire quantity of test item formulation were transferred to measuring cylinder. Finally, the volume was adjusted to required mark in measuring cylinder with vehicle to get a desired concentration of the test item.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was used as vehicle for the preparation of formulations as evidenced by the lab miscibility test results. Corn oil is the universally accepted vehicle for oral toxicity studies.
- Concentration in vehicle: 10, 30 and 90 mg/mL.
- Amount of vehicle (if gavage): 10mL/kg
- Lot/batch no. (if required): N12009002 & M12212004 (MP Biomedicals (India) Pvt. Ltd.) - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Homogeneity and dose formulation analysis for dose concentration verification was done by a validated analytical method UV-Vis spectrophotometer (study nº BIO-ANM 1305). The stability of the test item in dose formulations was established in this preliminary study. The test item formulations were stable at room temperature for 6 hours at the concentrations of 0.5 mg/mL and 200 mg/mL in corn oil. However, freshly prepared test item formulations were administered to the animals.
Sampling and analysis of all dose formulations were performed during week 1, month 2 and month 3 of the treatment. Approximately 5 mL of samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%. - Duration of treatment / exposure:
- 90 days
- Frequency of treatment:
- Daily.
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- G1-control group;
G1R- control recovery - Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Remarks:
- G2-low dose group
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Remarks:
- G3-mid dose group
- Dose / conc.:
- 900 mg/kg bw/day (nominal)
- Remarks:
- G4-high dose group;
G4R- high dose recovery - No. of animals per sex per dose:
- 10 (main groups)
5 (recovery groups) - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
- Fasting period before blood sampling for clinical biochemistry: overnight.
- Post-exposure recovery period in satellite groups: 28 days.
- Dose range finding studies: The doses of 100, 300 and 900 mg/kg/day were selected based on the results of a OECD 422 study (Study No. BIO-DTX 002) and a 14-Day Dose range finding study (BIO-DTX 001). In the 14-Day DRF study, 100, 300 and 1000 mg/kg/day were tested wherein one mortality was observed at 1000 mg/kg/day. Also as per BIO-DTX 002 study, although not considered adverse, the doses of 300 mg/kg/day [mid dose] and 600 mg/kg/day [high dose] revealed treatment related clinical signs of toxicity, reduction in body weight and feed consumption. - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity
- Cage side observations checked: clinical signs of toxicity, mortality and morbidity.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before initiation of the treatment and at weekly intervals thereafter during the study.
BODY WEIGHT: Yes
- Time schedule for examinations: on the Day 1 before test item administration and weekly thereafter.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
-Cage wise feed consumption was recorded weekly coinciding with the body weight recordings of the respective animals. Average feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats in each cage.
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during Week 12 for vehicle control and high dose main group animals (G1, and G4) and during Week 17 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1, G4, G1R and G4R
HAEMATOLOGY: Yes
- Time schedule for collection of blood: on experimental day 91 and 119 for main and recovery groups respectively.
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes
- How many animals: all of them
- Parameters checked: Hemoglobin concentration, hematocrit, erythrocyte count, total leukocyte count, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, mean platelet volume, reticulocyte count, absolute reticulocyte count, differential leucocytes count, absolute differential leucocytes count, prothrombin time and activated partial thromboplastin time.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on experimental day 91 and 119 for main and recovery groups respectively
- Animals fasted: Yes
- How many animals: all of them
- Parameters checked: Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, cholinesterase, total protein, albumin, total bilirubin, glucose, total cholesterol, creatinine, urea, triglycerides, phosphorous, calcium, high density lipoprotein, low density lipoprotein, blood urea nitrogen, globulin, albumin/globulin ratio, sodium, potassium and chloride.
PLASMA/SERUM HORMONES/LIPIDS: Yes (Thyroid Hormones T3 (Triiodothyronine), T4 (Thyroxine) and Thyroid Stimulating Hormone (TSH))
- Time of blood sample collection: at termination
- Animals fasted: Not specified
- How many animals: all of them
URINALYSIS: Yes
- Time schedule for collection of urine: on experimental day 91 and 119 for main and recovery groups respectively
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Blood, bilirubin, urobilinogen, ketones, protein, leuocytes, microalbumin, pH, nitrite and specific gravity.
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during Week 12 for vehicle control and high dose main group animals (G1 and G4) and during Week 17 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1, G4, G1R and G4R
- Battery of functions tested: home cage observations, handling observations, open field observations, sensory observations, neuromuscular observations, physiological observation (rectal temperature), grip strength and locomotor activity.
IMMUNOLOGY: No
OTHER:
- Vaginal smear examination from all the females on the day of sacrifice to determine the stages of estrus cycle. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
On day 91, all the animals of groups G1, G2, G3 and G4 were subjected to necropsy and detailed gross pathological examination. All the animals from recovery groups (G1R and G4R) were subjected to necropsy and detailed gross pathological examination on day 119. The animals were fasted over night (water was provided ad libitum) prior to scheduled necropsy. On the day of terminal sacrifice, the body weight of all the fasted animals were recorded prior to necropsy. The animals were euthanized using CO2 asphyxiation followed by exsanguination. Necropsy included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues.
HISTOPATHOLOGY: Yes
Histopathological examination was carried out on some preserved organs (see table 1 below) of vehicle control and high dose group (G1 and G4). The examination was extended to the lower dose groups and recovery groups as test item related microscopic changes were observed in the liver, kidneys and epididymides of high dose group animals (G4). A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen or any cell or stage specificity of testicular findings. - Other examinations:
- Bone marrow smear examination:
Bone marrow smear was prepared for all the animals at the time of necropsy. The bone marrow smear was fixed in methanol and stained by the May- Grunwald-Giemsa method but not evaluated as it was not warranted by haematology and histopathology findings. - Statistics:
- After verification, the data was subjected to statistical analysis using SPSS software, version 27. Body weight, percent change in body weight (with respect to Day 1 body weight), feed consumption, organ weights and ratios, hematological and clinical chemistry estimations, urinalysis parameters (urine volume, pH, specific gravity and urobilinogen) and FOB parameters (rearing, urination, defecation, excessive grooming, body temperature, movement count, hind limb foot splay and grip strength) was subjected to statistical analysis. One way ANOVA followed by Dunnett’s post test was done for different treatment groups. Student ‘t’ test was done for recovery groups comparing with the respective vehicle control group data. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05).
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- Wet perineum (slight) was observed in few animals of main group from Day 06 to Day 90 and recovery group animals from Day 15 to Day 90.
The incidence of slight wet perineum is due to administration of corn oil as the same was noted across all groups including control and is considered not an adverse observation. - Mortality:
- no mortality observed
- Description (incidence):
- No mortality/morbidity were noted in any of the treated group animals.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item related variations in the mean body weight or percent body weight change with respect to day 1 were observed at all the tested doses when compared to vehicle control group. However, statistically significant increase in mean body weight on Day 90 and percent body weight change with respect to day 1 during Days 1-90 was observed in G2 group males. Statistically significant decrease in mean body weight was observed on Day 43 in G4R female and percent body weight change with respect to day 1 during Days 1-8, 1-15, 1-36 and 1-43 was observed in G4R group females. These changes are considered as incidental in the absence of dose responsiveness and there were no concomitant changes noted in feed consumption.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No test item related changes in feed consumption were noted in any of the tested group animals in either sex.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No ophthalmoscopic abnormalities were noted.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No toxicologically significant treatment related changes in hematology parameters were noted. However, the following statistically significant variations were noted.
In males, increase in Platelet counts (G2); decrease in MPV (G2, G3 and G4) and relative basophils (G2 and G4) was observed.
In females, decrease in RBC (G3), Haemoglobin (G3), percent neutrophils (G3) and MCHC (G3, G4) was observed; increase in MPV (G4) was observed.
In recovery females, increase in MCH, APTT and decrease in relative basophils were observed in high dose group (G4).
Decrease in MPV in males is considered not related to test item as similar changes were not noted in other sex-females. All other variations noted were considered incidental due to lack of dose responsiveness and also, the variations noted could be due to random biological variations and were minimal in nature. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no toxicologically significant variations noted in clinical chemistry parameters at all the tested dose groups. However, the following statistically significant variations were noted.
In males, decrease in Glucose (G4), sodium (G4), chloride (G3, G4) was observed and increase in HDL (G4) was observed.
In females, increase in total cholesterol (G3, G4), HDL (G3, G4), LDL (G4) and ALT (G4) was observed.
The variations in lipoprotein and liver enzymes were associated with microscopic changes and hence considered to be due to administration of test item. Few other variations lacked dose responsiveness and hence considered not related to test item administration and incidental.
In recovery group, increase in phosphorous and albumin/globulin ratio was observed in G4R males; decrease in ALT and globulin was observed in G4R males and decrease in ALP was observed in G4R females. All the noted variations are considered incidental in the absence of similar changes at the end of recovery group. - Endocrine findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Statistically significant increase in T3 (G3F); increase in T4 (G2F and G4F), decrease in T4 (G2M) and decrease in TSH (G2M, G3M, G4M and G2F, G3F, G4F) was noted. Decrease in TSH in main group is considered incidental due to lack of dose dependency. Also, in the absence of associated microscopic of macroscopic changes in thyroid at the high dose, the noted changes are considered not to be test item related.
- Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No significant test item related changes in urinary parameters were noted. However, statistically significant increase in urine volume (G2, G3 and G4) was noted in males; statistically significant decrease in pH (G2, G3 and G4) and increase in specific gravity (G3, G4) was observed in females. The variations in above parameters were BIO-CTX 513 Study Report Page 31 of 398
associated with microscopic changes in the kidneys at the high dose group and considered to be secondary due to administration of test item. However, in the absence of severity of microscopic changes in the mid and low dose groups the changes are considered non-adverse in those groups. - Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- No test item related changes were observed in neurological/functional examination battery carried out during Week 12 for main group animals (G1 and G4) and Week 17 for recovery group animals (G1R and G4R).
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Absolute weights: Increase in weight of heart (G2M), epididymides (G2M), kidneys (G2M, G3M and G4M), liver (G2M, G3M and G4M); decrease in heart and brain weight (G4F) and increase in liver weight (G3F, G4F) was observed. In recovery groups, decrease in weight of spleen (G4RF) was observed.
Relative weights (relative to body weight): Increase in weight of kidneys (G4M), liver (G2M, G3M, G4M and G4RM), thyroid and parathyroid (G4M); increase in kidneys weight (G4F) and liver weight (G3F, G4F) was observed; increase in heart and thyroid and parathyroid weight was observed in G4R females.
The changes observed in kidneys, liver and epididymides are considered as test item related due to the microscopic changes observed at high dose groups and other variations are considered as incidental due to lack of dose response relationships. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No test item related gross pathological lesions were observed.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related changes were observed in liver, kidneys and epididymides. The changes in liver were observed in both the sexes and changes in kidneys and epididymides were observed in male animals.
In liver, minimal to mild, diffuse, hepatocellular hypertrophy (G4M-10/10; G4F-8/10) and minimal to mild, multifocal hepatocyte necrosis (G4M-3/10; G4F-3/10) was observed in high dose rats of both the sexes.
The changes in liver were found to be reversible at the end of 28 days treatment free recovery period as similar changes were not observed in recovery group of animals.
In kidneys (unilateral/bilateral), minimal to mild, focal/multifocal basophilic tubules were observed from mid dose group male rats (G3-2/10; G4-9/10). Tubular basophilia was characterized by bluish tinctorial change in the cytoplasm of tubular epithelium. Similar changes were not observed in recovery high dose group rats, hence this change was found to be reversible at the end of recovery period. Considering the magnitude of severity of lesions and number of incidences in mid dose, the changes at 300 mg/kg/day dose level could be considered as non-adverse effects.
In epididymides (unilateral/bilateral), minimal to moderate, focal inflammation was observed in high dose and high dose recovery male rats (G4-5/10; G4R-2/5). All these changes in epididymides were considered as adverse effects of test item administration. However, the lesion was focally confined and majority of epididymal lumen contained normal sperms. Also testes and accessory sex glands were found to be within normal histological limits.
Few microscopic findings observed in this study such as ultimobranchial cyst/s in thyroid gland, epithelial cyst/s in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected in laboratory rats (Elizabeth F. McInnes, 2012). - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Details on results:
- Vaginal smear examination revealed normal stages of estrous cyclicity both in main and recovery
females. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 300 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Key result
- Critical effects observed:
- no
- Conclusions:
- In a 90-day repeated dose toxicity study conducted in Sprague Dawley Rats, the NOAEL of the test item for both sexes is 300 mg/kg bw/day.
- Executive summary:
A study was conducted to assess the toxic potential of the test item when administered for a period of 90 consecutive days repeatedly to Sprague Dawley rats, according to OECD 408 Guideline (GLP study). Four groups (G1, G2, G3 and G4) of 10 animals of each sex and two recovery groups with 28-day recovery period (G1R and G4R) of 5 animals of each sex were tested, being G1 and G1R the vehicle control groups (corn oil). The treatment groups received the following test item concentrations: 100, 300 and 900 mg/kg bw/day. The vehicle and test item formulations were administered orally by gavage to the animals at an equivolume of 10 mL/kg bw. No clinical signs of toxicity and no mortality/morbidity were noted, as well as no changes in body weight, feed consumption and gross pathology in all groups. Besides, no effects in the ophthalmological and neurological/functional examinations were observed. No treatment related changes were observed in hematology, coagulation, clinical chemistry and urinalysis parameters. Increase in urine volume in males and decrease in pH in females and specific gravity in females were associated with microscopic changes in kidneys. No treatment related changes were observed in thyroid hormones (T3, T4 and TSH), vaginal smear examination and fasting body weights. No gross pathological changes were observed. Test item-related changes were observed in liver, kidneys and epididymides. The changes in liver were observed in both the sexes and changes in kidneys and epididymides were observed in male animals. In liver, minimal to mild, diffuse, hepatocellular hypertrophy (G4M-10/10; G4F-8/10) and minimal to mild, multifocal hepatocyte necrosis (G4M-3/10; G4F-3/10) was observed in high dose rats of both the sexes. The changes in liver were found to be reversible at the end of 28 days treatment free recovery period as similar changes were not observed in recovery group of animals. In kidneys (unilateral/bilateral), minimal to mild, focal/multifocal basophilic tubules were observed from mid dose group male rats (G3-2/10; G4-9/10). Tubular basophilia was characterized by bluish tinctorial change in the cytoplasm of tubular epithelium. Similar changes were not observed in recovery high dose group, hence this change was found to be reversible at the end of recovery period. Considering the magnitude of severity of lesions and number of incidences in mid dose, the changes at 300 mg/kg/day dose level could be considered as a non-adverse effect. In epididymides (unilateral/bilateral), minimal to moderate, focal inflammation was observed in high dose and high dose recovery male rats (G4-5/10; G4R-2/5). All these changes in epididymides were considered as adverse effects of test item administration. However, the lesion was focally confined and majority of epididymal lumen contained normal sperms. Also, testes and accessory sex glands were found to be within normal histological limits.
Therefore, based on the obtained results from this 90-day repeated dose toxicity study conducted in Sprague Dawley Rats, the NOAEL is estimated as 300 mg/kg bw/day under experimental conditions.
Referenceopen allclose all
Table 1.1. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Males
Group, Sex & Dose |
No. of Animals |
Clinical Signs of Toxicity |
Mortality (No. of incidence/ No. of Animals) |
G1, M & 0 |
3 |
N (3) |
0/3 |
G2, M & 100 |
3 |
N (3) |
0/3 |
G3, M & 300 |
3 |
73+(3), 110+(1), 117+(1) |
0/3 |
G4, M & 1000 |
3 |
73+(3), 110+(1), 25 (1), 1 (1) |
0/3 |
N: Normal; M: Male
1: Lethargy; 117: Piloerection; 110: Slight wet perineum; 73: Slight nasal discharge; 25: Aggression; +: Slight
Table 1.2. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Females
Group, Sex & Dose |
No. of Animals |
Clinical Signs of Toxicity/Detailed Clinical Examination |
Mortality (No. of incidence/ No. of animals) |
G1, F & 0 |
3 |
N (3) |
0/3 |
G2, F & 100 |
3 |
N (3) |
0/3 |
G3, F & 300 |
3 |
N (2), 110+(1) |
0/3 |
G4, F & 1000 |
3 |
73+(2), 110+(3), 82+(2) |
1/3 |
N: Normal; F: Female
110: Slight wet perineum; 73: Slight nasal discharge; 82: chromodacryorrhea; +: Slight
Table 2.1. Summary of body weight (g) record. Males
Group, Sex & Dose |
|
Body Weight (g) on Days |
||
1 |
7 |
14 |
||
G1, M & 0 |
Mean |
258.22 |
291.74 |
319.06 |
±SD |
25.75 |
21.45 |
14.54 |
|
n |
3 |
3 |
3 |
|
G2, M & 100 |
Mean |
263.15 |
291.93 |
322.87 |
±SD |
19.42 |
8.07 |
15.17 |
|
n |
3 |
3 |
3 |
|
G3, M & 300 |
Mean |
262.48 |
277.67 |
299.21 |
±SD |
13.91 |
17.65 |
19.73 |
|
n |
3 |
3 |
3 |
|
G4, M & 1000 |
Mean |
263.41 |
274.88 |
295.79 |
±SD |
14.46 |
14.36 |
15.47 |
|
n |
3 |
3 |
3 |
|
Table 2.2. Summary of body weight (g) record. Females
Group, Sex & Dose |
|
Body Weight (g) on Days |
||
1 |
7 |
14 |
||
G1, F & 0 |
Mean |
240.31 |
250.00 |
258.68 |
±SD |
12.54 |
18.60 |
15.78 |
|
n |
3 |
3 |
3 |
|
G2, F & 100 |
Mean |
239.22 |
249.31 |
262.99 |
±SD |
18.91 |
23.59 |
22.23 |
|
n |
3 |
3 |
3 |
|
G3, F & 300 |
Mean |
241.06 |
251.51 |
259.58 |
±SD |
5.62 |
3.95 |
12.87 |
|
n |
3 |
3 |
3 |
|
G4, F & 1000 |
Mean |
242.03 |
236.03 |
230.60 |
±SD |
8.39 |
22.43 |
32.54 |
|
n |
3 |
3 |
2# |
|
#: One female found dead on Day 12
Table 3.1. Summary of percent change in body weight (%) with respect to day 1. Males
Group, Sex & Dose |
Percent Change in Body Weight (%) during Days |
||
1 to 8 |
1 to 15 |
||
G1, M & 0 |
Mean |
13.19 |
24.13 |
±SD |
3.28 |
9.54 |
|
n |
3 |
3 |
|
G2, M & 100 |
Mean |
11.18 |
22.92 |
±SD |
4.99 |
5.94 |
|
n |
3 |
3 |
|
G3, M & 300 |
Mean |
5.75 |
13.94 |
±SD |
1.40 |
1.61 |
|
n |
3 |
3 |
|
G4, M & 1000 |
Mean |
4.44 |
12.30 |
±SD |
4.83 |
0.55 |
|
n |
3 |
3 |
|
Table 3.2. Summary of percent change in body weight (%) with respect to day 1. Females
Group, Sex & Dose |
Percent Change in Body Weight (%) during Days |
||
1 to 8 |
1 to 15 |
||
G1, F & 0 |
Mean |
3.95 |
7.61 |
±SD |
2.44 |
1.42 |
|
n |
3 |
3 |
|
G2, F & 100 |
Mean |
4.16 |
9.95 |
±SD |
3.68 |
4.21 |
|
n |
3 |
3 |
|
G3, F & 300 |
Mean |
4.35 |
7.66 |
±SD |
1.35 |
4.02 |
|
n |
3 |
3 |
|
G4, F & 1000 |
Mean |
-2.48 |
-6.61 |
±SD |
8.45 |
12.09 |
|
n |
3 |
2# |
|
#: One female found dead on Day 12
Table 4.1. Summary of average feed consumption (g/animal/day) record. Males
Group, Sex & Dose |
|
Feed Consumption (g/animal/day) |
|
Week 1 |
Week 2 |
||
G1, M & 0 |
Mean |
19.5 |
21.4 |
±SD |
- |
- |
|
n |
3 |
3 |
|
G2, M & 100 |
Mean |
18.8 |
21.6 |
±SD |
- |
- |
|
n |
3 |
3 |
|
G3, M & 300 |
Mean |
18.6 |
20.8 |
±SD |
- |
- |
|
n |
3 |
3 |
|
G4, M & 1000 |
Mean |
17.1 |
20.3 |
±SD |
- |
- |
|
n |
3 |
3 |
|
Table 4.2. Summary of average feed consumption (g/animal/day) record. Females
Group, Sex & Dose |
|
Feed Consumption (g/rat/day) |
|
Week 1 Feed Consumption |
Week 2 Feed Consumption |
||
G1, F & 0 |
Mean |
13.3 |
18.1 |
±SD |
- |
- |
|
n |
3 |
3 |
|
G2, F & 100 |
Mean |
11.4 |
17.5 |
±SD |
- |
- |
|
n |
3 |
3 |
|
G3, F & 300 |
Mean |
11.3 |
17.2 |
±SD |
- |
- |
|
n |
3 |
3 |
|
G4, F & 1000 |
Mean |
10.8 |
15.9 |
±SD |
- |
- |
|
n |
3 |
3 |
|
Table 5.1. Summary of haematology record. Males
Group, Sex & Dose |
|
Total Leucocyte Count |
Total Erythrocyte Count |
Hemoglobin |
Haematocrit |
Mean Corpuscular Volume |
Mean Corpuscular Hemoglobin |
Mean Corpuscular Hemoglobin Concentration |
Platelet Count |
(WBC) |
(RBC) |
(HGB) |
(HCT) |
(MCV) |
(MCH) |
(MCHC) |
(PLT) |
||
(103cells/µL) |
(106cells/µL) |
(g/dL) |
(%) |
(fL) |
(pg) |
(g/dL) |
(103cells/µL) |
||
G1, M & 0 |
Mean |
11.92 |
7.31 |
13.53 |
41.90 |
57.30 |
18.53 |
32.37 |
784.00 |
±SD |
5.61 |
0.23 |
0.50 |
1.41 |
0.53 |
0.12 |
0.31 |
127.43 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G2, M & 100 |
Mean |
9.08 |
7.37 |
13.87 |
43.33 |
58.77 |
18.83 |
31.97 |
880.33 |
±SD |
1.38 |
0.55 |
1.12 |
3.19 |
0.68 |
0.25 |
0.15 |
90.52 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G3, M & 300 |
Mean |
10.38 |
7.61 |
14.17 |
43.30 |
56.93 |
18.60 |
32.67 |
930.33 |
±SD |
1.06 |
0.47 |
1.14 |
2.72 |
0.47 |
0.36 |
0.67 |
46.46 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G4, M & 1000 |
Mean |
11.81 |
7.51 |
13.73 |
43.13 |
57.57 |
18.30 |
31.83 |
891.67 |
±SD |
0.90 |
0.59 |
0.72 |
1.70 |
2.48 |
0.70 |
0.57 |
135.74 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
Group, Sex & Dose |
|
Mean Platelet Volume |
Reticulocyte Count |
Neutrophils |
Lymphocytes |
Monocytes |
Eosinophils |
Basophils |
(MPV) |
(Retic) |
Neut |
Lymph |
Mono |
Eos |
Baso |
||
(fL) |
(%) |
(%) |
(%) |
(%) |
(%) |
(%) |
||
G1, M & 0 |
Mean |
6.40 |
2.23 |
22.80 |
73.03 |
2.63 |
0.40 |
0.20 |
±SD |
0.17 |
0.45 |
3.16 |
1.68 |
1.29 |
0.17 |
0.10 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G2, M & 100 |
Mean |
6.00* |
2.25 |
29.43 |
65.33 |
2.83 |
0.97 |
0.17 |
±SD |
0.17 |
0.82 |
1.35 |
2.30 |
0.97 |
0.40 |
0.06 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G3, M & 300 |
Mean |
5.93* |
2.35 |
23.90 |
70.60 |
3.80 |
0.43 |
0.27 |
±SD |
0.06 |
0.65 |
0.78 |
1.23 |
1.05 |
0.25 |
0.12 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G4, M & 1000 |
Mean |
6.03* |
2.82 |
20.90 |
74.13 |
2.73 |
0.73 |
0.27 |
±SD |
0.12 |
1.02 |
6.28 |
7.40 |
0.64 |
0.31 |
0.06 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
*: Statistically significant (P<0.05)
Group, Sex & Dose weight/day) |
|
Absolute Reticulocyte Count |
Absolute Neutrophils |
Absolute Lymphocytes |
Absolute Monocytes |
Absolute Eosinophils |
Absolute Basophils |
Prothrombin Time |
Activated Prothrombin Time |
(Retic) |
Neut |
Lymph |
Mono |
Eos |
Baso |
PT |
APTT |
||
(109cells/µL) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(Seconds) |
(Seconds) |
||
G1, M & 0 |
Mean |
162.50 |
2.62 |
8.74 |
0.36 |
0.05 |
0.03 |
25.27 |
28.33 |
±SD |
29.88 |
0.90 |
4.27 |
0.34 |
0.05 |
0.03 |
1.29 |
9.05 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G2, M & 100 |
Mean |
163.17 |
2.66 |
5.94 |
0.25 |
0.09 |
0.02 |
25.20 |
25.53 |
±SD |
47.98 |
0.35 |
1.05 |
0.06 |
0.04 |
0.01 |
6.71 |
7.71 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G3, M & 300 |
Mean |
177.23 |
2.48 |
7.34 |
0.39 |
0.05 |
0.03 |
24.33 |
21.13 |
±SD |
38.93 |
0.18 |
0.86 |
0.10 |
0.03 |
0.02 |
6.97 |
2.05 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G4, M & 1000 |
Mean |
208.23 |
2.45 |
8.78 |
0.32 |
0.08 |
0.03 |
33.07 |
27.70 |
±SD |
65.06 |
0.68 |
1.42 |
0.06 |
0.03 |
0.00 |
20.56 |
11.00 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
Table 5.2. Summary of haematology record. Females
Group, Sex & Dose |
|
Total Leucocyte Count |
Total Erythrocyte Count |
Hemoglobin |
Haematocrit |
Mean Corpuscular Volume |
Mean Corpuscular Hemoglobin |
Mean Corpuscular Hemoglobin Concentration |
Platelet Count |
(WBC) |
(RBC) |
(HGB) |
(HCT) |
(MCV) |
(MCH) |
(MCHC) |
(PLT) |
||
(103cells/µL) |
(106cells/µL) |
(g/dL) |
(%) |
(fL) |
(pg) |
(g/dL) |
(103cells/µL) |
||
G1, F & 0 |
Mean |
9.95 |
7.30 |
13.40 |
40.87 |
56.00 |
18.33 |
32.70 |
936.33 |
±SD |
2.17 |
0.04 |
0.53 |
0.84 |
1.39 |
0.83 |
0.70 |
44.74 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G2, F & 100 |
Mean |
10.02 |
7.22 |
13.37 |
40.73 |
56.57 |
18.53 |
32.80 |
1044.33 |
±SD |
1.48 |
0.35 |
0.23 |
0.23 |
2.50 |
1.15 |
0.70 |
118.29 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G3, F & 300 |
Mean |
13.14 |
7.31 |
13.53 |
41.43 |
56.73 |
18.53 |
32.60 |
918.00 |
±SD |
1.44 |
0.19 |
0.15 |
0.25 |
1.12 |
0.32 |
0.00 |
74.75 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
2 |
3 |
|
G4, F & 1000 |
Mean |
14.22 |
7.53 |
13.75 |
41.70 |
55.40 |
18.25 |
33.00 |
1122.00 |
±SD |
0.83 |
0.27 |
0.64 |
0.14 |
1.84 |
0.21 |
1.56 |
25.46 |
|
n# |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
#: One female was found dead on Day 12.
Group, Sex & Dose |
|
Mean Platelet Volume |
Reticulocyte Count |
Neutrophils |
Lymphocytes |
Monocytes |
Eosinophils |
Basophils |
(MPV) |
(Retic) |
Neut |
Lymph |
Mono |
Eos |
Baso |
||
(fL) |
(%) |
(%) |
(%) |
(%) |
(%) |
(%) |
||
G1, F & 0 |
Mean |
6.07 |
2.15 |
16.90 |
78.97 |
1.97 |
0.83 |
0.23 |
±SD |
0.12 |
0.73 |
3.70 |
3.92 |
0.29 |
0.25 |
0.12 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G2, F & 100 |
Mean |
6.10 |
2.15 |
17.13 |
78.77 |
1.87 |
0.93 |
0.27 |
±SD |
0.10 |
0.31 |
4.42 |
5.80 |
1.24 |
0.38 |
0.06 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G3, F & 300 |
Mean |
6.17 |
1.97 |
15.77 |
80.30 |
1.57 |
0.83 |
0.33 |
±SD |
0.21 |
0.61 |
4.25 |
5.19 |
0.42 |
0.60 |
0.15 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G4, F & 1000 |
Mean |
5.85 |
1.30 |
20.70 |
68.70 |
6.65 |
1.25 |
0.40 |
±SD |
0.07 |
0.55 |
11.17 |
4.38 |
5.87 |
0.07 |
0.00 |
|
n# |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
#: One female was found dead on Day 12.
Group, Sex & Dose |
|
Absolute Reticulocyte Count |
Absolute Neutrophils |
Absolute Lymphocytes |
Absolute Monocytes |
Absolute Eosinophils |
Absolute Basophils |
Prothrombin Time |
Activated Prothrombin Time |
(Retic) |
Neut |
Lymph |
Mono |
Eos |
Baso |
PT |
APTT |
||
(109cells/µL) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(Seconds) |
(Seconds) |
||
G1, F & 0 |
Mean |
157.17 |
1.74 |
7.81 |
0.20 |
0.09 |
0.03 |
31.10 |
13.80 |
±SD |
53.90 |
0.71 |
1.41 |
0.06 |
0.04 |
0.02 |
14.30 |
2.96 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G2, F & 100 |
Mean |
155.70 |
1.68 |
7.95 |
0.18 |
0.09 |
0.02 |
19.07 |
20.07 |
±SD |
25.95 |
0.25 |
1.69 |
0.08 |
0.04 |
0.01 |
6.80 |
3.76 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G3, F & 300 |
Mean |
143.10 |
2.10 |
10.51 |
0.20 |
0.11 |
0.04 |
14.50 |
22.80 |
±SD |
40.56 |
0.76 |
0.75 |
0.06 |
0.07 |
0.03 |
4.20 |
5.60 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G4, F & 1000 |
Mean |
98.65 |
2.99 |
9.75 |
0.92 |
0.18 |
0.05 |
22.10 |
15.60 |
±SD |
45.33 |
1.77 |
0.06 |
0.78 |
0.01 |
0.00 |
4.67 |
0.71 |
|
n# |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
#: One female was found dead on Day 12.
Table 6.1. Summary of clinical chemistry record. Males
Group, Sex & Dose |
|
Glucose |
Creatinine |
Total Cholesterol |
Triglycerides |
Total Protein |
Albumin |
Alanine aminotransferase |
Aspartate aminotransferase |
(GLU) |
(CRE) |
(CHO) |
(TRI) |
(TPR) |
(ALB) |
(ALT) |
(AST) |
||
mg/dL |
mg/dL |
mg/dL |
mg/dL |
g/dL |
g/dL |
U/L |
U/L |
||
G1, M & 0 |
Mean |
126.00 |
0.48 |
49.00 |
39.67 |
6.10 |
2.83 |
41.67 |
79.67 |
±SD |
10.58 |
0.01 |
4.36 |
14.05 |
0.00 |
0.05 |
7.51 |
11.50 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G2, M & 100 |
Mean |
110.67 |
0.42* |
44.00 |
33.67 |
6.13 |
2.87 |
34.33 |
68.33 |
±SD |
18.72 |
0.01 |
3.00 |
6.43 |
0.06 |
0.10 |
2.52 |
3.21 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G3, M & 300 |
Mean |
115.33 |
0.48 |
43.00 |
42.33 |
6.23 |
2.86 |
40.00 |
75.00 |
±SD |
5.03 |
0.03 |
12.12 |
13.80 |
0.15 |
0.03 |
5.57 |
7.00 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G4, M & 1000 |
Mean |
94.67* |
0.47 |
39.00 |
32.00 |
6.27 |
2.92 |
52.67 |
99.00 |
±SD |
6.11 |
0.02 |
1.73 |
11.79 |
0.31 |
0.18 |
12.66 |
8.89 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
*: Statistically significant (p<0.05)
Group, Sex & Dose |
|
Alkaline phosphatase |
Total Bilirubin |
Calcium |
Phosphorous |
Globulin |
Albumin/Globulin ratio |
Blood Urea Nitrogen |
Sodium |
Potassium |
Chloride |
(ALP) |
(BIT) |
(CAL) |
(PHO) |
(GLO) |
(A/G Ratio) |
(BUN) |
(Na) |
(K) |
(CLO) |
||
U/L |
mg/dL |
mg/dL |
mg/dL |
mg/dL |
mg/dL |
mg/dL |
mmol/L |
mmol/L |
mmol/L |
||
G1, M & 0 |
Mean |
166.33 |
0.05 |
8.73 |
5.30 |
3.27 |
0.87 |
10.76 |
150.40 |
3.45 |
105.97 |
±SD |
34.08 |
0.01 |
0.06 |
0.36 |
0.05 |
0.03 |
0.82 |
1.41 |
0.20 |
0.86 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G2, M & 100 |
Mean |
206.33 |
0.03* |
9.27* |
5.87 |
3.26 |
0.88 |
9.75 |
150.83 |
3.75 |
105.00 |
±SD |
42.25 |
0.01 |
0.21 |
0.25 |
0.16 |
0.07 |
2.15 |
3.24 |
0.03 |
0.60 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G3, M & 300 |
Mean |
174.33 |
0.02* |
9.10 |
5.30 |
3.38 |
0.85 |
10.27 |
149.03 |
3.78 |
104.70 |
±SD |
4.62 |
0.00 |
0.20 |
0.40 |
0.12 |
0.03 |
0.75 |
0.15 |
0.19 |
0.82 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G4, M & 1000 |
Mean |
136.67 |
0.04 |
8.97 |
6.30* |
3.35 |
0.87 |
10.97 |
148.80 |
3.62 |
107.00 |
±SD |
7.64 |
0.01 |
0.23 |
0.30 |
0.18 |
0.06 |
1.20 |
0.70 |
0.09 |
0.10 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
*: Statistically significant (p<0.05)
Table 6.2. Summary of clinical chemistry record. Females
Group, Sex & Dose |
|
Glucose |
Creatinine |
Total Cholesterol |
Triglycerides |
Total Protein |
Albumin |
Alanine aminotransferase |
Aspartate aminotransferase |
(GLU) |
(CRE) |
(CHO) |
(TRI) |
(TPR) |
(ALB) |
(ALT) |
(AST) |
||
mg/dL |
mg/dL |
mg/dL |
mg/dL |
g/dL |
g/dL |
U/L |
U/L |
||
G1, F & 0 |
Mean |
119.33 |
0.51 |
45.33 |
31.67 |
6.50 |
3.06 |
34.33 |
73.00 |
±SD |
18.01 |
0.04 |
5.13 |
4.16 |
0.26 |
0.09 |
9.50 |
3.46 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G2, F & 100 |
Mean |
107.00 |
0.48 |
54.67 |
26.67 |
6.47 |
3.14 |
29.00 |
70.00 |
±SD |
11.53 |
0.04 |
10.21 |
8.14 |
0.06 |
0.08 |
3.61 |
9.17 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G3, F & 300 |
Mean |
126.00 |
0.49 |
60.67 |
26.33 |
6.30 |
2.99 |
41.67 |
78.33 |
±SD |
1.73 |
0.04 |
7.02 |
3.79 |
0.44 |
0.19 |
13.28 |
13.28 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G4, F & 1000 |
Mean |
102.50 |
0.58 |
56.50 |
25.50 |
6.15 |
3.08 |
51.50 |
77.50 |
±SD |
19.09 |
0.09 |
10.61 |
6.36 |
0.49 |
0.28 |
16.26 |
7.78 |
|
n# |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
#: One female was found dead on Day 12.
Group, Sex & Dose |
|
Alkaline phosphatase |
Total Bilirubin |
Calcium |
Phosphorous |
Globulin |
Albumin/ Globulin ratio |
Blood Urea Nitrogen |
Sodium |
Potassium |
Chloride |
(ALP) |
(BIT) |
(CAL) |
(PHO) |
(GLO) |
(A/G Ratio) |
(BUN) |
(Na) |
(K) |
(CLO) |
||
U/L |
mg/dL |
mg/dL |
mg/dL |
mg/dL |
mg/dL |
mg/dL |
mmol/L |
mmol/L |
mmol/L |
||
G1, F & 0 |
Mean |
109.67 |
0.04 |
9.07 |
4.60 |
3.44 |
0.89 |
8.96 |
146.43 |
3.52 |
106.20 |
±SD |
39.72 |
0.01 |
0.06 |
0.72 |
0.24 |
0.07 |
0.37 |
0.72 |
0.17 |
0.80 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G2, F & 100 |
Mean |
98.00 |
0.02 |
9.13 |
4.37 |
3.33 |
0.95 |
8.23 |
146.23 |
3.59 |
106.57 |
±SD |
14.93 |
0.01 |
0.21 |
0.76 |
0.03 |
0.03 |
0.96 |
0.50 |
0.10 |
1.72 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G3, F & 300 |
Mean |
110.00 |
0.02 |
9.03 |
4.73 |
3.31 |
0.90 |
11.73 |
146.17 |
3.69 |
106.27 |
±SD |
53.36 |
0.00 |
0.55 |
0.29 |
0.24 |
0.01 |
2.57 |
1.07 |
0.56 |
0.95 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G4, F & 1000 |
Mean |
97.00 |
0.05 |
8.70 |
4.60 |
3.08 |
1.00 |
13.56 |
145.40 |
3.11 |
106.75 |
±SD |
7.07 |
0.01 |
0.71 |
0.14 |
0.22 |
0.01 |
6.30 |
2.55 |
0.45 |
2.90 |
|
n# |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
#: One female was found dead on Day 12.
Table 7.1. Summary of absolute organ weights (g) record. Males
Group, Sex & Dose |
Adrenals |
Thymus |
Spleen |
Epididymes |
Testes |
Heart |
Kidneys |
Brain |
Liver |
PSC |
Lungs |
|
G1, M & 0 |
Mean |
0.0533 |
0.3939 |
0.6615 |
1.1405 |
3.4398 |
1.2664 |
2.6892 |
2.1170 |
10.8868 |
2.4154 |
2.5611 |
±SD |
0.0033 |
0.0749 |
0.0502 |
0.0571 |
0.2415 |
0.1716 |
0.2351 |
0.1906 |
1.0098 |
0.3625 |
0.4313 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G2, M & 100 |
Mean |
0.0696* |
0.5771 |
0.6654 |
1.2033 |
3.3983 |
1.3397 |
3.1196 |
2.0742 |
11.5421 |
2.6900 |
2.8954 |
±SD |
0.0031 |
0.0522 |
0.1348 |
0.1277 |
0.2410 |
0.1489 |
0.2602 |
0.1081 |
1.4462 |
0.3132 |
0.5263 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G3, M & 300 |
Mean |
0.0659 |
0.5643 |
0.6782 |
1.1154 |
3.4666 |
1.1658 |
2.8486 |
2.1140 |
11.0042 |
2.4310 |
2.4830 |
±SD |
0.0038 |
0.0492 |
0.0340 |
0.0651 |
0.1936 |
0.0883 |
0.5473 |
0.1356 |
0.9703 |
0.1533 |
0.3730 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G4, M & 1000 |
Mean |
0.0639 |
0.4349 |
0.6730 |
1.1609 |
3.3178 |
1.1664 |
3.3758 |
2.2839 |
14.7867* |
2.9496 |
2.6389 |
±SD |
0.0099 |
0.1499 |
0.0652 |
0.0919 |
0.2031 |
0.0807 |
0.1692 |
0.1420 |
1.2961 |
0.4687 |
0.3236 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
PSC: Prostate+Seminal vesicles with coagulating glands
*: Statistically significant (P<0.05)
Table 7.2. Summary of absolute organ weights (g) record. Females
Group, Sex & Dose |
Adrenals |
Thymus |
Spleen |
Uterus |
Ovaries |
Heart |
Kidneys |
Brain |
Liver |
Lungs |
|
G1, F & 0 |
Mean |
0.0955 |
0.6085 |
0.6788 |
0.5831 |
0.2129 |
1.0115 |
2.6268 |
2.4659 |
9.4801 |
2.6740 |
±SD |
0.0127 |
0.1084 |
0.0535 |
0.0888 |
0.0232 |
0.0626 |
0.3510 |
0.0625 |
0.9003 |
0.1106 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G2, F & 100 |
Mean |
0.1144 |
0.5597 |
0.7401 |
0.7950 |
0.2709 |
1.0814 |
2.5443 |
2.4417 |
10.9313 |
2.2571 |
±SD |
0.0060 |
0.1446 |
0.0827 |
0.0542 |
0.0537 |
0.1494 |
0.3340 |
0.0392 |
1.7597 |
0.2692 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G3, F & 300 |
Mean |
0.0950 |
0.6081 |
0.7264 |
0.6463 |
0.2147 |
1.0944 |
2.3206 |
2.3258 |
11.2076 |
2.2677 |
±SD |
0.0187 |
0.0933 |
0.1095 |
0.2188 |
0.0534 |
0.1176 |
0.0929 |
0.0931 |
1.0288 |
0.1572 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G4, F & 1000 |
Mean |
0.1051 |
0.4002 |
0.5380 |
0.4603 |
0.2182 |
0.9071 |
2.7250 |
2.3067 |
11.3479 |
2.0137* |
±SD |
0.0072 |
0.1550 |
0.1348 |
0.0103 |
0.0501 |
0.1858 |
0.3120 |
0.1549 |
2.8030 |
0.0391 |
|
n# |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
#: One female was found dead on Day 12.
*: Statistically significant (P<0.05)
Table 8.1. Summary of fasting body weight (g) and organ weight relative to fasting body weight (%) record. Males
Group, Sex & Dose |
Fasting Body Weight (g) |
Adrenals |
Thymus |
Spleen |
Epididymes |
Testes |
Heart |
Kidneys |
Brain |
Liver |
PSC |
Lungs |
|
G1, M & 0 |
Mean |
292.59 |
0.0183 |
0.1341 |
0.2259 |
0.3899 |
0.9181 |
0.4320 |
0.9183 |
0.7231 |
3.7174 |
0.8286 |
0.8720 |
±SD |
15.73 |
0.0017 |
0.0194 |
0.0066 |
0.0095 |
0.4490 |
0.0420 |
0.0419 |
0.0413 |
0.1948 |
0.1445 |
0.0995 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G2, M & 100 |
Mean |
292.55 |
0.0239* |
0.1975 |
0.2265 |
0.4105 |
0.9116 |
0.4572 |
1.0651 |
0.7110 |
3.9360 |
0.9187 |
0.9880 |
±SD |
14.23 |
0.0022 |
0.0185 |
0.0374 |
0.0245 |
0.4278 |
0.0331 |
0.0384 |
0.0663 |
0.3243 |
0.0838 |
0.1557 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G3, M & 300 |
Mean |
279.24 |
0.0236* |
0.2018 |
0.2431 |
0.4004 |
1.2423 |
0.4175 |
1.0152 |
0.7570 |
3.9362 |
0.8730 |
0.8952 |
±SD |
15.41 |
0.0008 |
0.0068 |
0.0122 |
0.0345 |
0.0563 |
0.0224 |
0.1488 |
0.0244 |
0.1369 |
0.0817 |
0.1822 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G4, M & 1000 |
Mean |
273.49 |
0.0233 |
0.1584 |
0.2457 |
0.4259 |
1.2161 |
0.4262 |
1.2369* |
0.8370 |
5.4008* |
1.0791 |
0.9661 |
±SD |
11.22 |
0.0034 |
0.0508 |
0.0138 |
0.0506 |
0.1188 |
0.0141 |
0.1044 |
0.0813 |
0.2732 |
0.1731 |
0.1258 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
PSC: Prostate+Seminal vesicles with coagulating glands
*: Statistically significant (P<0.05)
Table 8.2. Summary of fasting body weight (g) and organ weight relative to fasting body weight (%) record. Females
Group, Sex & Dose |
Fasting Body Weight (g) |
Adrenals |
Thymus |
Spleen |
Uterus |
Ovaries |
Heart |
Kidneys |
Brain |
Liver |
Lungs |
|
G1, F & 0 |
Mean |
242.57 |
0.0393 |
0.2505 |
0.2803 |
0.2432 |
0.1312 |
0.4188 |
1.0796 |
1.0200 |
3.9043 |
1.1072 |
±SD |
18.41 |
0.0024 |
0.0351 |
0.0189 |
0.0571 |
0.0629 |
0.0428 |
0.0667 |
0.0707 |
0.0782 |
0.1041 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G2, F & 100 |
Mean |
249.96 |
0.0461 |
0.2218 |
0.2960 |
0.3205 |
0.1707 |
0.4312 |
1.0156 |
0.9813 |
4.3557 |
0.9013 |
±SD |
19.15 |
0.0061 |
0.0428 |
0.0226 |
0.0482 |
0.1024 |
0.0278 |
0.0686 |
0.0877 |
0.4035 |
0.0501 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G3, F & 300 |
Mean |
243.63 |
0.0389 |
0.2507 |
0.3004 |
0.2641 |
0.0892 |
0.4502 |
0.9566 |
0.9594 |
4.6072 |
0.9363 |
±SD |
17.03 |
0.0065 |
0.0457 |
0.0617 |
0.0815 |
0.0276 |
0.0533 |
0.0940 |
0.1031 |
0.4230 |
0.1246 |
|
n |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
|
G4, F & 1000 |
Mean |
220.90 |
0.0480 |
0.1778 |
0.2418 |
0.2118 |
0.0983 |
0.4094 |
1.2393 |
1.0533 |
5.1027* |
0.9232 |
±SD |
37.19 |
0.0048 |
0.0402 |
0.0203 |
0.0403 |
0.0061 |
0.0152 |
0.0674 |
0.1072 |
0.4099 |
0.1377 |
|
n |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
#: One female was found dead on Day 12.
*: Statistically significant (P<0.05)
Table 9. Summary of gross pathology record
Organs/ Lesions |
Group |
G1 |
G2 |
G3 |
G4 |
||||
Dose (mg/kg body weight/day) |
0 |
100 |
300 |
1000 |
|||||
Sex |
M |
F |
M |
F |
M |
F |
M |
F |
|
No. of Animals Terminally Sacrificed |
3 |
3 |
3 |
3 |
3 |
3 |
3 |
2 |
|
No. of Animals Found Dead |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|
No Abnormality Detected [External & Internal] |
3 |
3 |
3 |
3 |
3 |
3 |
2 |
2 |
|
Wet Perineum [External] |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
|
Autolyzed [Internal] |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
|
Table 4.1. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Males
Group, Sex & Dose (mg/kg body weight) |
No. of Animals |
Clinical Signs of Toxicity/ |
Mortality (No. of Mortality / No. of Animals dosed) |
G1, M & 0 |
12 |
N (12) |
0/12 |
G2, M & 100 |
12 |
N (12) |
0/12 |
G3, M & 300 |
12 |
N (8); 110 (3); 82 (1) |
0/12 |
G4, M & 600 |
12 |
N (6); 110 (6) |
0/12 |
M: Male; N: Normal; 110: Wet perineum; 82: Perinasal staining
Table 4.2. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Females
Group, Sex & Dose (mg/kg body weight) |
No. of Animals |
Clinical Signs of Toxicity/ |
Mortality (No. of Mortality / No. of Animals dosed) |
G1, F & 0 |
12 |
N (12) |
0/12 |
G2, F & 100 |
12 |
N (12) |
0/12 |
G3, F & 300 |
12 |
N (8); 110 (4) |
0/12 |
G4, F & 600 |
12 |
N (5); 110 (7) |
0/12 |
F: Female; N: Normal; 110: Wet perineum
Table 4.3. Summary of clinical signs of toxicity, detailed clinical examination and mortality record. Recovery groups
Group, Sex & Dose (mg/kg body weight) |
No. of Animals |
Clinical Signs of Toxicity/ |
Mortality (No. of Mortality / No. of Animals dosed) |
G1R, M & 0 |
5 |
N (5) |
0/5 |
G4R, M & 600 |
5 |
N (3); 110 (2) |
0/5 |
G1R, F & 0 |
5 |
N (5) |
0/5 |
G4R, F & 600 |
5 |
N (3); 110 (2) |
0/5 |
M: Male; F: Female; R: Recovery; N: Normal; 110: Wet perineum
Table 5.1. Summary of body weight (g) record. Males
Group, Sex & Dose (mg/kg body weight) |
Body Weight (g) on Day |
||||||
1 |
7 |
14 |
21 |
28 |
35 |
||
G1, M & 0 |
Mean |
381.03 |
389.68 |
399.42 |
410.44 |
421.01 |
430.64 |
±SD |
35.22 |
36.88 |
38.54 |
39.75 |
41.60 |
40.79 |
|
n |
12 |
12 |
12 |
12 |
12 |
12 |
|
G2, M & 100 |
Mean |
377.18 |
382.56 |
390.29 |
401.50 |
409.69 |
420.12 |
±SD |
34.26 |
34.69 |
35.09 |
34.76 |
33.37 |
33.81 |
|
n |
12 |
12 |
12 |
12 |
12 |
12 |
|
G3, M & 300 |
Mean |
378.65 |
383.40 |
390.25 |
397.82 |
405.35 |
416.56 |
±SD |
33.89 |
34.48 |
36.34 |
36.53 |
37.42 |
37.36 |
|
n |
12 |
12 |
12 |
12 |
12 |
12 |
|
G4, M & 600 |
Mean |
379.39 |
378.29 |
380.29 |
386.13 |
392.89 |
403.02 |
±SD |
32.03 |
31.84 |
31.22 |
33.21 |
34.10 |
34.33 |
|
n |
12 |
12 |
12 |
12 |
12 |
12 |
|
Table 5.2. Summary of body weight (g) record. Females
Group, Sex & Dose (mg/kg body weight) |
Body Weight (g) on Days |
|||||
1 |
7 |
14 |
21# |
28# |
||
G1, F & 0 |
Mean |
271.32 |
277.65 |
284.52 |
294.48 |
303.51 |
±SD |
14.70 |
15.30 |
15.77 |
18.17 |
19.76 |
|
n |
12 |
12 |
12 |
6 |
3 |
|
G2, F & 100 |
Mean |
271.24 |
276.96 |
278.30 |
283.87 |
296.55 |
±SD |
15.07 |
15.77 |
15.75 |
17.32 |
9.49 |
|
n |
12 |
12 |
12 |
7 |
3 |
|
G3, F & 300 |
Mean |
272.21 |
271.34 |
273.93 |
267.49 |
274.21 |
±SD |
16.11 |
16.02 |
15.95 |
18.29 |
20.14 |
|
n |
12 |
12 |
12 |
5 |
4 |
|
G4, F & 600 |
Mean |
271.70 |
272.07 |
270.96 |
266.87 |
265.26 |
±SD |
15.28 |
15.58 |
16.05 |
12.03 |
10.62 |
|
n |
12 |
12 |
12 |
7 |
5 |
|
#: The data of Day 21 and 28 body weight was not subjected to statistical analysis due to uneven number of variables
Table 5.3. Summary of body weight (g) record. Recovery groups
Group, Sex & Dose (mg/kg body weight) |
Body Weight (g) on Day |
||||||||||
1 |
7 |
14 |
21 |
28 |
35 |
42 |
49 |
56 |
63 |
||
G1R, M & 0 |
Mean |
375.04 |
387.85 |
396.37 |
407.72 |
419.05 |
427.32 |
438.02 |
449.49 |
455.22 |
462.27 |
±SD |
27.86 |
27.90 |
26.43 |
26.46 |
28.44 |
26.52 |
33.26 |
37.89 |
37.83 |
36.57 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, M & 600 |
Mean |
375.02 |
376.48 |
378.88 |
391.20 |
397.15 |
407.68 |
420.85 |
436.91 |
441.32 |
450.62 |
±SD |
24.55 |
29.70 |
32.63 |
35.14 |
34.76 |
32.71 |
40.13 |
47.18 |
46.16 |
42.45 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G1R, F & 0 |
Mean |
270.17 |
276.46 |
283.02 |
289.28 |
295.46 |
300.34 |
302.72 |
305.75 |
311.49 |
319.96 |
±SD |
11.70 |
11.50 |
11.66 |
11.90 |
9.99 |
7.71 |
7.29 |
6.66 |
8.64 |
6.48 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, F & 600 |
Mean |
269.74 |
270.61 |
275.06 |
277.47 |
277.96* |
283.45* |
288.21 |
290.14 |
294.73 |
301.14 |
±SD |
11.53 |
12.03 |
10.63 |
13.27 |
11.26 |
10.17 |
12.72 |
17.39 |
16.26 |
17.58 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
* Statistically significant (P<0.05) change than the vehicle control group
Table 6.1. Summary of percent change in body weight (%) with respect to day 1. Males
Group, Sex & Dose (mg/kg body weight) |
Percent Change in Body Weight (%) during Day |
|||||
1 to 7 |
1 to 14 |
1 to 21 |
1 to 28 |
1 to 35 |
||
G1, M & 0 |
Mean |
2.26 |
4.79 |
7.68 |
10.44 |
13.01 |
±SD |
1.06 |
1.27 |
1.82 |
2.40 |
2.46 |
|
n |
12 |
12 |
12 |
12 |
12 |
|
G2, M & 100 |
Mean |
1.43 |
3.49* |
6.50 |
8.72 |
11.50 |
±SD |
0.52 |
1.00 |
1.30 |
1.98 |
2.31 |
|
n |
12 |
12 |
12 |
12 |
12 |
|
G3, M & 300 |
Mean |
1.25* |
3.03* |
5.04* |
7.02* |
10.01* |
±SD |
0.45 |
0.65 |
1.14 |
1.20 |
1.43 |
|
n |
12 |
12 |
12 |
12 |
12 |
|
G4, M & 600 |
Mean |
-0.28* |
0.27* |
1.81* |
3.59* |
6.28* |
±SD |
1.22 |
1.66 |
3.00 |
3.20 |
3.38 |
|
n |
12 |
12 |
12 |
12 |
12 |
|
* Statistically significant (P<0.05) change than the vehicle control group.
Table 6.2. Summary of percent change in body weight (%) with respect to day 1. Females
Group, Sex & Dose (mg/kg body weight) |
Percent Change in Body Weight (%) during Day |
||||
1 to 7 |
1 to 14 |
1 to 21# |
1 to 28# |
||
G1, F & 0 |
Mean |
2.33 |
4.86 |
6.94 |
9.92 |
±SD |
0.58 |
1.05 |
1.30 |
1.82 |
|
n |
12 |
12 |
5 |
3 |
|
G2, F & 100 |
Mean |
2.11 |
2.61* |
4.48 |
5.99 |
±SD |
0.72 |
1.48 |
0.73 |
0.73 |
|
n |
12 |
12 |
7 |
3 |
|
G3, F & 300 |
Mean |
-0.31* |
0.66* |
2.23 |
3.66 |
±SD |
1.34 |
1.97 |
2.51 |
2.67 |
|
n |
12 |
12 |
5 |
4 |
|
G4, F & 600 |
Mean |
0.15* |
-0.27* |
0.62 |
0.14 |
±SD |
1.88 |
1.68 |
2.07 |
2.44 |
|
n |
12 |
12 |
7 |
5 |
|
#: The data of Day 21 and 28 body weight was not subjected to statistical analysis due to uneven number of variables
* Statistically significant (P<0.05) change than the vehicle control group.
Table 6.3. Summary of percent change in body weight (%) with respect to day 1. Recovery groups
Group, Sex & Dose (mg/kg body weight) |
Percent Change in Body Weight (%) during Day |
|||||||||
1 to 7 |
1 to 14 |
1 to 21 |
1 to 28 |
1 to 35 |
1 to 42 |
1 to 49 |
1 to 56 |
1 to 63 |
||
G1R, M & 0 |
Mean |
3.44 |
5.74 |
8.78 |
11.79 |
14.03 |
16.79 |
19.80 |
21.35 |
23.26 |
±SD |
1.59 |
1.48 |
1.80 |
2.36 |
2.12 |
1.55 |
2.75 |
3.02 |
3.02 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, M & 600 |
Mean |
0.32* |
0.98* |
4.23 |
5.84* |
8.68* |
12.11 |
16.33 |
17.52 |
20.06 |
±SD |
2.01 |
4.14 |
4.47 |
4.67 |
4.04 |
5.42 |
7.07 |
6.87 |
6.06 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G1R, F & 0 |
Mean |
2.33 |
4.77 |
7.09 |
9.41 |
11.25 |
12.13 |
13.28 |
15.40 |
18.55 |
±SD |
0.94 |
1.21 |
1.95 |
2.32 |
2.66 |
2.82 |
3.68 |
4.29 |
4.06 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, F & 600 |
Mean |
0.32* |
2.00* |
2.86* |
3.08* |
5.14* |
6.87* |
7.53* |
9.24* |
11.61* |
±SD |
1.19 |
1.69 |
2.15 |
2.83 |
3.00 |
2.95 |
3.70 |
3.18 |
3.50 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
* Statistically significant (P<0.05) change than the vehicle control group.
Table 7.1. Summary of average feed consumption (g/animal/day) record. Males
Group, Sex & Dose |
Feed Consumption (g/animal/day) |
||
Week 1 (Day 1 to 7) |
Week 2 (Day 7 to 14) |
||
G1, M & 0 |
Mean |
19.41 |
22.53 |
±SD |
1.01 |
2.12 |
|
n |
12 |
12 |
|
G2, M & 100 |
Mean |
17.96* |
23.03 |
±SD |
0.73 |
1.78 |
|
n |
12 |
12 |
|
G3, M & 300 |
Mean |
19.36 |
22.14 |
±SD |
0.50 |
1.24 |
|
n |
12 |
12 |
|
G4, M & 600 |
Mean |
17.97* |
20.47 |
±SD |
0.28 |
1.15 |
|
n |
12 |
12 |
|
* Statistically significant (P<0.05) change than the vehicle control group.
Table 7.2. Summary of average feed consumption (g/animal/day) record. Females
Group, Sex & Dose |
Feed Consumption (g/animal/day) |
||
Week 1 (Day 1 to 7) |
Week 2 (Day 7 to 14) |
||
G1, F & 0 |
Mean |
15.79 |
16.65 |
±SD |
0.32 |
0.74 |
|
n |
6 |
6 |
|
G1, F & 100 |
Mean |
14.14* |
16.81 |
±SD |
0.37 |
1.83 |
|
n |
6 |
6 |
|
G3, F & 300 |
Mean |
13.77* |
16.00 |
±SD |
0.38 |
0.80 |
|
n |
6 |
6 |
|
G4, F & 600 |
Mean |
13.64* |
15.77 |
±SD |
0.34 |
1.12 |
|
n |
6 |
6 |
|
* Statistically significant (P<0.05) change than the vehicle control group.
Table 7.3. Summary of average feed consumption (g/animal/day) record. Recovery groups
Group, Sex & Dose |
Feed Consumption (g/animal/day) |
||||||||||
Week 1 (Day 1 to 7) |
Week 2 (Day 7 to 14) |
Week 3 (Day 14 to 21) |
Week 4 (Day 21 to 28) |
Week 5 (Day 28 to 35) |
Week 6 (Day 35 to 42) |
Week 7 (Day 42 to 49) |
Week 8 (Day 49 to 56) |
Week 9 (Day 56 to 63) |
Week 10 (Day 63 to 67) |
||
G1R, M & 0 |
Mean |
19.00 |
20.97 |
21.80 |
22.95 |
23.37 |
23.78 |
24.10 |
24.64 |
25.32 |
24.10 |
±SD |
0.63 |
2.36 |
1.32 |
0.49 |
0.55 |
0.28 |
0.14 |
0.13 |
0.46 |
0.14 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, M & 600 |
Mean |
18.68 |
20.75 |
20.91 |
21.28 |
22.72 |
23.35 |
23.63 |
24.08 |
24.74 |
23.63 |
±SD |
1.66 |
1.55 |
1.48 |
1.82 |
0.65 |
0.39 |
0.28 |
0.12 |
0.23 |
0.28 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G1R, F & 0 |
Mean |
15.05 |
16.48 |
16.81 |
17.43 |
17.88 |
18.69 |
20.42 |
21.49 |
22.43 |
20.42 |
±SD |
1.66 |
0.07 |
0.08 |
0.32 |
0.30 |
0.65 |
0.17 |
0.13 |
0.46 |
0.17 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, F & 600 |
Mean |
14.34 |
16.10 |
16.46 |
16.76 |
17.43 |
18.15 |
19.88 |
20.73 |
22.07 |
19.88 |
±SD |
0.09 |
0.42 |
0.39 |
0.27 |
0.05 |
0.30 |
0.08 |
0.37 |
0.24 |
0.08 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
Table 8.1. Neurological/functional observation battery record. Open field and sensory observations. Males
Day 36 |
||||||
Parameters↓ |
Group & Sex |
G1 & M |
G2 & M |
G3 & M |
G4 & M |
|
Dose (mg/kg body weight) |
0 |
100 |
300 |
600 |
||
Number of Randomly selected Animals |
5 |
5 |
5 |
5 |
||
Open field Observation |
||||||
Mobility |
1 |
1 |
1 |
1 |
||
Gait |
1 |
1 |
1 |
1 |
||
Arousal |
3 |
3 |
3 |
3 |
||
Number of Rearing |
Mean |
3.8 |
3.4 |
3.0 |
3.0 |
|
±SD |
0.8 |
1.1 |
1.2 |
0.7 |
||
Numbers of Urination |
Mean |
2.8 |
2.4 |
1.6 |
2.8 |
|
±SD |
0.8 |
1.1 |
0.5 |
0.8 |
||
Number of Defecation |
Mean |
2.8 |
2.2 |
2.4 |
3.2 |
|
±SD |
1.3 |
1.3 |
0.5 |
1.3 |
||
Clonic involuntary movement |
1 |
1 |
1 |
1 |
||
Tonic involuntary movement |
1 |
1 |
1 |
1 |
||
Stereotype Behaviour |
1 |
1 |
1 |
1 |
||
Excessive Grooming |
Mean |
2.8 |
2.4 |
3.2 |
3.0 |
|
±SD |
0.8 |
0.5 |
0.8 |
0.7 |
||
Sensory Observations |
||||||
Approach Response |
1 |
1 |
1 |
1 |
||
Auditory Response |
2 |
2 |
2 |
2 |
||
Touch Response |
2 |
2 |
2 |
2 |
||
Pupil Reflex |
2 |
2 |
2 |
2 |
||
Tail Pinch Response |
2 |
2 |
2 |
2 |
||
Righting Reflex |
1 |
1 |
1 |
1 |
||
Physiological observation |
||||||
Body temperature (°F) |
Mean |
98.6 |
98.8 |
98.4 |
98.2 |
|
±SD |
0.8 |
0.8 |
0.8 |
0.9 |
||
Table 8.2. Neurological/functional observation battery record. Open field and sensory observations. Females
Lactation Day 13 |
||||||
Parameters↓ |
Group & Sex |
G1 & F |
G2 & F |
G3 & F |
G4 & F |
|
Dose (mg/kg body weight) |
0 |
100 |
300 |
600 |
||
Number of Randomly selected Animals |
5 |
5 |
5 |
5 |
||
Open field Observation |
||||||
Mobility |
1 |
1 |
1 |
1 |
||
Gait |
1 |
1 |
1 |
1 |
||
Arousal |
3 |
3 |
3 |
3 |
||
Number of Rearing |
Mean |
2.4 |
3.6* |
2.8 |
3.6* |
|
±SD |
0.5 |
0.5 |
0.8 |
0.5 |
||
Numbers of Urination |
Mean |
3.6 |
3.6 |
3.4 |
3.6 |
|
±SD |
0.5 |
1.1 |
0.9 |
1.1 |
||
Number of Defecation |
Mean |
3.4 |
3.0 |
2.8 |
3.2 |
|
±SD |
0.9 |
0.7 |
0.8 |
0.8 |
||
Clonic involuntary movement |
1 |
1 |
1 |
1 |
||
Tonic involuntary movement |
1 |
1 |
1 |
1 |
||
Stereotype Behaviour |
1 |
1 |
1 |
1 |
||
Excessive Grooming |
Mean |
2.8 |
2.8 |
2.8 |
3.4 |
|
±SD |
0.8 |
0.8 |
0.8 |
0.5 |
||
Sensory Observations |
||||||
Approach Response |
1 |
1 |
1 |
1 |
||
Auditory Response |
2 |
2 |
2 |
2 |
||
Touch Response |
2 |
2 |
2 |
2 |
||
Pupil Reflex |
2 |
2 |
2 |
2 |
||
Tail Pinch Response |
2 |
2 |
2 |
2 |
||
Righting Reflex |
1 |
1 |
1 |
1 |
||
Physiological observation |
||||||
Body temperature (°F) |
Mean |
98.7 |
98.9 |
98.5 |
99.0 |
|
±SD |
0.5 |
0.4 |
0.7 |
0.3 |
||
Table 8.3. Neurological/functional observation battery record. Open field and sensory observations. Recovery groups
Day 66 |
||||||
Parameters↓ |
Group & Sex |
G1R & M |
G4R & M |
G1R & F |
G4R & F |
|
Dose (mg/kg body weight) |
0 |
600 |
0 |
600 |
||
Number of Animals |
5 |
5 |
5 |
5 |
||
Open field Observation |
||||||
Mobility |
1 |
1 |
1 |
1 |
||
Gait |
1 |
1 |
1 |
1 |
||
Arousal |
3 |
3 |
3 |
3 |
||
Number of Rearing |
Mean |
3.0 |
2.8 |
2.8 |
3.0 |
|
±SD |
0.7 |
0.8 |
0.8 |
0.7 |
||
Numbers of Urination |
Mean |
3.0 |
2.6 |
3.0 |
3.0 |
|
±SD |
0.7 |
0.5 |
0.7 |
0.7 |
||
Number of Defecation |
Mean |
2.8 |
3.0 |
2.8 |
3.0 |
|
±SD |
0.8 |
0.7 |
0.8 |
1.0 |
||
Clonic involuntary movement |
1 |
1 |
1 |
1 |
||
Tonic involuntary movement |
1 |
1 |
1 |
1 |
||
Stereotype Behaviour |
1 |
1 |
1 |
1 |
||
Excessive Grooming |
Mean |
2.8 |
2.8 |
3.0 |
3.0 |
|
±SD |
0.8 |
0.8 |
0.7 |
0.7 |
||
Sensory Observations |
||||||
Approach Response |
1 |
1 |
1 |
1 |
||
Auditory Response |
2 |
2 |
2 |
2 |
||
Touch Response |
2 |
2 |
2 |
2 |
||
Pupil Reflex |
2 |
2 |
2 |
2 |
||
Tail Pinch Response |
2 |
2 |
2 |
2 |
||
Righting Reflex |
1 |
1 |
1 |
1 |
||
Physiological observation |
||||||
Body temperature (°F) |
Mean |
98.9 |
98.5 |
98.8 |
98.6 |
|
±SD |
0.5 |
0.7 |
0.6 |
1.0 |
||
Open field Observation: a. Mobility - 1=Normal, b. Gait - 1=Normal, c. Arousal - 3=Normal, g. Stereotypies - repetitive circling - 1= Absent, 2=Present, Sensory Observations:
a. Startle Response - 2=Normal, b. Touch Response - 2=Normal, c. Pupil Response 2=Normal, d. Response to Nociceptive stimuli - 2=Normal, e. Righting Reflex - 1=Present, 2=Slow, 3=Absent
* Statistically significant (P<0.05) change than the vehicle control group.
Table 9.1. Summary of haematology record. Males
Group, Sex & Dose (mg/kg body weight) |
Total Leucocyte Count |
Total Erythrocyte Count |
Hemoglobin |
Haematocrit |
Mean Corpuscular Volume |
Mean Corpuscular Hemoglobin |
Mean Corpuscular Hemoglobin Concentration |
Platelet Count |
Mean Platelet Volume |
Reticulocyte Count |
|
(WBC) |
(RBC) |
(HGB) |
(HCT) |
(MCV) |
(MCH) |
(MCHC) |
(PLT) |
(MPV) |
(Retic) |
||
(103cells/µL) |
(106cells/µL) |
(g/dL) |
(%) |
(fL) |
(pg) |
(g/dL) |
(103cells/µL) |
(fL) |
(%) |
||
G1, M & 0 |
Mean |
7.90 |
8.04 |
13.38 |
43.22 |
53.86 |
16.66 |
30.94 |
831.00 |
5.76 |
1.55 |
±SD |
1.57 |
0.58 |
0.86 |
2.27 |
1.14 |
0.41 |
0.70 |
95.01 |
0.30 |
0.13 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G2, M & 100 |
Mean |
7.20 |
7.53 |
12.44 |
41.10 |
54.64 |
16.52 |
30.26 |
797.00 |
5.90 |
1.68 |
±SD |
0.60 |
0.57 |
1.05 |
2.52 |
1.71 |
0.68 |
1.77 |
94.89 |
0.29 |
0.52 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G3, M & 300 |
Mean |
7.11 |
7.71 |
13.18 |
42.26 |
54.80 |
17.10 |
31.18 |
783.20 |
5.92 |
1.44 |
±SD |
0.90 |
0.26 |
0.59 |
0.94 |
1.99 |
0.92 |
0.72 |
65.48 |
0.26 |
0.74 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4, M & 600 |
Mean |
8.58 |
7.74 |
12.92 |
42.88 |
55.56 |
16.66 |
30.08 |
830.80 |
6.00 |
2.33 |
±SD |
1.14 |
0.71 |
1.55 |
3.16 |
2.35 |
0.67 |
2.27 |
152.49 |
0.25 |
1.60 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
Group, Sex & Dose (mg/kg body weight) |
Neutrophils |
Lymphocytes |
Monocytes |
Eosinophils |
Basophils |
|
(Neut) |
(Lymph) |
(Mono) |
(Eos) |
(Baso) |
||
(%) |
(%) |
(%) |
(%) |
(%) |
||
G1, M & 0 |
Mean |
24.34 |
70.60 |
2.86 |
1.80 |
0.20 |
±SD |
6.17 |
6.81 |
0.98 |
0.66 |
0.10 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
G2, M & 100 |
Mean |
27.48 |
66.72 |
3.66 |
1.68 |
0.22 |
±SD |
2.57 |
2.34 |
2.08 |
0.25 |
0.11 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
G3, M & 300 |
Mean |
27.00 |
66.68 |
3.72 |
2.14 |
0.22 |
±SD |
5.31 |
4.48 |
0.74 |
1.03 |
0.08 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
G4, M & 600 |
Mean |
23.38 |
71.96 |
2.98 |
1.32 |
0.24 |
±SD |
3.67 |
5.22 |
1.84 |
0.36 |
0.09 |
|
n |
5 |
5 |
5 |
5 |
5 |
Group, Sex & Dose (mg/kg body weight) |
|
Absolute Reticulocyte Count |
Absolute Neutrophils |
Absolute Lymphocytes |
Absolute Monocytes |
Absolute Eosinophils |
Absolute Basophils |
Prothrombin Time |
Activated Prothrombin Time |
(Retic) |
(Neut) |
(Lymph) |
(Mono) |
(Eos) |
(Baso) |
(PT) |
(APTT) |
||
(109cells/L) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(Seconds) |
(Seconds) |
||
G1, M & 0 |
Mean |
124.26 |
1.91 |
5.59 |
0.22 |
0.15 |
0.01 |
26.70 |
13.36 |
±SD |
3.05 |
0.51 |
1.37 |
0.08 |
0.07 |
0.01 |
0.97 |
2.24 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G2, M & 100 |
Mean |
124.68 |
1.99 |
4.79 |
0.27 |
0.12 |
0.02 |
28.30 |
16.16 |
±SD |
29.57 |
0.33 |
0.23 |
0.15 |
0.03 |
0.01 |
2.31 |
2.22 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G3, M & 300 |
Mean |
111.26 |
1.94 |
4.72 |
0.27 |
0.15 |
0.02 |
26.20 |
17.40 |
±SD |
56.81 |
0.53 |
0.54 |
0.07 |
0.05 |
0.01 |
1.29 |
4.09 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4, M & 600 |
Mean |
176.02 |
2.03 |
6.14 |
0.26 |
0.12 |
0.02 |
25.68 |
17.90* |
±SD |
109.06 |
0.54 |
0.59 |
0.20 |
0.04 |
0.01 |
1.38 |
1.42 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
* Statistically significant (P<0.05) change than the vehicle control group.
Table 9.2. Summary of haematology record. Females
Group, Sex & Dose (mg/kg body weight) |
Total Leucocyte Count |
Total Erythrocyte Count |
Hemoglobin |
Haematocrit |
Mean Corpuscular Volume |
Mean Corpuscular Hemoglobin |
Mean Corpuscular Hemoglobin Concentration |
Platelet Count |
Mean Platelet Volume |
Reticulocyte Count |
|
(WBC) |
(RBC) |
(HGB) |
(HCT) |
(MCV) |
(MCH) |
(MCHC) |
(PLT) |
(MPV) |
(Retic) |
||
(103cells/µL) |
(106cells/µL) |
(g/dL) |
(%) |
(fL) |
(pg) |
(g/dL) |
(103cells/µL) |
(fL) |
(%) |
||
G1, F & 0 |
Mean |
12.44 |
7.68 |
14.78 |
44.32 |
57.78 |
19.26 |
33.34 |
604.20 |
7.40 |
1.76 |
±SD |
3.97 |
0.57 |
0.86 |
3.30 |
1.85 |
0.67 |
0.62 |
247.85 |
0.76 |
1.01 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G2, F & 100 |
Mean |
9.89 |
6.89 |
13.36* |
40.82 |
59.28 |
19.46 |
32.78 |
671.40 |
7.16 |
2.07 |
±SD |
2.84 |
0.51 |
0.57 |
2.29 |
1.13 |
0.73 |
0.62 |
197.31 |
0.66 |
0.82 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G3, F & 300 |
Mean |
10.38 |
7.70 |
14.74 |
45.38 |
59.00 |
19.14 |
32.48 |
610.40 |
7.50 |
2.18 |
±SD |
2.05 |
0.29 |
0.44 |
1.37 |
1.07 |
0.44 |
0.87 |
232.46 |
1.07 |
1.12 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4, F & 600 |
Mean |
9.17 |
7.22 |
14.14 |
43.16 |
59.86 |
19.62 |
32.78 |
801.40 |
7.06 |
1.92 |
±SD |
2.17 |
0.65 |
1.05 |
3.08 |
2.55 |
0.76 |
0.69 |
145.93 |
0.58 |
0.98 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
Group, Sex & Dose (mg/kg body weight) |
Neutrophils |
Lymphocytes |
Monocytes |
Eosinophils |
Basophils |
|
(Neut) |
(Lymph) |
(Mono) |
(Eos) |
(Baso) |
||
(%) |
(%) |
(%) |
(%) |
(%) |
||
G1, F & 0 |
Mean |
27.94 |
66.88 |
2.96 |
1.78 |
0.24 |
±SD |
7.71 |
7.63 |
1.43 |
0.82 |
0.05 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
G2, F & 100 |
Mean |
31.52 |
63.44 |
2.52 |
2.16 |
0.20 |
±SD |
12.12 |
12.91 |
1.47 |
0.62 |
0.07 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
G3, F & 300 |
Mean |
24.40 |
71.14 |
1.62 |
2.38 |
0.26 |
±SD |
5.35 |
6.68 |
1.06 |
1.07 |
0.09 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
G4, F & 600 |
Mean |
22.38 |
71.52 |
3.92 |
1.80 |
0.24 |
±SD |
6.45 |
7.33 |
2.34 |
0.37 |
0.05 |
|
n |
5 |
5 |
5 |
5 |
5 |
Group, Sex & Dose (mg/kg body weight) |
Absolute Reticulocyte Count |
Absolute Neutrophils |
Absolute Lymphocytes |
Absolute Monocytes |
Absolute Eosinophils |
Absolute Basophils |
Prothrombin Time |
Activated Prothrombin Time |
|
(Retic) |
(Neut) |
(Lymph) |
(Mono) |
(Eos) |
(Baso) |
(PT) |
(APTT) |
||
(109cells/L) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(Seconds) |
(Seconds) |
||
G1, F & 0 |
Mean |
131.92 |
3.47 |
8.33 |
0.38 |
0.21 |
0.04 |
23.02 |
14.64 |
±SD |
69.35 |
1.62 |
2.65 |
0.24 |
0.10 |
0.01 |
4.81 |
2.57 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G2, F & 100 |
Mean |
140.98 |
3.11 |
6.28 |
0.25 |
0.22 |
0.02 |
21.80 |
16.98 |
±SD |
50.35 |
1.37 |
2.14 |
0.15 |
0.11 |
0.01 |
3.93 |
4.20 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G3, F & 300 |
Mean |
167.44 |
2.62 |
7.28 |
0.18 |
0.26 |
0.03 |
21.94 |
15.46 |
±SD |
86.60 |
1.04 |
0.76 |
0.16 |
0.16 |
0.02 |
3.01 |
3.26 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4, F & 600 |
Mean |
134.16 |
2.16 |
6.46 |
0.35 |
0.17 |
0.02 |
25.00 |
19.90 |
±SD |
51.84 |
1.21 |
0.99 |
0.21 |
0.06 |
0.00 |
8.24 |
9.87 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
* Statistically significant (P<0.05) change than the vehicle control group.
Table 9.3. Summary of haematology record. Recovery groups
Group, Sex & Dose (mg/kg body weight) |
Total Leucocyte Count |
Total Erythrocyte Count |
Hemoglobin |
Haematocrit |
Mean Corpuscular Volume |
Mean Corpuscular Hemoglobin |
Mean Corpuscular Hemoglobin Concentration |
Platelet Count |
Mean Platelet Volume |
Reticulocyte Count |
|
(WBC) |
(RBC) |
(HGB) |
(HCT) |
(MCV) |
(MCH) |
(MCHC) |
(PLT) |
(MPV) |
(Retic) |
||
(103cells/µL) |
(106cells/µL) |
(g/dL) |
(%) |
(fL) |
(pg) |
(g/dL) |
(103cells/µL) |
(fL) |
(%) |
||
G1R, M & 0 |
Mean |
8.71 |
8.19 |
14.48 |
42.66 |
52.14 |
17.70 |
33.96 |
962.60 |
6.92 |
1.47 |
±SD |
0.61 |
0.43 |
0.41 |
1.58 |
1.65 |
0.49 |
0.44 |
140.30 |
0.15 |
0.23 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, M & 600 |
Mean |
7.51* |
8.07 |
14.68 |
42.50 |
52.66 |
18.18 |
34.50 |
794.00 |
7.58* |
1.31 |
±SD |
0.59 |
0.34 |
0.46 |
1.31 |
0.96 |
0.52 |
0.46 |
85.12 |
0.44 |
0.16 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G1R, F & 0 |
Mean |
5.93 |
7.34 |
13.78 |
40.00 |
54.58 |
18.76 |
34.38 |
867.60 |
7.26 |
1.51 |
±SD |
1.05 |
0.25 |
0.36 |
0.78 |
1.38 |
0.22 |
0.58 |
150.83 |
0.32 |
0.34 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, F & 600 |
Mean |
5.10 |
7.47 |
14.12 |
40.42 |
54.12 |
18.94 |
35.02 |
844.80 |
7.56 |
1.55 |
±SD |
1.74 |
0.39 |
0.44 |
2.18 |
1.48 |
0.96 |
1.63 |
153.15 |
0.41 |
0.28 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
Group, Sex & Dose (mg/kg body weight) |
Neutrophils |
Lymphocytes |
Monocytes |
Eosinophils |
Basophils |
|
(Neut) |
(Lymph) |
(Mono) |
(Eos) |
(Baso) |
||
(%) |
(%) |
(%) |
(%) |
(%) |
||
G1R, M & 0 |
Mean |
20.92 |
74.34 |
2.96 |
1.32 |
0.20 |
±SD |
2.23 |
3.30 |
1.56 |
0.52 |
0.00 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
G4R, M & 600 |
Mean |
26.32 |
69.66 |
2.14 |
1.48 |
0.22 |
±SD |
7.17 |
7.97 |
1.15 |
0.40 |
0.11 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
G1R, F & 0 |
Mean |
24.28 |
70.96 |
2.46 |
1.80 |
0.22 |
±SD |
7.22 |
8.79 |
1.24 |
0.78 |
0.13 |
|
n |
5 |
5 |
5 |
5 |
5 |
|
G4R, F & 600 |
Mean |
24.40 |
70.32 |
2.26 |
2.72 |
0.16 |
±SD |
5.54 |
6.18 |
0.30 |
0.89 |
0.09 |
|
n |
5 |
5 |
5 |
5 |
5 |
Group, Sex & Dose (mg/kg body weight) |
Absolute Reticulocyte Count |
Absolute Neutrophils |
Absolute Lymphocytes |
Absolute Monocytes |
Absolute Eosinophils |
Absolute Basophils |
Prothrombin Time |
Activated Prothrombin Time |
|
(Retic) |
(Neut) |
(Lymph) |
(Mono) |
(Eos) |
(Baso) |
(PT) |
(APTT) |
||
(109cells/L) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(103cells/µL) |
(Seconds) |
(Seconds) |
||
G1R, M & 0 |
Mean |
120.24 |
1.82 |
6.48 |
0.26 |
0.11 |
0.02 |
25.14 |
21.48 |
±SD |
16.86 |
0.20 |
0.61 |
0.14 |
0.03 |
0.01 |
3.32 |
4.44 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, M & 600 |
Mean |
105.48 |
1.97 |
5.24* |
0.16 |
0.11 |
0.02 |
22.26 |
14.92* |
±SD |
13.61 |
0.55 |
0.76 |
0.08 |
0.04 |
0.01 |
2.59 |
3.67 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G1R, F & 0 |
Mean |
110.42 |
1.38 |
4.27 |
0.14 |
0.10 |
0.01 |
26.70 |
20.06 |
±SD |
23.36 |
0.19 |
1.19 |
0.05 |
0.03 |
0.01 |
2.11 |
4.15 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, F & 600 |
Mean |
114.88 |
1.24 |
3.60 |
0.11 |
0.13 |
0.01 |
27.28 |
17.46 |
±SD |
16.01 |
0.54 |
1.35 |
0.04 |
0.03 |
0.01 |
5.16 |
2.20 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
* Statistically significant (P<0.05) change than the vehicle control group.
Table 10.1. Summary of clinical chemistry record. Males
Group, Sex & Dose (mg/kg body weight) |
Glucose |
Urea |
Creatinine |
Total Cholesterol |
Triglycerides |
Total Protein |
Albumin |
Alanine aminotransferase |
Aspartate aminotransferase |
|
(GLU) |
(CRE) |
(CHO) |
(TRI) |
(TPR) |
(ALB) |
(ALT) |
(AST) |
|||
(mg/dL) |
(mg/dL) |
(mg/dL) |
(mg/dL) |
(mg/dL) |
(g/dL) |
(g/dL) |
(U/L) |
(U/L) |
||
G1, M & 0 |
Mean |
77.60 |
31.70 |
0.47 |
44.40 |
39.40 |
6.12 |
3.04 |
43.40 |
109.00 |
±SD |
8.32 |
1.02 |
0.17 |
10.16 |
10.74 |
0.22 |
0.13 |
12.70 |
40.98 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G2, M & 100 |
Mean |
81.60 |
30.26 |
0.51 |
45.80 |
48.60 |
5.88 |
3.04 |
51.40 |
116.80 |
±SD |
12.97 |
5.31 |
0.02 |
4.92 |
13.09 |
0.22 |
0.11 |
7.60 |
27.60 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G3, M & 300 |
Mean |
83.40 |
37.84 |
0.53 |
53.80 |
33.60 |
6.78 |
3.06 |
45.40 |
111.00 |
±SD |
11.28 |
6.95 |
0.05 |
24.47 |
7.27 |
1.31 |
0.19 |
7.16 |
10.98 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4, M & 600 |
Mean |
97.60 |
31.84 |
0.56 |
42.00 |
27.20 |
5.78 |
2.75 |
43.60 |
101.00 |
±SD |
28.01 |
3.36 |
0.06 |
5.70 |
6.14 |
0.46 |
0.35 |
4.88 |
13.51 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
Group, Sex & Dose (mg/kg body weight) |
Alkaline phosphatase |
Total Bilirubin |
Calcium |
Phosphorous |
Globulin |
Blood Urea Nitrogen |
Sodium |
Potassium |
Chloride |
|
(ALP) |
(BIT) |
(CAL) |
(PHO) |
(GLO) |
(BUN) |
(Na) |
(K) |
(CLO) |
||
(U/L) |
(mg/dL) |
(mg/dL) |
(mg/dL) |
(g/dL) |
(mg/dL) |
(mmol/L) |
(mmol/L) |
(mmol/L) |
||
G1, M & 0 |
Mean |
116.20 |
0.05 |
9.36 |
5.12 |
3.08 |
14.79 |
150.98 |
3.96 |
112.58 |
±SD |
34.17 |
0.01 |
0.23 |
0.57 |
0.10 |
0.47 |
1.72 |
0.51 |
1.46 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G2, M & 100 |
Mean |
101.60 |
0.03 |
9.46 |
5.36 |
2.84 |
14.12 |
149.48 |
4.15 |
112.34 |
±SD |
21.15 |
0.02 |
0.38 |
0.36 |
0.12 |
2.48 |
1.70 |
0.63 |
1.84 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G3, M & 300 |
Mean |
141.20 |
0.03* |
9.76 |
5.30 |
3.72 |
17.66 |
148.70 |
3.87 |
110.82 |
±SD |
60.95 |
0.01 |
0.59 |
1.41 |
1.41 |
3.25 |
4.53 |
0.47 |
1.51 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4, M & 600 |
Mean |
91.20 |
0.03 |
9.48 |
6.54 |
3.03 |
14.86 |
151.38 |
4.46 |
112.28 |
±SD |
23.76 |
0.02 |
0.35 |
0.76 |
0.48 |
1.57 |
2.14 |
0.36 |
3.23 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
* Statistically significant (P<0.05) change than the vehicle control group.
Table 10.2. Summary of clinical chemistry record. Females
Group, Sex & Dose (mg/kg body weight) |
Glucose |
Urea |
Creatinine |
Total Cholesterol |
Triglycerides |
Total Protein |
Albumin |
Alanine aminotransferase |
Aspartate aminotransferase |
|
(GLU) |
(CRE) |
(CHO) |
(TRI) |
(TPR) |
(ALB) |
(ALT) |
(AST) |
|||
(mg/dL) |
(mg/dL) |
(mg/dL) |
(mg/dL) |
(mg/dL) |
(g/dL) |
(g/dL) |
(U/L) |
(U/L) |
||
G1, F & 0 |
Mean |
80.60 |
48.44 |
0.48 |
67.60 |
106.40 |
6.68 |
3.03 |
110.80 |
143.40 |
±SD |
18.90 |
5.74 |
0.06 |
11.70 |
47.83 |
0.36 |
0.24 |
23.34 |
43.36 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G2, F & 100 |
Mean |
76.80 |
45.38 |
0.45 |
67.80 |
292.80 |
6.26 |
2.81 |
112.80 |
143.00 |
±SD |
24.14 |
8.53 |
0.07 |
10.33 |
238.17 |
0.42 |
0.24 |
22.44 |
69.42 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G3, F & 300 |
Mean |
87.80 |
50.10 |
0.42 |
72.80 |
151.00 |
6.88 |
3.12 |
96.20 |
89.00 |
±SD |
13.81 |
16.09 |
0.06 |
10.21 |
56.63 |
0.26 |
0.29 |
14.50 |
30.51 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4, F & 600 |
Mean |
85.80 |
47.94 |
0.51 |
77.00 |
76.80 |
7.20 |
3.37 |
90.20 |
105.40 |
±SD |
24.79 |
13.82 |
0.07 |
15.87 |
45.38 |
0.68 |
0.35 |
33.87 |
36.62 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
Group, Sex & Dose (mg/kg body weight) |
Alkaline phosphatase |
Total Bilirubin |
Calcium |
Phosphorous |
Globulin |
Blood Urea Nitrogen |
Sodium |
Potassium |
Chloride |
|
(ALP) |
(BIT) |
(CAL) |
(PHO) |
(GLO) |
(BUN) |
(Na) |
(K) |
(CLO) |
||
(U/L) |
(mg/dL) |
(mg/dL) |
(mg/dL) |
(g/dL) |
(mg/dL) |
(mmol/L) |
(mmol/L) |
(mmol/L) |
||
G1, F & 0 |
Mean |
170.40 |
0.02 |
10.28 |
9.25 |
3.65 |
22.60 |
154.46 |
3.90 |
122.36 |
±SD |
64.28 |
0.00 |
0.75 |
1.40 |
0.24 |
2.68 |
16.36 |
0.98 |
15.63 |
|
n |
5 |
5 |
5 |
4 |
5 |
5 |
5 |
5 |
5 |
|
G2, F & 100 |
Mean |
240.60 |
0.02 |
10.00 |
6.54 |
3.45 |
21.18 |
157.82 |
3.58 |
121.80 |
±SD |
93.37 |
0.00 |
1.08 |
2.93 |
0.27 |
3.98 |
17.56 |
0.17 |
14.22 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G3, F & 300 |
Mean |
296.80 |
0.02 |
10.30 |
6.58 |
3.76 |
23.38 |
140.22 |
3.78 |
107.34 |
±SD |
141.57 |
0.00 |
1.06 |
2.78 |
0.10 |
7.51 |
1.08 |
0.30 |
5.50 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4, F & 600 |
Mean |
183.80 |
0.03 |
10.52 |
6.62 |
3.83 |
22.37 |
150.82 |
3.68 |
111.00 |
±SD |
85.44 |
0.02 |
0.98 |
2.75 |
0.55 |
6.45 |
6.51 |
0.62 |
7.02 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
Table 10.3. Summary of clinical chemistry record. Recovery groups
Group, Sex & Dose (mg/kg body weight) |
Glucose |
Urea |
Creatinine |
Total Cholesterol |
Triglycerides |
Total Protein |
Albumin |
Alanine aminotransferase |
Aspartate aminotransferase |
|
(GLU) |
(CRE) |
(CHO) |
(TRI) |
(TPR) |
(ALB) |
(ALT) |
(AST) |
|||
(mg/dL) |
(mg/dL) |
(mg/dL) |
(mg/dL) |
(mg/dL) |
(g/dL) |
(g/dL) |
(U/L) |
(U/L) |
||
G1R, M & 0 |
Mean |
118.20 |
26.78 |
0.62 |
51.00 |
52.20 |
6.64 |
3.12 |
51.00 |
42.00 |
±SD |
28.34 |
6.15 |
0.05 |
8.46 |
15.07 |
0.43 |
0.20 |
7.68 |
7.52 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, M & 600 |
Mean |
103.60 |
30.60 |
0.60 |
45.00 |
33.60* |
6.72 |
3.10 |
49.60 |
49.80 |
±SD |
8.17 |
4.38 |
0.06 |
10.77 |
7.09 |
0.45 |
0.22 |
12.74 |
13.31 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G1R, F & 0 |
Mean |
107.80 |
29.74 |
0.66 |
59.80 |
48.40 |
7.00 |
3.36 |
40.20 |
42.20 |
±SD |
17.54 |
4.93 |
0.03 |
13.77 |
19.09 |
0.19 |
0.09 |
7.40 |
4.38 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, F & 600 |
Mean |
102.60 |
28.54 |
0.64 |
51.20 |
28.60 |
6.78 |
3.20 |
44.20 |
43.40 |
±SD |
13.39 |
4.21 |
0.05 |
11.23 |
7.86 |
0.43 |
0.22 |
3.11 |
3.91 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
Group, Sex & Dose (mg/kg body weight) |
Alkaline phosphatase |
Total Bilirubin |
Calcium |
Phosphorous |
Globulin |
Blood Urea Nitrogen |
Sodium |
Potassium |
Chloride |
|
(ALP) |
(BIT) |
(CAL) |
(PHO) |
(GLO) |
(BUN) |
(Na) |
(K) |
(CLO) |
||
(U/L) |
(mg/dL) |
(mg/dL) |
(mg/dL) |
(g/dL) |
(mg/dL) |
(mmol/L) |
(mmol/L) |
(mmol/L) |
||
G1R, M & 0 |
Mean |
116.40 |
0.02 |
9.84 |
7.56 |
3.52 |
12.50 |
147.54 |
3.06 |
111.76 |
±SD |
29.87 |
0.00 |
0.36 |
0.64 |
0.25 |
2.87 |
3.55 |
0.15 |
1.55 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, M & 600 |
Mean |
93.40 |
0.02 |
10.12 |
7.30 |
3.62 |
14.28 |
151.54 |
3.01 |
111.42 |
±SD |
34.30 |
0.00 |
0.33 |
0.28 |
0.30 |
2.04 |
1.64 |
0.10 |
1.33 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G1R, F & 0 |
Mean |
49.80 |
0.02 |
9.90 |
7.48 |
3.64 |
13.88 |
152.42 |
3.11 |
112.38 |
±SD |
14.58 |
0.00 |
0.14 |
0.13 |
0.25 |
2.30 |
0.72 |
0.30 |
1.28 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, F & 600 |
Mean |
61.80 |
0.02 |
9.82 |
7.22 |
3.58 |
13.32 |
151.46 |
3.15 |
112.10 |
±SD |
23.57 |
0.00 |
0.25 |
0.37 |
0.30 |
1.96 |
1.18 |
0.27 |
0.80 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
Table 11. Summary of vaginal smear examination for determination of oestrus cyclicity
Group & Dose |
Total No. of Females |
No. of Females with Regular Oestrus Cyclicity during Pre-mating and Mating |
No. of Females confirmed with pregnancy
|
No. of Females with Regular Oestrus Cyclicity on Lactation Day 14 |
No. of Females with Irregular Oestrus Cyclicity during Pre-mating, Mating and on Lactation day 14 |
G1 & 0 |
12 |
12 |
10 |
10 |
0 |
G2 & 100 |
12 |
12 |
11 |
11 |
0 |
G3 & 300 |
12 |
12 |
10 |
10 |
0 |
G4 & 600 |
12 |
12 |
10 |
10 |
0 |
Table 12. Summary record of gestation body weight (g)
Group & Dose |
Body weight (g) on Gestation Days |
||||
0 |
7 |
14 |
20 |
||
G1 & 0 |
Mean |
293.54 |
312.05 |
346.33 |
411.36 |
±SD |
18.46 |
20.26 |
19.17 |
18.87 |
|
n |
10 |
10 |
10 |
10 |
|
G2 & 100 |
Mean |
285.12 |
300.22 |
338.92 |
404.73 |
±SD |
18.74 |
20.01 |
18.03 |
20.92 |
|
n |
11 |
11 |
11 |
11 |
|
G3 & 300 |
Mean |
280.85 |
300.22 |
336.08 |
406.54 |
±SD |
15.00 |
15.55 |
15.71 |
26.97 |
|
n |
10 |
10 |
10 |
10 |
|
G4 & 600 |
Mean |
276.42 |
294.72 |
327.07 |
389.37 |
±SD |
16.05 |
12.25 |
15.04 |
14.98 |
|
n |
10 |
10 |
10 |
10 |
|
n: Number of dams (the data of non-pregnant animals is excluded for mean calculations)
Table 13. Summary record of percent change in body weight (%) during gestation period
Group & Dose |
Change in body weight (%) during Gestation |
|||
0 to 7 |
7 to 14 |
14 to 20 |
||
G1 & 0 |
Mean |
6.30 |
11.13 |
18.93 |
±SD |
1.87 |
4.58 |
5.33 |
|
n |
10 |
10 |
10 |
|
G2 & 100 |
Mean |
5.30 |
13.02 |
19.45 |
±SD |
1.74 |
3.22 |
2.49 |
|
n |
11 |
11 |
11 |
|
G3 & 300 |
Mean |
6.91 |
11.98 |
20.96 |
±SD |
0.80 |
1.64 |
5.69 |
|
n |
10 |
10 |
10 |
|
G4 & 600 |
Mean |
6.72 |
10.99 |
19.20 |
±SD |
2.27 |
2.64 |
5.69 |
|
n |
10 |
10 |
10 |
|
n: Number of dams (the data of non-pregnant animals is excluded for mean calculations)
Table 14. Summary of lactation body weight (g) record
Group, Sex & Dose |
Body weight (g) on Lactation Days |
||||
1 |
4 |
7 |
13 |
||
G1 & 0 |
Mean |
327.10 |
330.97 |
334.42 |
348.86 |
±SD |
16.26 |
16.38 |
15.95 |
16.17 |
|
n |
10 |
10 |
10 |
10 |
|
G2 & 100 |
Mean |
322.50 |
326.63 |
331.30 |
348.67 |
±SD |
18.78 |
18.07 |
17.20 |
15.61 |
|
n |
11 |
11 |
11 |
11 |
|
G3 & 300 |
Mean |
310.79 |
315.83 |
320.05 |
337.45 |
±SD |
18.55 |
17.65 |
18.21 |
18.54 |
|
n |
10 |
10 |
10 |
10 |
|
G4 & 600 |
Mean |
311.35 |
314.75 |
318.00 |
336.09 |
±SD |
18.53 |
18.37 |
18.57 |
19.69 |
|
n |
10 |
10 |
10 |
10 |
|
n: Number of dams
Table 15. Summary of percent change in body weight (%) during lactation
Group & Dose |
Change in body weight (%) during Lactation Day (LD) |
|||
1 to 4 |
4 to 7 |
7 to 13 |
||
G1 & 0 |
Mean |
1.18 |
1.05 |
4.35 |
±SD |
0.45 |
0.55 |
2.19 |
|
n |
10 |
10 |
10 |
|
G2 & 100 |
Mean |
1.30 |
1.45 |
5.29 |
±SD |
0.66 |
0.59 |
1.70 |
|
n |
11 |
11 |
11 |
|
G3 & 300 |
Mean |
1.65 |
1.33 |
5.46 |
±SD |
0.99 |
0.72 |
1.59 |
|
n |
10 |
10 |
10 |
|
G4 & 600 |
Mean |
1.10* |
1.03 |
5.70 |
±SD |
0.35 |
0.47 |
2.20 |
|
n |
10 |
10 |
10 |
|
n: Number of dams
* Statistically significant (P<0.05) change than the vehicle control group.
Table 16.1. Summary of absolute organ weights (g) record. Males
Group, Sex & Dose (mg/kg body weight) |
Adrenals |
Thymus |
Spleen |
Testes |
Epididymes |
Heart |
Kidneys |
Brain |
Liver |
Prostate |
Seminal vesicles with coagulating glands |
Thyroid along with parathyroid# |
|
G1, M & 0 |
Mean |
0.0778 |
0.3166 |
0.8525 |
3.5521 |
1.5253 |
1.4954 |
3.2095 |
2.1084 |
12.2106 |
1.5663 |
1.7826 |
0.0284 |
±SD |
0.0230 |
0.0685 |
0.0981 |
0.3053 |
0.2003 |
0.2606 |
0.5003 |
0.1619 |
1.6959 |
0.2473 |
0.3736 |
0.0062 |
|
n |
5 |
5 |
5 |
12 |
12 |
5 |
5 |
5 |
5 |
12 |
12 |
12 |
|
G2, M & 100 |
Mean |
0.0908 |
0.3110 |
0.8461 |
3.5704 |
1.5355 |
1.4677 |
3.2837 |
2.0750 |
12.9020 |
1.7886 |
1.5368 |
0.0291 |
±SD |
0.0067 |
0.0833 |
0.1153 |
0.3160 |
0.1338 |
0.0619 |
0.4710 |
0.3149 |
0.9458 |
0.3161 |
0.2519 |
0.0054 |
|
n |
5 |
5 |
5 |
12 |
12 |
5 |
5 |
5 |
5 |
12 |
12 |
12 |
|
G3, M & 300 |
Mean |
0.0718 |
0.4086 |
0.7552 |
3.4894 |
1.4944 |
1.4913 |
3.4137 |
2.2140 |
12.8520 |
1.7174 |
1.3662* |
0.0271 |
±SD |
0.0118 |
0.1711 |
0.1110 |
0.2024 |
0.1371 |
0.2276 |
0.3343 |
0.0545 |
1.3223 |
0.3604 |
0.4805 |
0.0055 |
|
n |
5 |
5 |
5 |
12 |
12 |
5 |
5 |
5 |
5 |
12 |
12 |
12 |
|
G4, M & 600 |
Mean |
0.0831 |
0.2943 |
0.8536 |
3.5823 |
1.4279 |
1.4830 |
3.5043 |
2.1889 |
15.3838* |
1.6183 |
1.5367 |
0.0270 |
±SD |
0.0158 |
0.0744 |
0.2004 |
0.3689 |
0.1220 |
0.1331 |
0.3005 |
0.1384 |
1.7056 |
0.3435 |
0.5163 |
0.0051 |
|
n |
5 |
5 |
5 |
12 |
12 |
5 |
5 |
5 |
5 |
12 |
12 |
12 |
|
G1R, M & 0 |
Mean |
0.0773 |
0.3618 |
0.7242 |
3.6039 |
1.6468 |
1.9188 |
3.5834 |
2.2569 |
13.2972 |
1.7121 |
1.8645 |
0.0261 |
±SD |
0.0092 |
0.0263 |
0.0591 |
0.3635 |
0.0920 |
0.2272 |
0.4585 |
0.0468 |
1.4254 |
0.1573 |
0.0884 |
0.0063 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, M & 600 |
Mean |
0.0789 |
0.3709 |
0.7171 |
3.6500 |
1.6955 |
1.7452 |
3.3656 |
2.2167 |
12.9538 |
1.6941 |
1.8200 |
0.0260 |
±SD |
0.0065 |
0.0291 |
0.0499 |
0.2753 |
0.1809 |
0.2607 |
0.0824 |
0.0687 |
1.0505 |
0.2033 |
0.2601 |
0.0061 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
#: Weighed post fixation
*: Statistically significant than the control group (p<0.05)
Table 16.2. Summary of absolute organ weights (g) record. Females
Group, Sex & Dose (mg/kg body weight) |
Adrenals |
Thymus |
Spleen |
Heart |
Kidneys |
Brain |
Liver |
Thyroid along with parathyroid# |
|
G1, F & 0 |
Mean |
0.1048 |
0.2882 |
0.6049 |
1.2601 |
2.5891 |
2.0211 |
14.5537 |
0.0349 |
±SD |
0.0236 |
0.0253 |
0.0295 |
0.0594 |
0.3982 |
0.0756 |
1.2896 |
0.0031 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
12 |
|
G2, F & 100 |
Mean |
0.1010 |
0.3114 |
0.6619 |
1.3266 |
2.7373 |
2.0318 |
15.2112 |
0.0352 |
±SD |
0.0207 |
0.0316 |
0.0991 |
0.0908 |
0.4297 |
0.0535 |
0.6016 |
0.0031 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
12 |
|
G3, F & 300 |
Mean |
0.0879 |
0.2689 |
0.6149 |
1.3114 |
2.5901 |
2.0801 |
15.7306 |
0.0350 |
±SD |
0.0104 |
0.0410 |
0.1610 |
0.1273 |
0.4058 |
0.1362 |
1.0902 |
0.0040 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
12 |
|
G4, F & 600 |
Mean |
0.0834 |
0.3016 |
0.6075 |
1.3731 |
2.4364 |
2.0034 |
15.5934 |
0.0350 |
±SD |
0.0139 |
0.0282 |
0.0727 |
0.1828 |
0.3454 |
0.2747 |
0.8368 |
0.0018 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
12 |
|
G1R, F & 0 |
Mean |
0.0985 |
0.3394 |
0.6029 |
1.1973 |
2.7865 |
2.4941 |
9.4607 |
0.0277 |
±SD |
0.0077 |
0.0363 |
0.0939 |
0.0312 |
0.0626 |
0.1286 |
0.5923 |
0.0008 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, F & 600 |
Mean |
0.0967 |
0.3621 |
0.6393 |
1.1068* |
2.8554 |
2.4116 |
9.2773 |
0.0273 |
±SD |
0.0068 |
0.0448 |
0.0296 |
0.0372 |
0.2076 |
0.0840 |
0.3590 |
0.0047 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
#: Weighed post fixation
*: Statistically significant than the control group (p<0.05)
Table 17.1. Summary of terminal body weight (g) and organ weight (%) relative to terminal body weight record. Males
Group, Sex & Dose (mg/kg body weight) |
Fasting Body Weight (g) |
Adrenals |
Thymus |
Spleen |
Testes |
Epididymes |
Heart |
Kidneys |
Brain |
Liver |
Prostate |
Seminal vesicles with coagulating glands |
Thyroid along with parathyroid |
|
G1, M & 0 |
Mean |
416.86 |
0.0199 |
0.0804 |
0.2185 |
0.8611 |
0.3676 |
0.3762 |
0.8114 |
0.5349 |
3.0979 |
0.3771 |
0.4286 |
0.0069 |
±SD |
44.25 |
0.0062 |
0.0170 |
0.0422 |
0.1152 |
0.0474 |
0.0280 |
0.0895 |
0.0299 |
0.4111 |
0.0559 |
0.0834 |
0.0016 |
|
n |
12 |
5 |
5 |
5 |
12 |
12 |
5 |
5 |
5 |
5 |
12 |
12 |
12 |
|
G2, M & 100 |
Mean |
410.05 |
0.0228 |
0.0770 |
0.2118 |
0.8756 |
0.3767 |
0.3696 |
0.8248 |
0.5228 |
3.2457 |
0.4377 |
0.3737 |
0.0072 |
±SD |
34.57 |
0.0014 |
0.0154 |
0.0203 |
0.0980 |
0.0423 |
0.0264 |
0.1062 |
0.0845 |
0.2526 |
0.0783 |
0.0444 |
0.0017 |
|
n |
12 |
5 |
5 |
5 |
12 |
12 |
5 |
5 |
5 |
5 |
12 |
12 |
12 |
|
G3, M & 300 |
Mean |
404.28 |
0.0180 |
0.1010 |
0.1882 |
0.8730 |
0.3725 |
0.3697 |
0.8498 |
0.5523 |
3.1965 |
0.4278 |
0.3395 |
0.0067 |
±SD |
43.34 |
0.0035 |
0.0402 |
0.0253 |
0.1111 |
0.0445 |
0.0333 |
0.0672 |
0.0248 |
0.2120 |
0.0867 |
0.1157 |
0.0014 |
|
n |
12 |
5 |
5 |
5 |
12 |
12 |
5 |
5 |
5 |
5 |
12 |
12 |
12 |
|
G4, M & 600 |
Mean |
395.73 |
0.0208 |
0.0739 |
0.2141 |
0.9181 |
0.3649 |
0.3641 |
0.8614 |
0.5383 |
3.7942* |
0.4115 |
0.3907 |
0.0069 |
±SD |
39.18 |
0.0061 |
0.0263 |
0.0753 |
0.1657 |
0.0561 |
0.0300 |
0.0834 |
0.0455 |
0.5655 |
0.0858 |
0.1405 |
0.0013 |
|
n |
12 |
5 |
5 |
5 |
12 |
12 |
5 |
5 |
5 |
5 |
12 |
12 |
12 |
|
G1R, M & 0 |
Mean |
457.78 |
0.0169 |
0.0792 |
0.1583 |
0.7890 |
0.3607 |
0.4224 |
0.7818 |
0.4951 |
2.9004 |
0.3762 |
0.4093 |
0.0057 |
±SD |
32.41 |
0.0015 |
0.0055 |
0.0091 |
0.0796 |
0.0244 |
0.0746 |
0.0774 |
0.0387 |
0.1774 |
0.0492 |
0.0395 |
0.0016 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, M & 600 |
Mean |
446.24 |
0.0178 |
0.0835 |
0.1621 |
0.8226 |
0.3812 |
0.3916 |
0.7600 |
0.5008 |
2.9084 |
0.3805 |
0.4147 |
0.0058 |
±SD |
43.73 |
0.0022 |
0.0077 |
0.0215 |
0.0841 |
0.0388 |
0.0467 |
0.0763 |
0.0534 |
0.1148 |
0.0403 |
0.0937 |
0.0012 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
*: Statistically significant than the control group (p<0.05)
Table 17.2. Summary of terminal body weight (g) and organ weight (%) relative to terminal body weight record. Females
Group, Sex & Dose (mg/kg body weight) |
Fasting Body Weight (g) |
Adrenals |
Thymus |
Spleen |
Heart |
Kidneys |
Brain |
Liver |
Thyroid along with parathyroid |
|
G1, F & 0 |
Mean |
335.46 |
0.0323 |
0.0890 |
0.1870 |
0.3894 |
0.7987 |
0.6248 |
4.4983 |
0.0103 |
±SD |
17.75 |
0.0072 |
0.0080 |
0.0128 |
0.0225 |
0.1160 |
0.0376 |
0.4458 |
0.0007 |
|
n |
10 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
10 |
|
G2, F & 100 |
Mean |
335.68 |
0.0300 |
0.0927 |
0.1971 |
0.3952 |
0.8142 |
0.6053 |
4.5315 |
0.0105 |
±SD |
14.74 |
0.0056 |
0.0086 |
0.0294 |
0.0293 |
0.1169 |
0.0242 |
0.2128 |
0.0011 |
|
n |
11 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
11 |
|
G3, F & 300 |
Mean |
321.23 |
0.0274 |
0.0843 |
0.1906 |
0.4083 |
0.8052 |
0.6491 |
4.8949 |
0.0111 |
±SD |
19.34 |
0.0032 |
0.0171 |
0.0441 |
0.0370 |
0.1097 |
0.0618 |
0.2250 |
0.0016 |
|
n |
10 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
10 |
|
G4, F & 600 |
Mean |
322.66 |
0.0261 |
0.0950 |
0.1913 |
0.4327 |
0.7671 |
0.6322 |
4.8979 |
0.0109 |
±SD |
19.22 |
0.0047 |
0.0157 |
0.0346 |
0.0869 |
0.1540 |
0.1329 |
0.5877 |
0.0010 |
|
n |
10 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
10 |
|
G1R, F & 0 |
Mean |
309.32 |
0.0318 |
0.1101 |
0.1955 |
0.3875 |
0.9013 |
0.8070 |
3.0633 |
0.0090 |
±SD |
9.19 |
0.0020 |
0.0149 |
0.0342 |
0.0188 |
0.0268 |
0.0491 |
0.2507 |
0.0005 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
|
G4R, F & 600 |
Mean |
291.01 |
0.0333 |
0.1249 |
0.2202 |
0.3805 |
0.9828 |
0.8303 |
3.1921 |
0.0094 |
±SD |
13.04 |
0.0029 |
0.0185 |
0.0167 |
0.0061 |
0.0846 |
0.0516 |
0.1624 |
0.0015 |
|
n |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
5 |
Table 18. Summary of serum thyroxine (T4) hormone levels (ng/ml) record - adult males
Group, Sex & Dose |
Serum T4 Levels (ng/mL) |
|
G1, M & 0 |
Mean |
68.214 |
±SD |
1.654 |
|
n |
12 |
|
G2, M & 100 |
Mean |
66.284 |
±SD |
3.473 |
|
n |
12 |
|
G3, M & 300 |
Mean |
64.914* |
±SD |
2.161 |
|
n |
12 |
|
G4, M & 600 |
Mean |
65.095 |
±SD |
3.031 |
|
n |
12 |
*: Statistically significant than the control group (p<0.05)
Table 19. Summary of serum thyroxine (T4) hormone levels (ng/ml) record - lactation day 13 pups
Group & Dose |
Serum T4 Levels (ng/mL) |
|
G1 & 0 |
Mean |
70.035 |
±SD |
5.040 |
|
n |
10 |
|
G2 & 100 |
Mean |
70.175 |
±SD |
7.644 |
|
n |
11 |
|
G3 & 300 |
Mean |
68.226 |
±SD |
4.748 |
|
n |
10 |
|
G4 & 600 |
Mean |
70.150 |
±SD |
5.715 |
|
n |
10 |
n: Number of dams (pooled sample from two pups were selected for analysis from each dam)
Table 20. Summary of gross pathology findings: sire and dam
Parameters |
Sex |
Male |
Female |
||||||||||
Group |
G1 |
G1R |
G2 |
G3 |
G4 |
G4R |
G1 |
G1R |
G2 |
G3 |
G4 |
G4R |
|
Dose (mg/kg body weight) |
0 |
0 |
100 |
300 |
600 |
600 |
0 |
0 |
100 |
300 |
600 |
600 |
|
No of rats |
12 |
5 |
12 |
12 |
12 |
5 |
12 |
5 |
12 |
12 |
12 |
5 |
|
No. of dead animals during treatment period or recovery period |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
No. of moribund sacrificed animals |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
No. of terminally sacrificed |
12 |
5 |
12 |
12 |
12 |
5 |
12 |
5 |
12 |
12 |
12 |
5 |
|
No of showing gross pathology |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
||
Table 21. Summary of gross pathology findings :pups
Group |
G1 |
G2 |
G3 |
G4 |
||||
Dose (mg/kg body weight/day) |
0 |
100 |
300 |
600 |
||||
Sex |
Male |
Female |
Male |
Female |
Male |
Female |
Male |
Female |
No. of dead pups |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
No. of showing gross pathology v No. of pups showing external pathology |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
v No. of pups showing visceral pathology |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
No. of pups sacrificed on lactation day 4 |
0 |
13 |
0 |
11 |
0 |
15 |
0 |
8 |
No. of showing gross pathology v No. of pups showing external pathology |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
v No. of pups showing visceral pathology |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
No. of pups sacrificed on lactation day 13 |
45 |
60 |
67 |
31 |
61 |
46 |
57 |
37 |
No. of showing gross pathology v No. of pups showing external pathology |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
v No. of pups showing visceral pathology |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Table 22. Summary of histopathology findings
Sex |
Male |
Female |
|||
Group |
G1 |
G4 |
G1 |
G4 |
|
Dosage (mg/kg/day) |
0 |
600 |
0 |
600 |
|
ORGAN |
SEVERITY |
|
|
|
|
KIDNEYS |
5 |
5 |
5 |
5 |
|
Basophilic tubules; unilateral |
Minimal |
1 |
1 |
0 |
0 |
Sub-urothelium ; Mononuclear cells infiltrate; unilateral |
Slight |
0 |
0 |
0 |
1 |
THYROID |
5 |
5 |
5 |
5 |
|
Ultimobranchial cyst |
Not graded |
2 |
0 |
2 |
2 |
Ectopic Tissue: Thymus |
Not graded |
0 |
2 |
0 |
1 |
PROSTATE GLAND |
12 |
12 |
X |
X |
|
Interstitium, Mononuclear cells infiltrate |
Minimal |
2 |
1 |
X |
X |
Slight |
0 |
1 |
X |
X |
|
Concretions |
Minimal |
0 |
2 |
X |
X |
Slight |
0 |
1 |
X |
X |
|
UTERUS |
X |
X |
5 |
5 |
|
Placental scar |
Not graded |
X |
X |
5 |
5 |
Note: The numeral indicates number of animal/ tissues; X: Organs not subjected for histopathological examination due to sex difference.
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
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.
Table 1: Effect of 1,8-cineole (CIN) on hematological parameters in male and female Wistar rats treated orally for 50 days
Parameters |
Males |
Females |
||||||
control |
100 mg/kg |
500 mg/kg |
1000 mg/kg |
control |
100 mg/kg |
500 mg/kg |
1000 mg/kg |
|
RBC(106/μL) |
5.31±0.20 |
5.79±0.31 |
6.33±0.49 |
6.16±0.39 |
5.63±0.33 |
5.14±0.13 |
5.61±0.32 |
5.89±0.42 |
Hb(g/dL) |
11.30±0.41 |
12.29±0.48 |
12.76±0.99 |
13.06±0.67 |
12.39±0.59 |
11.88±0.22 |
12.39±0.46 |
13.20±0.78 |
Ht(%) |
27.95±1.10 |
31.72±1.80 |
34.04±2.90 |
34.69±2.16 |
31.78±2.16 |
28.91±0.77 |
31.57±1.72 |
33.62±2.54 |
MCV(fL) |
52.50±1.07 |
54.80±0.20 |
53.71±0.77 |
56.14±0.40* |
56.00±0.68 |
56.33±0.33 |
56.11±0.58 |
56.90±0.56 |
MCH(pg) |
21.36±0.57 |
21.37±0.35 |
20.24±0.79 |
21.26±0.32 |
22.12±0.37 |
23.13±0.28 |
22.26±0.40 |
22.56±0.35 |
MCHC(g/dL) |
40.49±0.37 |
39.12±0.74 |
37.76±1.25* |
37.77±0.49* |
39.42±0.75 |
41.13±0.46 |
39.50±0.69 |
39.69±0.76 |
RDW(%) |
13.81±0.09 |
13.67±0.27 |
14.57±0.24 |
14.56±0.23 |
13.84±0.22 |
13.77±0.10 |
14.04±0.21 |
13.73±0.11 |
WBC(103/μL) |
12.99±0.74 |
11.06±1.06 |
11.11±1.72 |
12.69±0.65 |
9.93±0.65 |
8.73±0.96 |
12.28±1.05 |
8.81±0.49 |
Platelets(103/μL) |
479.50±30.50 |
490.40±28.68 |
688.30±28.13* |
666.40±38.02* |
540.00±30.42 |
472.60±18.24 |
609.00±43.58 |
623.10±29.29 |
MPV(fL) |
7.50±0.27 |
6.72±0.08* |
6.70±0.17* |
6.32±0.06* |
6.32±0.13 |
6.35±0.07 |
6.56±0.26 |
6.22±0.06 |
Lymphocytes(%) |
57.32±1.23 |
47.43±6.10 |
61.66±3.49 |
59.56±1.57 |
67.23±2.16 |
65.70±1.53 |
63.91±1.66 |
63.26±1.77 |
Monocytes(%) |
11.98±0.40 |
15.53±1.54 |
15.40±3.07 |
13.51±0.78 |
10.59±0.73 |
10.73±0.49 |
11.70±0.83 |
11.96±1.12 |
Granulocytes(%) |
30.70±0.96 |
37.04±4.62 |
22.94±1.83 |
26.93±1.30 |
22.18±1.67 |
23.57±1.16 |
24.39±1.00 |
24.78±0.90 |
RBC: red blood cell (RBC) count, Hb: hemoglobin, Ht: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration, RDW: red cell distribution width, WBC: white blood cell, MPV: mean platelet volume. Values represent the mean ± S.E.M (n = 10/group). *Statistically different from control group (ANOVA followed by Dunnett's test, p < 0.05).
Table 2: Histopathological changes observed in male and female Wistar rats treated with 1,8-cineole (CIN) for 50 consecutive days
Organs |
Males |
Females |
||||||
control |
100 mg/kg |
500 mg/kg |
1000 mg/kg |
control |
100 mg/kg |
500 mg/kg |
1000 mg/kg |
|
Heart |
N |
N |
N |
N |
N |
N |
N |
N |
Lung |
N |
ELI (+) |
ELI (+) |
ELI (+) |
N |
ELI (+) |
ELI (++) |
ELI (+) |
Liver |
N |
LI (+) |
LI (+) |
LI (+) |
N |
N |
LI (+) |
LI (+) |
Kidney |
N |
N |
N |
IGS (+) |
N |
N |
IGS (+) |
IGS (++) |
Adrenal |
N |
N |
N |
N |
N |
N |
N |
N |
Spleen |
N |
N |
N |
N |
N |
N |
N |
N |
Stomach |
N |
N |
N |
N |
N |
N |
N |
N |
Intestine |
N |
N |
N |
N |
N |
N |
N |
N |
Pancreas |
N |
N |
N |
N |
N |
N |
N |
N |
Brain |
N |
N |
N |
N |
N |
N |
N |
N |
Testicle |
N |
N |
N |
N |
- |
- |
- |
- |
Prostate |
N |
N |
N |
N |
- |
- |
- |
- |
Uterus |
- |
- |
- |
- |
N |
ELI (++) |
ELI (++) |
ELI (+) |
Ovary |
- |
- |
- |
- |
N |
N |
N |
N |
Histopathology changes: ELI (eosinophilic and lymphocytic infiltrate), LI (lymphocytic infiltrate) and IGS (increase of the glomerular space). Scores: (N) within normal range, (+) weak, (++) moderate or (+++) intense.
Table 1: The weight and relative organ weight of liver, spleen and kidney in the subacute toxicity study
Dose |
Liver |
Spleen |
Kidney |
|||
mg/kg |
g |
% |
g |
% |
g |
% |
Group I: 0 |
0.95±0.03 |
4.15±0.37 |
0.09±0.02 |
0.44±0.14 |
0.25±0.03 |
1.16±0.04 |
Group II: 21.38 |
0.95±0.07 |
4.59±0.33 |
0.08±0.01 |
0.41±0.02 |
0.25±0.06 |
1.18±0.12 |
Group III: 64.15 |
1.06±0.11 |
4.55±0.52 |
0.08±0.02 |
0.37±0.11 |
0.28±0.09 |
1.22±0.36 |
Group IV: 192.45 |
0.95±0.08 |
4.39±0.26 |
0.08±0.01 |
0.39±0.04 |
0.28±0.05 |
1.29±0.22 |
Values are mean±S.D.; n=20. Values are absolute wet weight of organ (g) and relative organ weight (%, per body weight). No significant difference from the control group at P<0.05 (using one-way ANOVA followed by Dunnett’s test for multiple comparisons with the control group). |
||||||
Table 2. Summary of clinical signs of toxicity, detailed clinical examination and mortality record.
| Group, Sex & Dose (mg/kg/day) | No. of Animals | Clinical Signs of Toxicity | Mortality (No. of Incidences/ No. of Animals) |
| G1, M & 0 | 10 | N | 0/10 |
| G2, M & 100 | 10 | N | 0/10 |
| G3, M & 300 | 10 | N | 0/10 |
| G4, M & 1000 | 10 | N | 0/10 |
| G1R, M & 0 | 5 | N | 0/5 |
| G4R, M & 1000 | 5 | N | 0/5 |
| G1, F & 0 | 10 | N | 0/10 |
| G2, F & 100 | 10 | N | 0/10 |
| G3, F & 300 | 10 | N | 0/10 |
| G4, F & 1000 | 10 | N | 0/10 |
| G1R, F & 0 | 5 | N | 0/5 |
| G4R, F & 1000 | 5 | N | 0/5 |
M: Male; F: Female; R: Recovery; N: Normal
Table 3. Summary of body weights (g) record
| Group, Sex & Dose (mg/kg/day) | Body Weight (g) on Days | ||||||||||||||||||
| 1 | 8 | 15 | 22 | 29 | 36 | 43 | 50 | 57 | 64 | 71 | 78 | 85 | 90 | 97 | 104 | 111 | 118 | ||
| G1, M & 0 | Mean | 162.27 | 190.41 | 221.11 | 253.30 | 276.29 | 300.40 | 321.20 | 336.15 | 366.71 | 366.71 | 374.72 | 386.92 | 392.26 | 396.61 | - | - | - | - |
| ±SD | 13.03 | 19.47 | 25.09 | 31.56 | 31.09 | 38.56 | 45.50 | 51.24 | 50.88 | 52.69 | 55.50 | 57.27 | 59.61 | 58.65 | - | - | - | - | |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | - | - | - | - | |
| G2, M & 100 | Mean | 161.43 | 193.13 | 227.94 | 261.0 | 285.37 | 316.18 | 339.17 | 361.22 | 380.28 | 394.67 | 407.91 | 424.12 | 434.43 | 442.45* | - | - | - | - |
| ±SD | 12.76 | 19.69 | 19.65 | 21.72 | 20.11 | 21.21 | 25.99 | 26.75 | 30.12 | 32.50 | 36.18 | 34.53 | 34.59 | 35.36 | - | - | - | - | |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | - | - | - | - | |
| G3, M & 300 | Mean | 163.38 | 195.71 | 231.02 | 265.62 | 288.89 | 315.40 | 338.69 | 359.84 | 377.44 | 389.36 | 403.24 | 414.64 | 424.66 | 429.68 | - | - | - | - |
| ±SD | 12.98 | 18.71 | 23.19 | 22.78 | 23.81 | 24.42 | 23.00 | 24.68 | 22.50 | 24.46 | 25.45 | 28.55 | 29.32 | 28.48 | - | - | - | - | |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | - | - | - | - | |
| G4, M & 900 | Mean | 162.93 | 193.09 | 225.18 | 254.77 | 276.71 | 301.65 | 321.40 | 340.51 | 359.76 | 373.15 | 382.40 | 391.19 | 395.19 | 398.31 | - | - | - | - |
| ±SD | 12.81 | 12.86 | 16.11 | 15.75 | 18.30 | 22.41 | 24.77 | 26.91 | 27.93 | 30.16 | 32.14 | 29.53 | 29.36 | 28.93 | - | - | - | - | |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | - | - | - | - | |
| G1R, M & 0 | Mean | 159.30 | 184.47 | 218.0 | 248.35 | 272.75 | 296.31 | 320.25 | 335.39 | 358.13 | 378.50 | 392.22 | 407.96 | 412.70 | 418.40 | 427.49 | 434.28 | 441.88 | 444.29 |
| ±SD | 9.93 | 11.46 | 12.46 | 23.19 | 26.12 | 34.65 | 37.09 | 34.38 | 32.92 | 29.02 | 27.50 | 28.38 | 30.14 | 31.88 | 29.46 | 27.22 | 24.67 | 25.08 | |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
| G4R, M & 900 | Mean | 160.60 | 188.86 | 220.33 | 249.79 | 269.53 | 288.39 | 314.48 | 335.13 | 354.79 | 366.61 | 376.38 | 385.58 | 393.84 | 400.17 | 410.09 | 422.14 | 425.49 | 429.90 |
| ±SD | 12.19 | 16.68 | 25.69 | 26.78 | 29.55 | 30.60 | 34.70 | 30.94 | 29.79 | 31.81 | 29.03 | 28.54 | 27.49 | 28.17 | 26.16 | 24.93 | 23.73 | 23.98 | |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
| Group, Sex & Dose (mg/kg/day) | Body Weight (g) on Days | ||||||||||||||||||
| 1 | 8 | 15 | 22 | 29 | 36 | 43 | 50 | 57 | 64 | 71 | 78 | 85 | 90 | 97 | 104 | 111 | 118 | ||
| G1, F & 0 | Mean | 147.50 | 166.89 | 187.10 | 204.53 | 216.18 | 230.93 | 242.18 | 250.37 | 259.85 | 264.68 | 266.47 | 270.88 | 275.37 | 278.84 | - | - | - | - |
| ±SD | 9.43 | 13.48 | 17.35 | 18.27 | 18.82 | 23.52 | 24.54 | 24.31 | 28.19 | 27.32 | 25.86 | 26.31 | 27.14 | 31.62 | - | - | - | - | |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | - | - | - | - | |
| G2, F & 100 | Mean | 147.99 | 169.09 | 189.20 | 208.40 | 219.71 | 232.75 | 242.92 | 250.24 | 255.91 | 261.80 | 264.47 | 269.04 | 271.85 | 273.76 | - | - | - | |
| ±SD | 9.02 | 11.09 | 17.38 | 20.51 | 20.51 | 23.07 | 26.47 | 27.65 | 27.43 | 30.55 | 30.70 | 30.80 | 30.82 | 30.35 | - | - | - | - | |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | - | - | - | - | |
| G3, F & 300 | Mean | 147.33 | 168.30 | 189.12 | 205.54 | 217.85 | 232.31 | 242.24 | 248.57 | 257.76 | 263.86 | 267.32 | 271.61 | 274.50 | 276.59 | - | - | - | - |
| ±SD | 8.54 | 6.95 | 11.40 | 9.35 | 11.89 | 14.40 | 15.07 | 15.97 | 17.85 | 21.27 | 20.76 | 19.54 | 18.83 | 20.13 | - | - | - | - | |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | - | - | - | - | |
| G4, F & 900 | Mean | 146.45 | 159.92 | 176.56 | 192.83 | 201.83 | 215.89 | 224.34 | 230.98 | 241.38 | 245.25 | 247.23 | 250.37 | 252.59 | 256.03 | - | - | - | |
| ±SD | 9.97 | 12.55 | 16.08 | 18.54 | 21.27 | 23.74 | 25.48 | 25.36 | 27.47 | 32.74 | 31.63 | 32.72 | 32.08 | 33.02 | - | - | - | - | |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | - | - | - | - | |
| G1R, F & 0 | Mean | 145.84 | 165.91 | 182.85 | 199.17 | 121.80 | 228.42 | 237.44 | 246.14 | 253.15 | 259.31 | 261.08 | 264.93 | 270.0 | 272.16 | 276.13 | 277.62 | 279.66 | 282.05 |
| ±SD | 9.33 | 11.56 | 12.69 | 18.71 | 14.05 | 8.76 | 4.06 | 11.66 | 16.09 | 17.33 | 18.04 | 19.04 | 26.09 | 27.29 | 28.12 | 27.79 | 27.20 | 27.63 | |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
| G4R, F & 900 | Mean | 145.15 | 156.99 | 171.33 | 186.34 | 197.56 | 210.93 | 219.88* | 230.47 | 238.77 | 246.66 | 247.56 | 251.14 | 255.86 | 257.93 | 260.29 | 268.88 | 270.27 | 272.29 |
| ±SD | 9.83 | 9.48 | 12.63 | 15.21 | 18.72 | 16.73 | 14.30 | 14.67 | 17.07 | 14.32 | 15.02 | 13.06 | 12.17 | 12.45 | 14.24 | 17.37 | 16.47 | 16.37 | |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
Table 4. Summary of feed consumption (g/rat/day) record
Group, Sex & Dose (mg/kg/day) | Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | |
G1, M & 0 | Mean | 18.19 | 19.61 | 20.12 | 21.38 | 21.88 | 21.56 | 21.24 | 21.45 | 20.90 | 21.22 | 20.47 | 20.17 | 22.32 |
±SD | 1.35 | 1.69 | 2.29 | 2.43 | 3.05 | 3.75 | 3.83 | 4.08 | 4.14 | 3.87 | 3.76 | 3.49 | 2.90 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
G2, M & 100 | Mean | 19.34 | 20.11 | 21.32 | 21.53 | 22.68 | 22.79 | 22.66 | 22.07 | 21.68 | 21.53 | 22.01 | 21.78 | 23.99 |
±SD | 1.78 | 1.04 | 1.10 | 0.67 | 1.88 | 1.10 | 1.06 | 1.35 | 1.61 | 1.29 | 0.74 | 0.98 | 2.03 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
G3, M & 300 | Mean | 19.43 | 17.79 | 22.28 | 24.08 | 24.38 | 24.29 | 24.44 | 24.04 | 22.80 | 23.04 | 22.59 | 22.73 | 24.32 |
±SD | 2.50 | 3.09 | 2.53 | 1.94 | 2.18 | 1.73 | 0.98 | 1.00 | 1.85 | 1.81 | 0.89 | 1.17 | 1.23 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
G4, M & 900 | Mean | 18.39 | 19.35 | 21.16 | 22.36 | 22.49 | 22.27 | 23.41 | 23.62 | 21.12 | 22.82 | 21.93 | 22.30 | 22.83 |
±SD | 1.79 | 1.90 | 1.13 | 1.35 | 1.73 | 2.16 | 1.38 | 1.80 | 2.19 | 2.53 | 2.56 | 2.19 | 1.62 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
Group, Sex & Dose (mg/kg/day) | Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | |
G1, F & 0 | Mean | 15.22 | 15.60 | 15.39 | 15.73 | 16.21 | 16.19 | 15.70 | 14.65 | 14.16 | 14.54 | 13.59 | 13.48 | 14.62 |
±SD | 1.18 | 1.82 | 1.27 | 1.98 | 2.48 | 2.53 | 2.23 | 2.01 | 1.97 | 2.17 | 1.91 | 2.07 | 2.48 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
G2, F & 100 | Mean | 15.74 | 16.19 | 15.77 | 16.03 | 15.51 | 15.29 | 14.84 | 13.73 | 13.53 | 13.536 | 13.57 | 13.98 | 14.48 |
±SD | 1.74 | 1.80 | 2.16 | 2.56 | 2.47 | 2.80 | 2.31 | 1.76 | 2.04 | 2.33 | 2.09 | 1.63 | 2.45 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
G3, F & 300 | Mean | 16.19 | 16.20 | 16.22 | 16.23 | 16.19 | 15.49 | 14.82 | 14.51 | 14.19 | 14.21 | 13.81 | 13.76 | 14.28 |
±SD | 1.03 | 1.41 | 1.37 | 1.66 | 1.56 | 1.47 | 1.45 | 1.48 | 1.06 | 1.37 | 1.18 | 1.04 | 0.86 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
G4, F & 900 | Mean | 14.82 | 15.39 | 14.82 | 14.88 | 15.82 | 15.84 | 15.67 | 15.90 | 15.53 | 15.70 | 15.26 | 15.3. | 15.59 |
±SD | 0.57 | 1.17 | 0.93 | 0.78 | 1.50 | 2.10 | 2.21 | 2.17 | 1.82 | 1.45 | 1.14 | 0.69 | 1.28 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
Group, Sex & Dose (mg/kg/day) | Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | |
G1R, M & 0 | Mean | 17.62 | 19.64 | 19.4619.46 | 21.69 | 20.97 | 22.66 | 23.45 | 23.08 | 23.19 | 23.83 |
±SD | 0.87 | 1.94 | 0.54 | 1.34 | 1.50 | 1.28 | 2.92 | 2.20 | 1.41 | 0.74 | |
n | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | |
G4R, M & 900 | Mean | 18.18 | 18.90 | 20.03 | 21.49 | 21.16 | 22.45 | 22.92 | 22.95 | 22.16 | 22.18 |
±SD | 1.59 | 1.75 | 2.50 | 1.62 | 2.01 | 2.41 | 2.37 | 1.32 | 1.21 | 2.66 | |
n | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | |
G1R, F & 0 | Mean | 14.91 | 15.06 | 14.86 | 15.04 | 15.01 | 15.17 | 14.35 | 13.86 | 13.40 | 14.37 |
±SD | 0.53 | 1.22 | 1.49 | 1.37 | 1.87 | 1.39 | 1.63 | 2.11 | 2.62 | 2.19 | |
n | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | |
G4R, F & 9000 | Mean | 14.73 | 14.91 | 15.61 | 16.34 | 16.96 | 16.91 | 16.07 | 15.66 | 14.93 | 15.50 |
±SD | 1.08 | 0.76 | 0.96 | 1.19 | 1.41 | 1.75 | 1.35 | 1.67 | 1.90 | 1.98 | |
n | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
Group, Sex & Dose (mg/kg/day) | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | |
G1R, M & 0 | Mean | 22.80 | 22.81 | 23.80 | 25.86 | 26.10 | 26.61 | 26.95 |
±SD | 0.93 | 1.56 | 2.10 | 0.53 | 1.91 | 2.05 | 3.54 | |
n | 3 | 3 | 3 | 3 | 3 | 3 | 3 | |
G4R, M & 900 | Mean | 22.71 | 22.64 | 24.06 | 24.84 | 26.55 | 26.86 | 27.49 |
±SD | 3.60 | 3.90 | 3.27 | 1.95 | 1.04 | 1.52 | 1.92 | |
n | 3 | 3 | 3 | 3 | 3 | 3 | 3 | |
G1R, F & 0 | Mean | 13.47 | 15.09 | 16.51 | 17.54 | 18.29 | 18.58 | 19.22 |
±SD | 2.61 | 1.95 | 1.31 | 0.91 | 0.97 | 1.26 | 1.47 | |
n | 3 | 3 | 3 | 3 | 3 | 3 | 3 | |
G4R, F & 900 | Mean | 14.32 | 15.17 | 15.53 | 17.31 | 19.08 | 19.45 | 19.62 |
±SD | 1.54 | 1.93 | 2.57 | 3.83 | 4.14 | 4.06 | 3.43 | |
n | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
Table 5. Summary of neurological/functional observation battery (FOB) record
Week 12 | ||||||
Parameters↓ | Group & Sex | G1 & M | G4 & M | G1 & F | G4 & F | |
Dose (mg/kg/day) | 0 | 900 | 0 | 900 | ||
Number of Animals | 10 | 10 | 10 | 10 | ||
Open field Observation | ||||||
Mobility | 1 | 1 | 1 | 1 | ||
Gait | 1 | 1 | 1 | 1 | ||
Arousal | 3 | 3 | 3 | 3 | ||
Number of Rearing | Mean | 6.1 | 5.8 | 7.6 | 7.3 | |
±SD | 1.4 | 1.5 | 1.7 | 1.7 | ||
Numbers of Urination | Mean | 1.1 | 1.0 | 1.3 | 1.2 | |
±SD | 0.6 | 0.5 | 0.8 | 0.8 | ||
Number of Defecation | Mean | 1.1 | 1.1 | 1.2 | 1.2 | |
±SD | 0.3 | 0.3 | 0.4 | 0.8 | ||
Clonic involuntary movement | 1 | 1 | 1 | 1 | ||
Tonic involuntary movement | 1 | 1 | 1 | 1 | ||
Stereotype Behaviour | 1 | 1 | 1 | 1 | ||
Excessive Grooming | Mean | 3.9 | 4.0 | 4.8 | 4.5 | |
±SD | 0.9 | 0.8 | 0.8 | 1.3 | ||
Sensory Observations | ||||||
Approach Response | 2 | 2 | 2 | 2 | ||
Auditory Response | 2 | 2 | 2 | 2 | ||
Touch Response | 2 | 2 | 2 | 2 | ||
Pupil Reflex | 2 | 2 | 2 | 2 | ||
Tail Pinch Response | 2 | 2 | 2 | 2 | ||
Righting Reflex | 1 | 1 | 1 | 1 | ||
Physiological observation |
| |||||
Body temperature (°F) | Mean | 96.9 | 96.8 | 98.5 | 98.4 | |
±SD | 0.7 | 0.7 | 0.8 | 0.9 | ||
Open field Observation: Mobility - 1=Normal, Gait - 1=Normal, Arousal - 3=Normal, Clonic involuntary movements - 1=None/Normal, Tonic involuntary movements - 1=None/Normal, Stereotype Behaviour - 1=Absent, Sensory Observations: Approach response - 2=Normal, Auditory Response - 2=Normal, Touch Response - 2=Normal, Pupil Reflex - 2=Normal, Tail Pinch Response - 2=Normal, Righting Reflex - 1=Present | ||||||
*: Statistically significant (P<0.05).
Table 6. Summary of actimeter reading record
#Week 12/17 | |||
Group, Sex & Dose (mg/kg/day) | Actimeter Readings | ||
Movement Counts (no.) | |||
G1, M & 0 | Mean | 1669.1 | |
±SD | 169.7 | ||
n | 10 | ||
G4, M & 900 | Mean | 1702.1 | |
±SD | 226.5 | ||
n | 10 | ||
G1, F & 0 | Mean | 1901.4 | |
±SD | 214.5 | ||
n | 10 | ||
G4, F & 900 | Mean | 1950.2 | |
±SD | 248.9 | ||
n | 10 | ||
G1R, M & 0 | Mean | 1685.2 | |
±SD | 144.1 | ||
n | 5 | ||
G4R, M & 900 | Mean | 1715.6 | |
±SD | 153.0 | ||
n | 5 | ||
G1R, F & 0 | Mean | 1881.8 | |
±SD | 202.3 | ||
n | 5 | ||
G4R, F & 900 | Mean | 1982.6 | |
±SD | 176.4 | ||
n | 5 | ||
*: Statistically significant (P<0.05)
#: Week 12 for main group and Week 17 for recovery group.
Table 7. Summary of haematology record
Group, Sex & Dose (mg/kg/day) |
| Total Leucocyte Count | Total Erythrocyte Count | Hemoglobin | Haematocrit | Mean Corpuscular Volume | Mean Corpuscular Hemoglobin | Mean Corpuscular Hemoglobin Concentration |
(WBC) | (RBC) | (HGB) | (HCT) | (MCV) | (MCH) | (MCHC) | ||
(103 cells/µL) | (106 cells/µL) | (g/dL) | (%) | (fL) | (pg) | (g/dL) | ||
G1, M & 0 | Mean | 11.62 | 9.27 | 15.83 | 48.16 | 51.98 | 17.10 | 32.89 |
±SD | 3.86 | 0.32 | 0.79 | 1.56 | 1.77 | 0.74 | 0.86 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G2,M & 100 | Mean | 10.26 | 8.97 | 15.69 | 47.84 | 53.39 | 17.53 | 32.83 |
±SD | 2.16 | 0.42 | 0.43 | 1.74 | 2.07 | 0.70 | 0.89 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G3, M & 300 | Mean | 14.76 | 8.98 | 15.59 | 47.59 | 53.03 | 17.39 | 21.77 |
±SD | 4.87 | 0.24 | 0.74 | 1.83 | 2.49 | 1.02 | 0.82 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G4, M & 900 | Mean | 14.13 | 9.05 | 15.36 | 47.87 | 52.99 | 17.00 | 32.07 |
±SD | 3.95 | 0.74 | 1.08 | 3.32 | 1.66 | 0.65 | 0.62 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G1R, M & 0 | Mean | 10.89 | 9.40 | 15.80 | 49.02 | 52.12 | 16.80 | 32.24 |
±SD | 2.52 | 0.51 | 1.08 | 3.46 | 1.24 | 0.75 | 1.35 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
G4R, M & 900 | Mean | 12.79 | 8.84 | 15.36 | 47.86 | 54.26 | 17.44 | 32.14 |
±SD | 2.64 | 0.58 | 0.75 | 1.92 | 2.13 | 0.46 | 0.58 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
Group, Sex & Dose (mg/kg/day) |
| Platelet Count | Mean Platelet Volume | Reticulocyte Count | Neutrophils | Lymphocytes | Monocytes | Eosinophils | Basophils |
(PLT) | (MPV) | (Retic) | (Neut) | (Lymph) | (Mono) | (Eos) | (Baso) | ||
(103 cells/µL) | (fL) | (%) | (%) | (%) | (%) | (%) | (%) | ||
G1, M & 0 | Mean | 826.00 | 8.19 | 2.45 | 26.63 | 67.05 | 2.82 | 1.84 | 0.40 |
±SD | 97.94 | 0.70 | 0.37 | 7.75 | 7.92 | 1.64 | 1.83 | 0.17 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G2, M & 100 | Mean | 1023.60* | 7.42* | 2.36 | 25.29 | 68.70 | 3.39 | 1.27 | 0.27* |
±SD | 137.76 | 0.20 | 0.37 | 5.85 | 5.28 | 1.17 | 0.54 | 0.05 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G3, M & 300 | Mean | 1000.60 | 7.50* | 2.27 | 21.96 | 72.11 | 3.52 | 1.05 | 0.29 |
±SD | 189.67 | 0.37 | 0.55 | 5.48 | 5.97 | 0.61 | 0.38 | 0.09 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G4, M & 900 | Mean | 973.0 | 7.24* | 2.11 | 25.84 | 67.93 | 3.84 | 0.93 | 0.26* |
±SD | 222.22 | 0.22 | 0.66 | 9.76 | 9.47 | 1.01 | 0.35 | 0.10 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G1R, M & 0 | Mean | 977.00 | 7.90 | .254 | 27.44 | 67.28 | 2.54 | 1.64 | 0.30 |
±SD | 151.27 | 0.29 | 0.97 | 6.33 | 7.38 | 0.68 | 0.71 | 0.07 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
G4R, M & 900 | Mean | 905.60 | 7.82 | 2.58 | 23.14 | 70.94 | 3.14 | 1.24 | 0.32 |
±SD | 145.96 | 0.26 | 0.69 | 4.85 | 4.34 | 1.23 | 0.23 | 0.04 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
Group, Sex & Dose (mg/kg/day) |
| Absolute Reticulocyte Count | Absolute Neutrophils | Absolute Lymphocytes | Absolute Monocytes | Absolute Eosinophils | Absolute Basophils | Prothrombin Time | Activated Prothrombin Time |
(Retic) | (Neut) | (Lymph) | (Mono) | (Eos) | (Baso) | (PT) | (APTT) | ||
(109 cells/L) | (103 cells/µL) | (103 cells/µL) | (103 cells/µL) | (103 cells/µL) | (103 cells/µL) | (Seconds) | (Seconds) | ||
G1, M & 0 | Mean | 226.78 | 2.86 | 8.01 | 0.33 | 0.20 | 0.05 | 18.03 | 25.36 |
±SD | 30.93 | 0.62 | 3.65 | 0.21 | 0.21 | 0.04 | 1.78 | 4.91 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G2, M & 100 | Mean | 211.40 | 2.56 | 7.08 | 0.35 | 0.13 | 0.03 | 16.82 | 25.88 |
±SD | 32.0 | 0.63 | 1.76 | 0.17 | 0.07 | 0.01 | 1.09 | 3.12 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G3, M & 300 | Mean | 203.53 | 3.18 | 10.68 | 0.53 | 0.16 | 0.05 | 16.99 | 21.33 |
±SD | 49.03 | 1.33 | 3.81 | 0.22 | 0.10 | 0.03 | 1.36 | 4.0 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G4, M & 900 | Mean | 187.07 | 3.54 | 9.73 | 0.53 | 0.12 | 0.04 | 16.84 | 23.07 |
±SD | 46.42 | 1.45 | 3.70 | 0.15 | 0.04 | 0.02 | 0.93 | 4.22 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G1R, M & 0 | Mean | 238.50 | 2.98 | 7.33 | 0.28 | 0.18 | 0.04 | 18.90 | 19.20 |
±SD | 93.33 | 1.06 | 1.85 | 0.10 | 0.11 | 0.01 | 1.46 | 1.02 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
G4R, M & 900 | Mean | 226.12 | 2.91 | 9.13 | 0.39 | 0.15 | 0.04 | 17.42 | 24.76 |
±SD | 52.59 | 0.66 | 2.37 | 0.146 | 0.03 | 0.01 | 1.01 | 5.92 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
Group, Sex & Dose (mg/kg/day) |
| Total Leucocyte Count | Total Erythrocyte Count | Hemoglobin | Haematocrit | Mean Corpuscular Volume | Mean Corpuscular Hemoglobin | Mean Corpuscular Hemoglobin Concentration |
(WBC) | (RBC) | (HGB) | (HCT) | (MCV) | (MCH) | (MCHC) | ||
(103 cells/µL) | (106 cells/µL) | (g/dL) | (%) | (fL) | (pg) | (g/dL) | ||
G1, F & 0 | Mean | 9.11 | 8.57 | 15.18 | 45.97 | 53.71 | 17.73 | 33.03 |
±SD | 2.30 | 0.59 | 0.75 | 2.21 | 1.68 | 0.65 | 0.38 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G2, F & 100 | Mean | 11.09 | 8.34 | 15.35 | 46.61 | 55.92 | 18.40 | 32.91 |
±SD | 2.94 | 0.40 | 0.71 | 2.14 | 1.78 | 0.64 | 0.35 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G3, F & 300 | Mean | 11.12 | 7.98* | 14.21* | 43.87 | 54.98 | 17.80 | 32.36* |
±SD | 3.57 | 0.40 | 1.03 | 2.96 | 2.95 | 1.12 | 0.43 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G4, F & 900 | Mean | 10.62 | 8.40 | 14.82 | 45.80 | 54.61 | 17.66 | 32.38* |
±SD | 3.13 | 0.50 | 0.54 | 2.08 | 2.75 | 0.77 | 0.63 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G1R, F & 0 | Mean | 9.14 | 8.36 | 14.72 | 44.76 | 53.56 | 17.62 | 32.92 |
±SD | 1.23 | 0.34 | 0.34 | 1.63 | 2.51 | 0.58 | 0.68 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
G4R, F & 900 | Mean | 11.48 | 8.37 | 15.38 | 45.86 | 55.24 | 18.54* | 33.58 |
±SD | 3.75 | 0.37 | 0.65 | 2.56 | 1.10 | 0.63 | 1.00 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
Group, Sex & Dose (mg/kg/day) |
| Platelet Count | Mean Platelet Volume | Reticulocyte Count | Neutrophils | Lymphocytes | Monocytes | Eosinophils | Basophils |
(PLT) | (MPV) | (Retic) | (Neut) | (Lymph) | (Mono) | (Eos) | (Baso) | ||
(103 cells/µL) | (fL) | (%) | (%) | (%) | (%) | (%) | (%) | ||
G1, F & 0 | Mean | 975.80 | 7.64 | 2.68 | 24.93 | 69.54 | 2.74 | 1.54 | 0.34 |
±SD | 239.56 | 0.85 | 0.67 | 6.11 | 6.36 | 0.71 | 0.70 | 0.28 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G2, F & 100 | Mean | 886.30 | 7.92 | 2.91 | 27.11 | 65.39 | 3.65 | 2.38 | 0.28 |
±SD | 170.70 | 0.70 | 0.56 | 11.05 | 16.71 | 1.78 | 4.10 | 0.08 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G3, F & 300 | Mean | 991.00 | 8.08 | 3.20 | 16.80* | 76.53 | 1.77 | 0.94 | 0.28 |
±SD | 172.43 | 0.20 | 1.00 | 3.63 | 10.34 | 1.15 | 0.71 | 0.09 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G4, F & 900 | Mean | 915.0 | 8.67* | 2.57 | 21.48 | 73.31 | 3.12 | 1.04 | 0.21 |
±SD | 225.09 | 1.17 | 0.84 | 4.29 | 5.50 | 0.97 | 0.71 | 0.07 | |
n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | |
G1R, F & 0 | Mean | 889.40 | 8.32 | 3.06 | 19.16 | 73.94 | 2.52 | 2.80 | 0.38 |
±SD | 322.60 | 0.54 | 0.64 | 5.73 | 8.44 | 0.93 | 2.30 | 0.11 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | |
G4R, F & 900 | Mean | 882.40 | 7.92 | 2.76 | 25.62 | 68.74 | 3.18 | 1.20 | 0.24* |
±SD | 215.60 | 0.22 | 0.79 | 4.38 | 5.03 | 1.31 | 0.72 | 0.05 | |
n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
*: Statistically significant (P<0.05).
Table 8. Summary of clinical chemistry record
Group, Sex & Dose (mg/kg/day) |
|
Glucose |
Urea |
Creatinine | Total Cholesterol |
Triglycerides | High- density lipoprotein | Low- density lipoprotein | Total Protein |
Albumin | Alanine aminotransferase | Aspartate aminotransferase |
| (GLU) |
| (CRE) | (CHO) | (TRI) | (HDL) | (LDL) | (TPR) | (ALB) | (ALT) | (AST) | |
|
| (mg/dL) | (mg/dL) | (mg/dL) | (mg/dL) | (mg/dL) | (mg/dL) | (mg/dL) | g/dL) | (g/dL) | (U/L) | (U/L) |
| Mean | 91.20 | 26.76 | 0.58 | 51.10 | 55.80 | 11.90 | 10.51 | 6.79 | 3.32 | 69.00 | 125.50 |
G1, M & 0 | ±SD | 5.07 | 2.72 | 0.04 | 4.77 | 17.62 | 2.23 | 1.95 | 0.27 | 0.20 | 16.31 | 25.54 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 99.20 | 25.42 | 0.55 | 54.60 | 56.70 | 13.40 | 10.35 | 6.62 | 3.34 | 62.10 | 116.90 |
G2, M & 100 | ±SD | 12.47 | 4.82 | 0.03 | 10.44 | 17.68 | 1.86 | 2.48 | 0.11 | 0.10 | 13.76 | 15.78 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 85.10 | 27.36 | 0.58 | 56.30 | 68.90 | 13.88 | 11.29 | 6.80 | 3.39 | 66.40 | 124.90 |
G3, M & 300 | ±SD | 6.23 | 6.81 | 0.05 | 11.01 | 34.88 | 2.87 | 2.40 | 0.22 | 0.14 | 12.95 | 15.98 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 80.50* | 24.75 | 0.54 | 53.30 | 53.80 | 15.70* | 8.74 | 6.79 | 3.37 | 113.70 | 140.40 |
G4, M & 900 | ±SD | 7.86 | 5.06 | 0.02 | 10.67 | 16.29 | 2.27 | 2.46 | 0.32 | 0.21 | 130.47 | 90.92 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 120.40 | 34.64 | 0.54 | 50.00 | 36.60 | 12.76 | 7.04 | 6.96 | 3.21 | 81.00 | 138.60 |
G1R, M & 0 | ±SD | 14.64 | 4.45 | 0.04 | 4.47 | 11.10 | 1.32 | 1.87 | 0.21 | 0.23 | 11.64 | 22.03 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| Mean | 122.20 | 31.44 | 0.50 | 48.00 | 39.00 | 12.46 | 5.34 | 6.42 | 3.38 | 52.80* | 116.20 |
G4R, M & 900 | ±SD | 11.52 | 8.00 | 0.03 | 9.41 | 11.85 | 1.78 | 1.68 | 0.50 | 0.22 | 5.89 | 13.75 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
Group, Sex & Dose (mg/kg/day) |
| Alkaline phosphatase | Total Bilirubin |
Calcium |
Phosphorous |
Cholinesterase |
Globulin | Albumin/ Globulin ratio | Blood Urea Nitrogen |
Sodium |
Potassium |
Chloride |
| (ALP) | (BIT) | (CAL) | (PHO) | (CHE) | (GLO) | (A/G Ratio) | (BUN) | (Na) | (K) | (CLO) | |
|
| (U/L) | (mg/dL) | (mg/dL) | (mg/dL) | (U/L) | (g/dL) |
| (mg/dL) | (mmol/L) | (mmol/L) | (mmol/L) |
| Mean | 324.10 | 0.07 | 10.04 | 4.91 | 115.70 | 3.47 | 0.97 | 12.51 | 142.04 | 3.30 | 108.95 |
G1, M & 0 | ±SD | 240.83 | 0.01 | 0.22 | 0.41 | 58.97 | 0.36 | 0.14 | 1.27 | 0.94 | 0.33 | 1.46 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 248.80 | 0.06 | 10.09 | 4.58 | 146.40 | 3.28 | 1.02 | 11.88 | 141.58 | 3.49 | 108.00 |
G2, M & 100 | ±SD | 69.24 | 0.02 | 0.23 | 0.31 | 72.11 | 0.10 | 0.05 | 2.25 | 1.21 | 0.19 | 1.56 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 294.80 | 0.07 | 10.24 | 5.09 | 146.40 | 3.41 | 1.00 | 12.79 | 140.69 | 3.50 | 106.09* |
G3, M & 300 | ±SD | 115.49 | 0.02 | 0.26 | 0.39 | 40.50 | 0.17 | 0.07 | 3.18 | 1.42 | 0.28 | 1.18 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 207.90 | 0.08 | 10.30 | 5.27 | 103.80 | 3.42 | 1.00 | 11.57 | 140.34* | 3.55 | 105.76* |
G4, M & 900 | ±SD | 81.85 | 0.02 | 0.28 | 0.68 | 20.02 | 0.31 | 0.12 | 2.36 | 1.37 | 0.32 | 2.04 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 172.40 | 0.09 | 10.18 | 4.93 | 96.20 | 3.75 | 0.86 | 16.19 | 143.62 | 3.56 | 110.52 |
G1R, M & 0 | ±SD | 77.00 | 0.02 | 0.19 | 0.38 | 12.03 | 0.27 | 0.11 | 2.08 | 3.44 | 0.34 | 2.17 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| Mean | 112.40 | 0.08 | 10.18 | 5.70* | 81.60 | 3.04* | 1.13* | 14.69 | 141.62 | 3.48 | 108.04 |
G4R, M & 900 | ±SD | 26.26 | 0.01 | 0.15 | 0.46 | 20.23 | 0.39 | 0.15 | 3.74 | 1.99 | 0.10 | 1.71 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
Group, Sex & Dose (mg/kg/day) |
|
Glucose |
Urea |
Creatinine | Total Cholesterol |
Triglycerides | High- density lipoprotein | Low- density lipoprotein | Total Protein |
Albumin | Alanine aminotransferase | Aspartate aminotransferase |
| (GLU) |
| (CRE) | (CHO) | (TRI) | (HDL) | (LDL) | (TPR) | (ALB) | (ALT) | (AST) | |
|
| (mg/dL) | (mg/dL) | (mg/dL) | (mg/dL) | (mg/dL) | (mg/dL) | (mg/dL) | (g/dL) | (g/dL) | (U/L) | (U/L) |
| Mean | 122.40 | 28.56 | 0.60 | 51.10 | 36.90 | 14.81 | 6.32 | 6.91 | 3.59 | 45.90 | 100.50 |
G1, F & 0 | ±SD | 14.84 | 4.62 | 0.02 | 5.30 | 6.31 | 1.77 | 1.21 | 0.30 | 0.19 | 6.64 | 11.10 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 111.80 | 29.31 | 0.58 | 55.60 | 40.90 | 16.62 | 6.29 | 7.07 | 3.54 | 49.60 | 110.90 |
G2, F & 100 | ±SD | 9.33 | 5.28 | 0.03 | 6.95 | 22.43 | 1.81 | 0.85 | 0.41 | 0.24 | 9.40 | 9.94 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 119.60 | 29.85 | 0.61 | 64.70* | 48.90 | 19.96* | 6.10 | 6.71 | 3.55 | 46.40 | 104.60 |
G3, F & 300 | ±SD | 20.59 | 5.11 | 0.07 | 10.31 | 18.41 | 2.78 | 0.88 | 0.50 | 0.28 | 10.45 | 13.63 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 106.50 | 31.37 | 0.60 | 84.00* | 47.00 | 25.36* | 7.91* | 6.99 | 3.45 | 59.30* | 109.80 |
G4, F & 900 | ±SD | 13.84 | 8.29 | 0.06 | 17.52 | 16.64 | 3.10 | 1.53 | 0.27 | 0.22 | 15.69 | 17.29 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 114.20 | 37.42 | 0.59 | 69.40 | 51.20 | 18.14 | 4.60 | 7.30 | 3.47 | 80.20 | 146.80 |
G1R, F & 0 | ±SD | 14.34 | 3.01 | 0.03 | 12.99 | 12.54 | 3.21 | 1.30 | 0.35 | 0.16 | 54.93 | 70.32 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| Mean | 118.20 | 35.82 | 0.60 | 72.80 | 52.40 | 19.64 | 3.42 | 7.16 | 3.50 | 59.00 | 109.40 |
G4R, F & 900 | ±SD | 13.44 | 5.29 | 0.03 | 8.41 | 18.65 | 1.34 | 1.39 | 0.42 | 0.20 | 30.32 | 29.37 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5
|
Group, Sex & Dose (mg/kg/day) |
| Alkaline phosphatase | Total Bilirubin |
Calcium |
Phosphorous |
Cholinesterase |
Globulin | Albumin/ Globulin ratio | Blood Urea Nitrogen |
Sodium |
Potassium |
Chloride |
| (ALP) | (BIT) | (CAL) | (PHO) | (CHE) | (GLO) | (A/G Ratio) | (BUN) | (Na) | (K) | (CLO) | |
|
| (U/L) | (mg/dL) | (mg/dL) | (mg/dL) | (U/L) | (g/dL) |
| (mg/dL) | mmol/L) | (mmol/L) | mmol/L) |
| Mean | 187.20 | 0.07 | 10.31 | 4.40 | 643.90 | 3.32 | 1.09 | 13.35 | 139.15 | 3.38 | 105.93 |
G1, F & 0 | ±SD | 152.67 | 0.01 | 0.26 | 0.56 | 294.30 | 0.25 | 0.10 | 2.16 | 2.46 | 0.34 | 2.15 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 197.00 | 0.05 | 10.35 | 4.80 | 692.80 | 3.54 | 1.01 | 13.70 | 140.50 | 3.30 | 107.24 |
G2, F & 100 | ±SD | 144.17 | 0.02 | 0.35 | 0.66 | 253.73 | 0.34 | 0.11 | 2.47 | 1.45 | 0.28 | 1.36 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 222.80 | 0.07 | 10.14 | 5.43 | 723.30 | 3.16 | 1.13 | 13.95 | 140.06 | 3.52 | 106.20 |
G3, F & 300 | ±SD | 157.51 | 0.03 | 0.35 | 2.09 | 212.96 | 0.28 | 0.09 | 2.39 | 0.69 | 0.68 | 1.76 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 184.40 | 0.09 | 10.04 | 4.85 | 572.50 | 3.54 | 0.98 | 14.66 | 138.55 | 3.59 | 105.14 |
G4, F & 900 | ±SD | 75.46 | 0.02 | 0.33 | 0.72 | 202.47 | 0.29 | 0.12 | 3.87 | 1.44 | 0.33 | 2.22 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 109.20 | 0.07 | 10.44 | 3.77 | 805.80 | 3.83 | 0.91 | 17.48 | 143.56 | 3.55 | 110.96 |
G1R, F & 0 | ±SD | 30.66 | 0.04 | 0.27 | 0.65 | 369.28 | 0.43 | 0.12 | 1.41 | 5.99 | 0.21 | 5.81 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| Mean | 66.60* | 0.05 | 10.32 | 4.21 | 609.40 | 3.66 | 0.96 | 16.74 | 141.04 | 3.54 | 108.68 |
G4R, F & 900 | ±SD | 18.04 | 0.04 | 0.16 | 0.39 | 159.80 | 0.29 | 0.08 | 2.47 | 3.37 | 0.23 | 4.24 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
*: Statistically significant (P<0.05).
Table 9. Summary of urinalysis record
Examination | Group & Sex | G1 & M | G2 & M | G3 & M | G4 & M | G1R & M | G4R & M | |
Dose (mg/kg/day) | 0 | 100 | 300 | 900 | 0 | 900 | ||
Number of Animals | 10 | 10 | 10 | 10 | 5 | 5 | ||
Physical |
Colour | Pale Yellow | 7 | 8 | 9 | 10 | 5 | 3 |
Yellow | 3 | 2 | 1 | - | - | 2 | ||
Appearance | Clear | 5 | 7 | 6 | 8 | 3 | 4 | |
Turbid | 5 | 3 | 4 | 2 | 2 | 1 | ||
Volume (mL) | Mean | 3.7 | 5.2* | 6.0* | 4.8* | 5.4 | 4.2 | |
±SD | 0.9 | 0.9 | 0.8 | 1.0 | 0.8 | 1.1 | ||
Chemical | pH | Mean | 5.9 | 6.3 | 6.1 | 6.6 | 7.0 | 6.9 |
±SD | 0.3 | 0.7 | 0.8 | 1.0 | 0.4 | 0.2 | ||
Specific Gravity | Mean | 1.028 | 1.025 | 1.026 | 1.022 | 1.019 | 1.022 | |
±SD | 0.003 | 0.007 | 0.006 | 0.009 | 0.004 | 0.003 | ||
Urobilinogen (mg/dL) | Mean | 0.3 | 0.6 | 0.6 | 0.4 | 0.2 | 0.2 | |
±SD | 0.3 | 0.4 | 0.4 | 0.3 | 0.0 | 0.0 | ||
Bilirubin (mg/dL) | Neg | 4 | 9 | 10 | 10 | 5 | 5 | |
1 | 6 | 1 | - | - | - | - | ||
Ketones (mg/dL) | 5 | 10 | 10 | 10 | 9 | 5 | 4 | |
Neg | - | - | - | 1 | - | 1 | ||
Blood (Ery/µL) | Neg | 8 | 8 | 6 | 8 | 3 | - | |
Ca10 | - | - | 2 | 2 | 1 | 1 | ||
Ca25 | - | 2 | 2 | - | 1 | 1 | ||
Ca80 | - | - | - | - | - | 3 | ||
>=Ca200 | 2 | - | - | - | - | - | ||
Proteins (mg/dL) | Neg | 9 | 9 | 8 | 8 | 4 | 3 | |
Trace | 1 | 1 | 2 | 2 | 1 | 2 | ||
Nitrite | Neg | 2 | 6 | 6 | 5 | 2 | 4 | |
Pos | 8 | 4 | 4 | 5 | 3 | 1 | ||
Leucocytes (Leu/µL) | Neg | - | - | - | - | - | 1 | |
Ca70 | 8 | 7 | 9 | 6 | 2 | 3 | ||
Ca15 | 2 | 3 | 1 | 2 | 1 | 1 | ||
Ca125 | - | - | - | 2 | 2 | - | ||
Glucose (mg/dL) | Neg | 10 | 10 | 10 | 10 | 5 | 5 | |
Micro albumin (MALB) (mg/dL) | Neg | - | - | - | - | 4 | 3 | |
15 | 9 | 9 | 8 | 8 | - | - | ||
>15 | 1 | 1 | 2 | 2 | 1 | 2 | ||
Microscopic | Epithelial cells | 0 | 9 | 10 | 9 | 7 | 5 | 4 |
0-1 | 1 | - | 1 | 3 | - | 1 | ||
Casts | Absent | 10 | 10 | 10 | 10 | 5 | 5 | |
Crystals | Present | 6 | 9 | 8 | 9 | 4 | 4 | |
Absent | 4 | 1 | 2 | 1 | 1 | 1 | ||
M: Male; R: Recovery; Neg: Negative; Ca: Calculated Approximately; Pos: Positive; SD: Standard Deviation;
>: more than; >=: more than equal to; *: Statistically significant (P<0.05).
Examination | Group & Sex | G1 & F | G2 & F | G3 & F | G4 & F | G1R & F | G4R & F | |
Dose (mg/kg/day) | 0 | 100 | 300 | 900 | 0 | 900 | ||
Number of Animals | 10 | 10 | 10 | 10 | 5 | 5 | ||
Physical |
Colour | Pale Yellow | 7 | 9 | 9 | 8 | 4 | 4 |
Yellow | 3 | 1 | 1 | 2 | 1 | 1 | ||
Appearance | Clear | 7 | 7 | 7 | 7 | 2 | 4 | |
Turbid | 3 | 3 | 3 | 3 | 3 | 1 | ||
Volume (mL) | Mean | 5.4 | 5.1 | 5.2 | 5.2 | 4.1 | 5.8 | |
±SD | 1.1 | 1.1 | 1.0 | 1.0 | 1.5 | 0.8 | ||
Chemical |
pH | Mean | 6.8 | 6.0* | 5.8* | 5.7* | 7.5 | 7.4 |
±SD | 1.3 | 0.3 | 0.3 | 0.3 | 0.9 | 0.7 | ||
Specific Gravity | Mean | 1.018 | 1.025 | 1.029* | 1.027* | 1.011 | 1.011 | |
±SD | 0.011 | 0.007 | 0.002 | 0.003 | 0.008 | 0.007 | ||
Urobilinogen (mg/dL) | Mean | 0.7 | 0.5 | 0.5 | 0.3 | 0.2 | 0.2 | |
±SD | 0.7 | 0.4 | 0.4 | 0.3 | 0.0 | 0.0 | ||
Bilirubin (mg/dL) | Neg | 6 | 10 | 9 | 9 | 5 | 5 | |
1 | 3 | - | 1 | 1 | - | - | ||
3 | 1 | - | - | - | - | - | ||
Ketones (mg/dL) | 5 | - | 5 | 9 | 9 | - | - | |
Neg | 10 | 5 | 1 | 1 | 5 | 5 | ||
Blood (Ery/µL) | Neg | 10 | 10 | 10 | 9 | 4 | 3 | |
Ca10 | - | - | - | - | - | 1 | ||
Ca25 | - | - | - | 1 | 1 | - | ||
>=Ca200 | - | - | - | - | - | 1 | ||
Proteins (mg/dL) | Neg | 6 | 10 | 9 | 10 | 4 | 5 | |
Trace | - | - | 1 | - | 1 | - | ||
>=300 | 4 | - | - | - | - | - | ||
Nitrite | Neg | - | 7 | 6 | 6 | 3 | 4 | |
Pos | 10 | 3 | 4 | 4 | 2 | 1 | ||
Leucocytes (Leu/µL) | Neg | 5 | 4 | 7 | 5 | 5 | 2 | |
Ca70 | 4 | - | 2 | - | - | 1 | ||
Ca15 | 1 | 6 | 1 | 5 | - | 2 | ||
Glucose (mg/dL) | Neg | 10 | 10 | 10 | 10 | 5 | 5 | |
Micro albumin (MALB) (mg/dL) | Neg | - | 3 | - | - | 4 | 5 | |
15 | 6 | 7 | 9 | 10 | - | - | ||
>15 | 4 | - | 1 | - | 1 | - | ||
Microscopic |
Epithelial cells | 0 | 9 | 8 | 7 | 8 | 4 | 4 |
0-1 | 1 | 2 | 2 | 2 | 1 | 1 | ||
1-2 | - | - | 1 | - | - | - | ||
Casts | Absent | 10 | 8 | 9 | 10 | 5 | 5 | |
Present | - | 2 | 1 | - | - | - | ||
Crystals | Present | 9 | 8 | 8 | 8 | 2 | 4 | |
Absent | 1 | 2 | 2 | 2 | 3 | 1 | ||
F: Female; R: Recovery; Neg: Negative; Ca: Calculated Approximately; Pos: Positive; SD: Standard Deviation;
>: more than; >=: more than equal to; *: Statistically significant (P<0.05).
Table 10. Summary of absolute organ weights (g) record
Group, Sex & Dose (mg/kg/day) |
|
Adrenals |
Thymus |
Spleen |
Testes |
Epididymides |
Heart |
Kidneys |
Brain |
Liver |
Lungs | Prostate+Seminal vesicles with coagulating glands |
Pituitary gland | Thyroid along with parathyroid |
| Mean | 0.0717 | 0.3237 | 1.1281 | 3.3101 | 1.4224 | 1.3720 | 2.6351 | 1.9898 | 10.5653 | 2.5310 | 3.3263 | 0.0124 | 0.0246 |
G1, M & 0 | ±SD | 0.0132 | 0.0978 | 0.2560 | 0.6579 | 0.2555 | 0.1965 | 0.4921 | 0.1291 | 1.6980 | 0.5450 | 0.4602 | 0.0020 | 0.0041 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 0.0685 | 0.3284 | 1.1589 | 3.8643 | 1.6817* | 1.6379* | 3.1053* | 2.0522 | 13.1887* | 2.4943 | 3.7447 | 0.0131 | 0.0289 |
G2, M & 100 | ±SD | 0.0141 | 0.1375 | 0.2474 | 0.6155 | 0.1984 | 0.1830 | 0.3266 | 0.1282 | 1.5662 | 0.4500 | 0.4570 | 0.0015 | 0.0052 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 0.0673 | 0.3548 | 1.0815 | 3.6159 | 1.5958 | 1.4890 | 3.1387* | 2.1144 | 14.0619* | 2.6114 | 3.6953 | 0.0141 | 0.0269 |
G3, M & 300 | ±SD | 0.0100 | 0.1032 | 0.4396 | 0.2127 | 0.1332 | 0.1296 | 0.3314 | 0.1315 | 1.4380 | 0.3926 | 0.3080 | 0.0027 | 0.0058 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 0.0701 | 0.3205 | 1.1086 | 3.6564 | 1.6070 | 1.4488 | 3.1079* | 2.1063 | 15.4205* | 2.4722 | 3.2250 | 0.0134 | 0.0300 |
G4, M & 900 | ±SD | 0.0113 | 0.1332 | 0.3081 | 0.5840 | 0.2112 | 0.0937 | 0.4666 | 0.1174 | 1.6603 | 0.3768 | 0.4481 | 0.0034 | 0.0057 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 0.0752 | 0.2550 | 1.1777 | 3.6784 | 1.5221 | 1.5348 | 2.8835 | 2.0108 | 11.3269 | 2.4340 | 3.2613 | 0.0116 | 0.0239 |
G1R, M & 0 | ±SD | 0.0051 | 0.0411 | 0.2236 | 0.1838 | 0.0933 | 0.1511 | 0.3292 | 0.0414 | 1.3938 | 0.4550 | 0.2542 | 0.0012 | 0.0047 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| Mean | 0.0667 | 0.3660 | 1.0481 | 3.5028 | 1.7419 | 1.6061 | 3.0824 | 2.0967 | 12.7888 | 2.0015 | 3.2248 | 0.0121 | 0.0200 |
G4R, M & 900 | ±SD | 0.0147 | 0.1115 | 0.2485 | 0.1594 | 0.2594 | 0.1498 | 0.2825 | 0.0760 | 1.0952 | 0.1319 | 0.3587 | 0.0013 | 0.0030 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (P<0.05)
Group, Sex & Dose (mg/kg/day) |
|
Adrenals |
Thymus |
Spleen | Ovaries with oviduct | Uterus with cervix |
Heart |
Kidneys |
Brain |
Liver |
Lungs |
Pituitary gland | Thyroid along with parathyroid |
| Mean | 0.0853 | 0.3221 | 0.8847 | 0.1586 | 0.6965 | 1.1095 | 1.7353 | 2.0057 | 8.4037 | 2.1134 | 0.0143 | 0.0264 |
G1, F & 0 | ±SD | 0.0188 | 0.0872 | 0.2711 | 0.0387 | 0.1812 | 0.2229 | 0.2357 | 0.1300 | 1.2470 | 0.4321 | 0.0031 | 0.0033 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 0.0791 | 0.3647 | 0.9061 | 0.1493 | 0.6671 | 1.0395 | 1.7015 | 1.9991 | 8.4241 | 2.1260 | 0.0155 | 0.0251 |
G2, F & 100 | ±SD | 0.0160 | 0.0979 | 0.1928 | 0.0357 | 0.2512 | 0.1344 | 0.3592 | 0.1193 | 0.9087 | 0.2894 | 0.0034 | 0.0058 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 0.0833 | 0.3377 | 0.9809 | 0.1492 | 0.6918 | 1.0748 | 1.8992 | 2.0433 | 10.2615* | 2.1953 | 0.0151 | 0.0257 |
G3, F & 300 | ±SD | 0.0158 | 0.0800 | 0.1810 | 0.0273 | 0.1529 | 0.1113 | 0.2359 | 0.1316 | 1.0587 | 0.4226 | 0.0024 | 0.0047 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 0.0789 | 0.2534 | 0.9143 | 0.1431 | 0.5492 | 0.9355* | 1.7696 | 1.8469* | 10.9091* | 2.0295 | 0.0126 | 0.0273 |
G4, F & 900 | ±SD | 0.0203 | 0.0523 | 0.2757 | 0.0264 | 0.1707 | 0.1196 | 0.2817 | 0.1546 | 2.2129 | 0.4828 | 0.0019 | 0.0044 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 0.0701 | 0.2300 | 0.9156 | 0.1311 | 0.7088 | 0.9873 | 1.7486 | 1.8492 | 8.2091 | 1.9013 | 0.0164 | 0.0195 |
G1R, F & 0 | ±SD | 0.0163 | 0.0765 | 0.1111 | 0.0133 | 0.1404 | 0.1961 | 0.1796 | 0.0865 | 1.7489 | 0.3388 | 0.0024 | 0.0024 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| Mean | 0.0745 | 0.3067 | 0.7530* | 0.1453 | 0.6207 | 1.1506 | 1.6861 | 1.9187 | 8.4539 | 2.1402 | 0.0149 | 0.0236 |
G4R, F & 900 | ±SD | 0.0029 | 0.0412 | 0.0949 | 0.0118 | 0.1972 | 0.1326 | 0.1372 | 0.0923 | 0.8259 | 0.4407 | 0.0019 | 0.0039 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (P<0.05).
Table 11. Summary of organ weight relative to fasting body weight (%) record
Group, Sex & Dose (mg/kg/day) |
|
Fasting Body Weight (g) |
Adrenals |
Thymus |
Spleen |
Testes |
Epididymides |
Heart |
Kidneys |
Brain |
Liver |
Lungs |
Prostate+Seminal vesicles with coagulating glands |
Pituitary gland |
Thyroid along with parathyroid |
| Mean | 373.70 | 0.0194 | 0.0863 | 0.3036 | 0.8957 | 0.3847 | 0.3684 | 0.7061 | 0.5413 | 2.8264 | 0.6873 | 0.8966 | 0.0034 | 0.0066 |
G1, M & 0 | ±SD | 57.68 | 0.0038 | 0.0224 | 0.0612 | 0.1672 | 0.0644 | 0.0274 | 0.0886 | 0.0682 | 0.1154 | 0.1678 | 0.0932 | 0.0007 | 0.0010 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 417.39 | 0.0163 | 0.0777 | 0.2781 | 0.9323 | 0.4047 | 0.3927 | 0.7460 | 0.4939 | 3.1589* | 0.6014 | 0.9024 | 0.0031 | 0.0069 |
G2, M & 100 | ±SD | 37.60 | 0.0024 | 0.0284 | 0.0553 | 0.1715 | 0.0509 | 0.0319 | 0.0740 | 0.0378 | 0.2319 | 0.1134 | 0.1226 | 0.0004 | 0.0012 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 403.74 | 0.0167 | 0.0884 | 0.2711 | 0.9003 | 0.3966 | 0.3697 | 0.7778 | 0.5246 | 3.4867* | 0.6494 | 0.9195 | 0.0035 | 0.0067 |
G3, M & 300 | ±SD | 28.24 | 0.0027 | 0.0258 | 0.1154 | 0.0914 | 0.0388 | 0.0339 | 0.0678 | 0.0282 | 0.3181 | 0.1049 | 0.1025 | 0.0007 | 0.0014 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 371.81 | 0.0188 | 0.0871 | 0.2978 | 0.9920 | 0.4346 | 0.3909 | 0.8353* | 0.5685 | 4.1527* | 0.6673 | 0.8708 | 0.0036 | 0.0081* |
G4, M & 900 | ±SD | 28.81 | 0.0024 | 0.0401 | 0.0749 | 0.1876 | 0.0673 | 0.0277 | 0.0993 | 0.0399 | 0.3857 | 0.1067 | 0.1282 | 0.0008 | 0.0015 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 418.85 | 0.0180 | 0.0611 | 0.2815 | 0.8791 | 0.3637 | 0.3661 | 0.6884 | 0.4809 | 2.6967 | 0.5852 | 0.7789 | 0.0028 | 0.0057 |
G1R, M & 0 | ±SD | 22.82 | 0.0012 | 0.0107 | 0.0548 | 0.0399 | 0.0208 | 0.0261 | 0.0717 | 0.0194 | 0.1989 | 0.1338 | 0.0510 | 0.0003 | 0.0012 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| Mean | 403.76 | 0.0166 | 0.0917 | 0.2610 | 0.8690 | 0.4336 | 0.3972 | 0.7645 | 0.5209 | 3.1672* | 0.4964 | 0.7995 | 0.0030 | 0.0050 |
G4R, M & 900 | ±SD | 22.35 | 0.0042 | 0.0320 | 0.0658 | 0.0479 | 0.0766 | 0.0178 | 0.0736 | 0.0398 | 0.2139 | 0.0352 | 0.0872 | 0.0002 | 0.0009 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (P<0.05).
Group, Sex & Dose (mg/kg/day) |
|
Fasting Body Weight (g) |
Adrenals |
Thymus |
Spleen |
Ovaries with oviduct |
Uterus with cervix |
Heart |
Kidneys |
Brain |
Liver |
Lungs |
Pituitary gland |
Thyroid along with parathyroid |
| Mean | 263.10 | 0.0327 | 0.1223 | 0.3367 | 0.0601 | 0.2669 | 0.4208 | 0.6602 | 0.7680 | 3.1949 | 0.8024 | 0.0054 | 0.0102 |
G1, F & 0 | ±SD | 29.73 | 0.0078 | 0.0285 | 0.0962 | 0.0121 | 0.0731 | 0.0620 | 0.0601 | 0.0690 | 0.3124 | 0.1282 | 0.0011 | 0.0017 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 252.69 | 0.0314 | 0.1448 | 0.3587 | 0.0591 | 0.2709 | 0.4108 | 0.6682 | 0.7963 | 3.3433 | 0.8463 | 0.0061 | 0.0100 |
G2, F & 100 | ±SD | 27.71 | 0.0059 | 0.0379 | 0.0643 | 0.0124 | 0.1122 | 0.0221 | 0.0987 | 0.0597 | 0.2474 | 0.1281 | 0.0009 | 0.0022 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 257.98 | 0.0323 | 0.1304 | 0.3817 | 0.0577 | 0.2689 | 0.4176 | 0.7380 | 0.7966 | 3.9825* | 0.8555 | 0.0059 | 0.0100 |
G3, F & 300 | ±SD | 20.07 | 0.0057 | 0.0261 | 0.0705 | 0.0091 | 0.0600 | 0.0410 | 0.0914 | 0.0859 | 0.3378 | 0.1776 | 0.0010 | 0.0016 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 234.66 | 0.0333 | 0.1083 | 0.3967 | 0.0622 | 0.2359 | 0.3997 | 0.7542* | 0.7956 | 4.6220* | 0.8853 | 0.0054 | 0.0116 |
G4, F & 900 | ±SD | 30.89 | 0.0059 | 0.0186 | 0.1282 | 0.0163 | 0.0756 | 0.0268 | 0.0702 | 0.0917 | 0.5053 | 0.2816 | 0.0007 | 0.0008 |
| n | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| Mean | 262.42 | 0.0265 | 0.0889 | 0.3536 | 0.0503 | 0.2700 | 0.3743 | 0.6672 | 0.7120 | 3.1050 | 0.7280 | 0.0063 | 0.0074 |
G1R, F & 0 | ±SD | 27.33 | 0.0042 | 0.0328 | 0.0640 | 0.0067 | 0.0475 | 0.0434 | 0.0397 | 0.0916 | 0.3421 | 0.1376 | 0.0011 | 0.0007 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| Mean | 251.23 | 0.0297 | 0.1225 | 0.3013 | 0.0581 | 0.2459 | 0.4585* | 0.6713 | 0.7676 | 3.3624 | 0.8623 | 0.0059 | 0.0094* |
G4R, F & 900 | ±SD | 15.16 | 0.0017 | 0.0197 | 0.0483 | 0.0067 | 0.0728 | 0.0541 | 0.0415 | 0.0830 | 0.2311 | 0.2288 | 0.0007 | 0.0012 |
| n | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (P<0.05).
Table 12. Summary of histopathology findings
Route of administration | Oral (gavage) | ||||
Treatment | Vehicle | Pine Oil | |||
Dose (mg/kg/day) | 0 | 900 | |||
Group | G1 | G4 | |||
Sex | M | F | M | F | |
Number of Animals | 10 | 10 | 10 | 10 | |
Adrenals | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Aorta | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Brain | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Caecum | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Cervix | Number examined | X | 10 | X | 10 |
Within normal limits | X | 10 | X | 10 | |
Coagulating glands | Number examined | 10 | X | 10 | X |
Within normal limits | 10 | X | 10 | X | |
Colon | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Duodenum | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Epididymides | Number examined | 10 | X | 10 | X |
Within normal limits | 9 | X | 4 | X | |
Cell debris, lumen | Minimal | 1 | X | 2 | X |
Inflammation | Minimal | - | X | 1 | X |
Mild | - | X | 3 | X | |
Moderate | - | X | 1 | X | |
Esophagus | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Eyes with optic nerve | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Femur bone with marrow and joint | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Heart | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 9 | |
Infiltrate, mononuclear cell | Minimal | - | - | - | 1 |
Ileum with Peyer’s patches | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Jejunum | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Kidneys | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 7 | 8 | 1 | 8 | |
Infiltrate, mononuclear cell, interstitium | Minimal | 2 | 1 | - | 2 |
Dilatation, tubule | Minimal | 1 | - | 2 | - |
Basophilia, tubule | Minimal | - | - | 5 | - |
Mild | - | - | 4 | - | |
Dilatation, pelvis | Minimal | - | 1 | - | - |
Route of administration | Oral (gavage) | ||||
Treatment | Vehicle | Pine Oil | |||
Dose (mg/kg/day) | 0 | 900 | |||
Group | G1 | G4 | |||
Sex | M | F | M | F | |
Number of Animals | 10 | 10 | 10 | 10 | |
Liver | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | - | 2 | |
Hypertrophy, hepatocyte | Minimal | - | - | 7 | 6 |
Mild | - | - | 3 | 2 | |
Necrosis, hepatocyte | Minimal | - | - | 1 | 2 |
Mild | - | - | 2 | 1 | |
Lungs | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 9 | 9 | 9 | 10 | |
Macrophages, increased, alveoli | Minimal | 1 | - | - | - |
Infiltrate, mononuclear cell, alveoli | Minimal | - | 1 | 1 | - |
Mandibular lymph nodes | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Mesenteric lymph nodes | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Mammary gland | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Ovaries | Number examined | X | 10 | X | 10 |
Within normal limits | X | 10 | X | 10 | |
Oviduct | Number examined | X | 10 | X | 10 |
Within normal limits | X | 10 | X | 10 | |
Pancreas | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Parathyroid | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Pituitary gland | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Prostate gland | Number examined | 10 | X | 10 | X |
Within normal limits | 10 | X | 9 | X | |
Cell debris, acini | Minimal | - | X | 1 | X |
Rectum | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Salivary glands | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Sciatic nerve | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Seminal vesicles | Number examined | 10 | X | 10 | X |
Within normal limits | 10 | X | 10 | X | |
Skeletal muscle | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Skin | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Spinal cord | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Route of administration | Oral (gavage) | ||||
Treatment | Vehicle | Pine Oil | |||
Dose (mg/kg/day) | 0 | 900 | |||
Group | G1 | G4 | |||
Sex | M | F | M | F | |
Number of Animals | 10 | 10 | 10 | 10 | |
Spleen | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Stomach | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Testes | Number examined | 10 | X | 10 | X |
Within normal limits | 9 | X | 10 | X | |
Degeneration/atrophy, tubule | Mild | 1 | X | - | X |
Thymus | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 9 | 9 | 9 | 7 | |
Cyst, epithelial | Present | 1 | 1 | 1 | 2 |
Involution | Present | - | - | - | 1 |
Thyroid | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 9 | 8 | 8 | 10 | |
Cyst, ultimobranchial | Present | 1 | 2 | 1 | - |
Ectopic tissue, thymus | Present | - | - | 1 | - |
Trachea | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Urinary bladder | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Uterus | Number examined | X | 10 | X | 10 |
Within normal limits | X | 9 | X | 10 | |
Dilatation, lumen | Minimal | X | 1 | X | - |
Vagina | Number examined | X | 10 | X | 10 |
Within normal limits | X | 10 | X | 10 | |
Route of administration | Oral (gavage) | ||||
Treatment | Pine Oil | ||||
Dose (mg/kg/day) | 100 | 300 | |||
Group | G2 | G3 | |||
Sex | M | F | M | F | |
Number of Animals | 10 | 10 | 10 | 10 | |
Liver | Number examined | 10 | 10 | 10 | 10 |
Within normal limits | 10 | 10 | 10 | 10 | |
Kidneys | Number examined | 10 | X | 10 | X |
Within normal limits | 10 | X | 8 | X | |
Basophilia, tubule | Minimal | - | X | 2 | X |
Dilatation, tubule | Minimal | - | X | 1 | X |
Epididymides | Number examined | 10 | X | 10 | X |
Within normal limits | 10 | X | 10 | X | |
Route of administration | Oral (gavage) | ||||
Treatment | Vehicle Control Recovery | High dose Recovery | |||
Dose (mg/kg/day) | 0 | 900 | |||
Group | G1R | G4R | |||
Sex | M | F | M | F | |
Number of Animals | 5 | 5 | 5 | 5 | |
Liver | Number examined | 5 | 5 | 5 | 5 |
Within normal limits | 5 | 5 | 5 | 5 | |
Kidneys | Number examined | 5 | X | 5 | X |
Within normal limits | 4 | X | 2 | X | |
Dilatation, tubule | Minimal | 1 | X | 1 | X |
Cyst | Present | - | X | 1 | X |
Dilatation, pelvis | Minimal | - | X | 1 | X |
Infiltrate, mononuclear cell, interstitium | Minimal | - | X | 1 | X |
Epididymides | Number examined | 5 | X | 5 | X |
Within normal limits | 5 | X | 3 | X | |
Inflammation | Mild | - | X | 1 | X |
Moderate | - | X | 1 | X | |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 300 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- Two studies (OECD 422 and OECD 408) are available with a Klimisch score of 1.
Also some available supporting studies on components/analogue substances with Klimisch score of 2 have been included in the dossier.
Repeated dose toxicity: inhalation - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- 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:
- supporting study
- 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.
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- 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)
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Details on species / strain selection:
- Fischer 344/NHSD
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Harlan Sprague-Dawley , Inc.
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 8 weeks old
- Weight at study initiation: 171.4 g (male) and 140.6 g (female)
- Fasting period before study: not specified
- Housing: rats were housed one per cage in stainless steel, wire-mesh cages both during and between exposures
- Diet (e.g. ad libitum): ad libitum during the non exposure periods. Animals were fed powdered Certified Agway Pro Lab RMH 3200 (Agway, Inc.).
- Water (e.g. ad libitum): ad libitum during the non exposure periods. Water (Municipal Authority of Westmoreland County, Greensburg, PA, U.S.A.) was provided by an 8-oz amber glass bottle.
- Acclimation period: animals were acclimated to the exposure chamber (air-only exposure) for 2 days prior to the initiation of exposure.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.9 to 24.0ºC
- Humidity (%):47 to 53%
- Air changes (per hr): 12.5/hour
- Photoperiod (hrs dark / hrs light): 12 h of fluorescent light and 12 h of darkness each 24 h. - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- whole body
- Vehicle:
- air
- Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Liquid cumene was metered from a piston pump (Fluid Metering) into a heated glass evaporator similar to that described by Carpenter CP, Kinkead ER, Geary DL Jr, Sullivan LJ, King JM. (1975) and Snellings and Dodd (1990). For the target concentration of 1,200 ppm, two glass evaporators were joined in series. Glass marbles were packed in the lower evaporator.
- Temperature, humidity, pressure in air chamber: the daily mean temperatures within the chambers ranged from 21.9 to 24.0ºC and the relative humidity ranged from 47 to 53%.
- Air flow rate: 900 L/min
- Air change rate: 12.5 air changes/h
TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography (Perkin-Elmer 8500 gas chromatograph with flame ionization detector)
- Samples taken from breathing zone: yes
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Prior to the initiation of exposures, chamber uniformity of vapor for the target concentrations of cumene was determined by sampling five locations distributed throughout the chambers. During the 6-h exposures, chamber concentrations of cumene vapor were analyzed approximately once every 25-30 min by GC-FID.
- Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- 6 h per day, 5 days per week.
- Dose / conc.:
- 0 ppm (nominal)
- Dose / conc.:
- 100 ppm (nominal)
- Dose / conc.:
- 500 ppm (nominal)
- Dose / conc.:
- 1 200 ppm (nominal)
- No. of animals per sex per dose:
- 21
- Control animals:
- yes
- Details on study design:
- - Dose selection rationale: Target concentrations of cumene vapor were selected based on the results from a 2-week pilot inhalation study in rats (Chevron Environmental Health Center, 1989).
- Positive control:
- no
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: preceding, during, and following each exposure.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first exposure, weekly during the exposure period and preceding sacrifice.
BODY WEIGHT: Yes
- Time schedule for examinations: prior to the first exposure, weekly during the exposure period and preceding sacrifice.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: weekly
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule: weekly
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the last week of exposure
- Dose groups that were examined: all animals
HAEMATOLOGY: Yes
- Time schedule for collection of blood: on 5 rats/sex/group prior to initiation of exposures and on 10 rats per sex per group during the week following conclusion of exposures
- Anaesthetic used for blood collection: methoxyflurane
- Animals fasted: Animals had access to water but not food prior to blood collection.
- How many animals: 15 per sex per dose
- Parameters examined: erythrocyte, platelet, leukocyte, differential leukocyte, and reticulocyte (male rats only) counts; hemoglobin; hematocrit; mean corpuscular volume, hemoglobin and hemoglobin concentration
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on 5 rats/sex/group prior to initiation of exposures and on 10 rats per sex per group during the week following conclusion of exposures
- Animals fasted: Animals had access to water but not food prior to blood collection.
- How many animals: 15 per sex per dose
- Parameters examined: glucose; urea nitrogen; creatinine; total protein; albumin; globulin; total, direct, and indirect bilirubin; calcium; phosphorus; sodium; potassium; chloride; aspartate and alanine aminotransferases; and gammaglutamyltransferase
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: functional observational battery: prior to the first exposure and on the weekend following study Weeks 1, 2, 4, 9, and 13. Motor activity: prior to the first exposure and on the weekend following study Weeks 4, 9, and 13.
- Dose groups that were examined: 10 rats per sex per group (behavioral evaluations) and 15 rats per sex per group (Motor activity).
- Battery of functions tested: Data for ambulatory activity, fine motor activity, rearing activity, and the sum of these individual types of activity (total activity) were collected.
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes.
Six rats per sex per group were perfusion-fixed for light microscopic evaluation of the nervous system tissues. The brain and spinal cord were further immersed in the same fixative prior to processing for microscopic evaluation.
The remaining animals underwent complete necropsy evaluations. The liver, kidneys, lungs, adrenals, gonads, and brain were weighed at necropsy. The ratios of organ weight to body weight and organ weight to brain weight were calculated.
HISTOPATHOLOGY: Yes. - Other examinations:
- The epididymides of 15 male rats per group (excluding perfused animals) were evaluated for sperm count and sperm morphology (Wyrobek and Bruce, 1975).
The left testis of each male rat was evaluated for spermatid counting according to the methods of Johnson et al. (1980) and Blazak et al. (1985).
The right testis from male rats of the 1,200-ppm and control groups was prepared for evaluation of the stages of spermatogenesis as described by Land and Chapin (1985). - Statistics:
- Results of quantitative continuous variables were intercompared among the cumene concentration groups and control group by Levene’s test for equal variances, analysis of variance (ANOVA) and t test. When Levene’s test indicated homogeneous variances and the ANOVA was significant, the pooled t test was used for pairwise comparisons. When Levene’s test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by the separate variance t test (Dixon, 1985). Comparisons to the control group were made with use of Fisher’s exact probability test. The fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Cage-side clinical observations included ataxia in the 1,200-ppm rats following the first 2-3 weeks and increased incidences of periocular tissue swelling, urine stains, urogenital area wetness, and/or perinasal encrustation, primarily in the 500- and 1200-ppm groups.
Also, rats in the 1,200-ppm group appeared hypoactive, exhibited blepharospasm, and showed a delayed or absent startle reflex. Rats in the 500-ppm group were hypoactive during exposure. - Mortality:
- no mortality observed
- Description (incidence):
- There were no exposure-related deaths during the study.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There were no differences in mean body weights between the cumene-exposed and controls groups.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Food consumption was decreased at Week 1 for the female rats at 500- and 1,200-ppm (mean 13.8 and 13.2 g per animal per day, respectively) as compared with control values (mean 14.6 g per animal per day).
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- A consistent increase in water consumption was observed in the male rats in the 500- and 1,200-ppm groups for Weeks 2 through 13. The magnitude of the increase for both exposure groups was approx. 40% above the control value at the end of the exposure regimen. There were also increased water consumption values for the female rats in the same exposure groups during Weeks 7, 8, and 10.
- Ophthalmological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Cataracts were observed in both the control and treated groups in 14-55% of the animals per sex. Because a high incidence of cataracts (19% of male and female rats combined) were observed in the control animals, the cataracts in the exposure groups were considered uninterpretable.
- Haematological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Exposure-related increases in total leukocytes, lymphocytes, and platelets were observed in rats in the 500- and 1,200-ppm groups.
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Serum chemistry changes observed in both male and female rats, primarily of the 1,200-ppm group, were increases in total protein, albumin, globulin, calcium, and phosphorus. Female rats of the 500 and 1,200-ppm groups had lower serum glucose values compared with the control group.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- effects observed, treatment-related
- Description (incidence and severity):
- There were no exposure-related changes in the functional observational battery in rats exposed repeatedly to cumene vapor.
Total motor activity (fine movement, rearing and ambulation combined) was decreased at Week 13 in male rats that were exposed to 500 or 1,200 ppm. In particular, ambulatory activity was statistically decreased at Weeks 4, 9, and 13. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- When evaluated as absolute weights or weight ratios relative to body weight or brain weight, statistically significant increases were observed for liver, kidneys, and adrenal glands of both male and female rats of the 1,200-ppm groups. In the 500-ppm group, the weight of the liver was increased for male and female rats, and female rats had a mild increase in kidney weight. Male rats had a mild increase in adrenal gland weight.
There were no cumene-related differences in weights of lungs, testes, ovaries, or brain at any exposure level. There were no organ weight changes for rats of the 100-ppm group. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Cumene-related gross lesions observed during necropsy were limited to periocular swelling in rats in the 500- and 1,200-ppm groups.
- Neuropathological findings:
- no effects observed
- Description (incidence and severity):
- There were no cumene-related microscopic alterations in the tissues of the peripheral and central nervous system.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- The only cumene exposure-related finding was in the kidneys of the male rats in the 500- and 1,200-ppm groups. The incidences of epithelial cell hypertrophy/hyperplasia of the proximal tubules and interstitial nephritis were higher in the rats of the 500- and 1,200-ppm groups. Also, it was found a greater amount of hyaline droplet formation within the proximal tubules of the male rats of the 500- and 1,200-ppm groups.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Description (incidence and severity):
- There were no cumene-related differences in the counts of testicular sperm heads or epididymal spermatozoa.
The total percentage of normal epididymal sperm across all groups was >96%, indicating no treatment-related effect on epididymal sperm morphology. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 100 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- behaviour (functional findings)
- clinical biochemistry
- clinical signs
- gross pathology
- haematology
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Key result
- Critical effects observed:
- no
- Conclusions:
- Based on the findings of this study, the NOEL of cumene after 13 weeks of vapour inhalation exposure to rats was determined to be 100 ppm.
- Executive summary:
A 13-week repeated dose toxicity study (inhalation route) was performed on cumene following a method similar to OECD guideline 413. Groups of 21 male and 21 female rats were exposed to the test item by whole body inhalation at concentrations of 0, 100, 500 and 1200 ppm, 6 hours per day, 5 days per week for 13 weeks. During the study, clinical observations, food and water consumption, bodyweight, organ weight, haematology, clinical chemistry, neurobehavior, ophthalmology, gross pathology, histopathology and spermatogenesis evaluations were undertaken. There were no exposure-related changes in the functional observational battery and neuropathological findings (peripheral and central nervous system). Motor activity decreases were seen only in male rats exposed to 500 or 1,200 ppm. The 500-and or 1,200-ppm groups showed decreases in food consumption, an increase in water consumption, and changes in several hematologic and clinical chemistry parameters. There were no exposure-related ophthalmologic findings or effects on spermatogenesis. Weights of liver, kidneys, and adrenal glands were increased in the 500 and 1,200 ppm groups. Renal proximal tubular cell hypertrophy, hyperplasia, and hyaline drop formation were observed in the male rats at 500 and 1,200 ppm. In conclusion, exposure to cumene vapor resulted in mild toxicity at 1,200 ppm, minimal effects at 500 ppm, and no observable effects at 100 ppm.
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- 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)
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Details on species / strain selection:
- Fischer 344/NHSD
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Harlan Sprague-Dawley , Inc.
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 7 weeks old
- Weight at study initiation: 168.4 g (male) and 130.4 g (female)
- Fasting period before study: not specified
- Housing: rats were housed one per cage in stainless steel, wire-mesh cages both during and between exposures
- Diet (e.g. ad libitum): ad libitum during the non exposure periods. Animals were fed powdered Certified Agway Pro Lab RMH 3200 (Agway, Inc.).
- Water (e.g. ad libitum): ad libitum during the non exposure periods. Water (Municipal Authority of Westmoreland County, Greensburg, PA, U.S.A.) was provided by an 8-oz amber glass bottle.
- Acclimation period: animals were acclimated to the exposure chamber (air-only exposure) for 2 days prior to the initiation of exposure.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.9 to 24.0ºC
- Humidity (%):47 to 53%
- Air changes (per hr): 13/hour
- Photoperiod (hrs dark / hrs light): 12 h of fluorescent light and 12 h of darkness each 24 h. - Route of administration:
- inhalation: vapour
- Type of inhalation exposure:
- whole body
- Vehicle:
- air
- Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Liquid cumene was metered from a piston pump (Fluid Metering) into a heated glass evaporator similar to that described by Carpenter CP, Kinkead ER, Geary DL Jr, Sullivan LJ, King JM. (1975) and Snellings and Dodd (1990). For the target concentration of 1,200 ppm, two glass evaporators were joined in series.
- Temperature, humidity, pressure in air chamber: the daily mean temperatures within the chambers ranged from 21.9 to 24.0ºC and the relative humidity ranged from 47 to 53%.
- Air flow rate: 200 L/min
- Air change rate: 13 air changes/h
TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography (Perkin-Elmer 8500 gas chromatograph with flame ionization detector)
- Samples taken from breathing zone: yes
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Prior to the initiation of exposures, chamber uniformity of vapor for the target concentrations of cumene was determined by sampling five locations distributed throughout the chambers. During the 6-h exposures, chamber concentrations of cumene vapor were analyzed approximately once every 25-30 min by GC-FID.
- Duration of treatment / exposure:
- 13 weeks exposure plus a 4-week recovery period without exposure.
- Frequency of treatment:
- 6 h per day, 5 days per week.
- Dose / conc.:
- 0 ppm (nominal)
- Dose / conc.:
- 50 ppm (nominal)
- Dose / conc.:
- 100 ppm (nominal)
- Dose / conc.:
- 500 ppm (nominal)
- Dose / conc.:
- 1 200 ppm (nominal)
- No. of animals per sex per dose:
- 15
- Control animals:
- yes
- Details on study design:
- - Dose selection rationale: Target concentrations of cumene vapor were selected based on the results from a 2-week pilot inhalation study in rats (Chevron Environmental Health Center, 1989).
- Positive control:
- no
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: preceding, during, and following each exposure.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first exposure, weekly during the exposure period and the recovery period, and preceding sacrifice.
BODY WEIGHT: Yes
- Time schedule for examinations: prior to the first exposure, weekly during the exposure period and the recovery period, and preceding sacrifice.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the preexposure period, at weeks 4,9, and 13, and during postexposure Week 4.
- Dose groups that were examined: all animals
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Motor activity: prior to the first exposure and on the weekend following study Weeks 4, 9, and 13. Tone-pip auditory brain stem responses (ABR): during postexposure week 1
- Dose groups that were examined: 15 rats per sex per group (Motor activity) and 10 rats per sex per group (ABR).
- Battery of functions tested: ABR / motor activity
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes.
Animals underwent complete necropsy evaluations. The liver, kidneys, lungs, adrenals, gonads, and brain were weighed at necropsy. The ratios of organ weight to body weight and organ weight to brain weight were calculated.
HISTOPATHOLOGY: Yes. - Statistics:
- Results of quantitative continuous variables were intercompared among the cumene concentration groups and control group by Levene’s test for equal variances, analysis of variance (ANOVA) and t test. When Levene’s test indicated homogeneous variances and the ANOVA was significant, the pooled t test was used for pairwise comparisons. When Levene’s test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by the separate variance t test (Dixon, 1985). Comparisons to the control group were made with use of Fisher’s exact probability test. The fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- There were treatment-related cage-side clinical observations during the study.
- Mortality:
- no mortality observed
- Description (incidence):
- There were no exposure-related deaths during the study.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There were no differences in mean body weights between the cumene-exposed and controls groups.
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- There were no cumene-related ophthalmologic findings.
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- There were no significant differences in motor activity between control and cumene-exposed animals.
The evaluation of ABR responses at postexposure week 1 found no changes in the auditory functions of cumene-exposed animals. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- When evaluated as absolute weights or weight ratios relative to body weight or brain weight, statistically significant increases were observed for liver, kidneys, and adrenal glands of both male and female rats of the 1,200-ppm groups. In the 500-ppm group, the weight of the liver was increased for male and female rats. Male rats had a mild increase in adrenal gland weight.
There were no cumene-related differences in weights of lungs, testes, ovaries, or brain at any exposure level. There were no organ weight changes for rats of the 100-ppm group. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Cumene-related gross lesions observed during necropsy were limited to periocular swelling in rats in the 500- and 1,200-ppm groups.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- The only cumene exposure-related finding was in the kidneys of the male rats in the 500- and 1,200-ppm groups. The incidences of epithelial cell hypertrophy/hyperplasia of the proximal tubules and interstitial nephritis were higher in the rats of the 500- and 1,200-ppm groups. Also, it was found a greater amount of hyaline droplet formation within the proximal tubules of the male rats of the 500- and 1,200-ppm groups.
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 100 ppm
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- clinical signs
- gross pathology
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Key result
- Critical effects observed:
- no
- Conclusions:
- Based on the findings of this study, the NOEL of cumene after 13 weeks of vapour inhalation exposure to rats was determined to be 100 ppm.
- Executive summary:
A 13-week repeated dose toxicity study (inhalation route) was performed on cumene following a method similar to OECD guideline 413. Groups of 15 male and 15 female rats were exposed to the test item by whole body inhalation at concentrations of 0, 50, 100, 500 and 1200 ppm, 6 hours per day, 5 days per week for 13 weeks. During the study, clinical observations, bodyweight, organ weight, neurobehavior, ophthalmology, gross pathology and histopathology evaluations were undertaken. There were no exposure-related changes in the motor activity, auditory brain stem response and brain measurements. There were no exposure-related ophthalmologic findings. Weights of liver, kidneys, and adrenal glands were increased in the 500 and 1,200 ppm groups. Renal proximal tubular cell hypertrophy, hyperplasia, and hyaline drop formation were observed in the male rats at 500 and 1,200 ppm. In conclusion, exposure to cumene vapor resulted in mild toxicity at 1,200 ppm, minimal effects at 500 ppm, and no observable effects at 50 and 100 ppm.
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
Key study: Test method OECD 422: The test substance Pine oil 50% was tested in a combined repeated dose toxicity study with the reproduction /developmental toxicity screening test according to OECD guideline 422, in accordance with GLP principles. Sprague Dawley rats were administered with test item at the dose levels of 100, 300 and 600 mg/kg body weight previously selected according to the results of a dose range finding study performed with the same species at doses of 100, 300 and 1000 mg/kg bw for a period of 14 days. The treatment of male and female rats with test substance at dose levels up to 600 mg/kg-bw/day by repeated oral (gavage) administration revealed no adverse effects for parental toxicity. The No Observed Effect Level (NOEL) was determined to be 100 mg/kg bw/day based on treatment related clinical signs of toxicity and reduction in body weight found at the dose of 300 mg/kg bw/day, and the No Observed Adverse Effect Level (NOAEL) was established at 600 mg/kg bw/day, as the treatment related clinical sings of toxicity observed at doses of 300 and 600 mg/kg bw/day were found recovered later during the treatment period in all cases and also no treatment related histopathological findings were noticed in this study.
Key study: Test method OECD 408: A study was conducted to assess the toxic potential of the test item when administered for a period of 90 consecutive days repeatedly to Sprague Dawley rats, according to OECD 408 Guideline (GLP study). Four groups (G1, G2, G3 and G4) of 10 animals of each sex and two recovery groups with 28-day recovery period (G1R and G4R) of 5 animals of each sex were tested, being G1 and G1R the vehicle control groups (corn oil). The treatment groups received concentrations of 100, 300 and 900 mg/kg bw/day. No clinical signs of toxicity and no mortality/morbidity were noted, as well as no changes in body weight, feed consumption and gross pathology in all groups. Besides, no effects in the ophthalmological and neurological/functional examinations were observed. No treatment related changes were observed in hematology, coagulation, clinical chemistry and urinalysis parameters. Increase in urine volume in males and decrease in pH in females and specific gravity in females were associated with microscopic changes in kidneys. No treatment related changes were observed in thyroid hormones (T3, T4 and TSH), vaginal smear examination and fasting body weights. No gross pathological changes were observed. Test item-related changes were observed in liver, kidneys and epididymides. The changes in liver were observed in both the sexes and changes in kidneys and epididymides were observed in male animals. The changes in liver were found to be reversible at the end of 28 days treatment free recovery period as similar changes were not observed in recovery group of animals. In kidneys, effects were observed in mid and high dose group male rats which were not observed in recovery high dose group, hence this change was found to be reversible at the end of recovery period. Furthermore, considering the magnitude of severity of lesions and number of incidences in mid dose, changes at this dose level could be considered as a non-adverse effect. In epididymides, effects were observed in high dose and high dose recovery male rats. All these changes in epididymides were considered as adverse effects of test item administration. However, the lesion was focally confined and majority of epididymal lumen contained normal sperms. Also, testes and accessory sex glands were found to be within normal histological limits. This is a clearly indication that effects found in epididymides does not reveal any concern in relation with reproductive toxicity. Therefore, based on the obtained results, the NOAEL is estimated as 300 mg/kg bw/day.
Supporting study: Dose range finding study according to test method OECD 407: A dose range finding study was performed for the test substance pine oil 50% in accordance with OECD guideline 407 in order to evaluate its toxic potential after repeated exposure and select the appropriate doses for the combined repeated dose toxicity study with the reproduction /developmental toxicity screening test (OECD 422). The test item was added to Sprague-Dawley rats (3 per sex per dose) at doses of 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day for a period of 14 days. The No Observed Effect Level (NOEL) was set at 100 mg/kg bw/day and Low Observed Effect Level (LOEL) at 300 mg/kg bw/day, as the dose of 100 mg/kg bw/day did not reveal any treatment related effects in either sex, the dose of 300 mg/kg bw/day revealed treatment related clinical signs of toxicity and reduction in body weight and feed consumption and the dose of 1000 mg/kg bw/day revealed treatment related clinical signs of toxicity, mortality, reduction in body weight and feed consumption.
Supporting studies with individual components or analogue substances:
Terpineol (44 days, rats, oral):
A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of terpineol (CAS 8000-41-7) by oral administration in rats was conducted according to OECD guideline 422, in accordance with GLP principles. Terpineol was diluted with corn oil and added to Sprague-Dawley SPF rats (12 per sex per dose) at doses of 0, 100, 300 and 1000 mg/kg bw/day, from day 14 before mating up to 30 days after mating for males (total 44 days) and up to 4 days of lactation for females (total 41-51 days). Based on results, the NOEL for repeated exposure was determined to be 100 mg/kg bw/day in male and female rats.
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.
cineole (30 days, mouse, oral):
A short term repeated dose toxicity study (oral route) was performed on 1,8-cineole according to OECD guideline 407. The substance was emulsified in 2% Tween 80 and administered daily to mice by oral gavage in doses of 0 (control, only 2% Tween 80), 21.38, 64.15 and 192.45mg/kg bw/day during 30 days.Animal behavior, body weight and mortality were recordedduring the study. After being sacrificed on day 31,organs/tissues were examined macroscopically and gross lesions were recorded. Relative weight of liver, spleen and double kidneys was calculated.Heart, liver, spleen, lungs, kidney, testis and ovary of treatment groups and control group were examined microscopically. Also, ultrastructural observation by electron microscopy was performed on those organ cells with effects. There were no significant differences in body weight and relative organ weight between the control group and treatment groups. The histopathological examinations showed that granular degeneration and vacuolar degeneration appeared in liver and kidney tissue after administration of high dose (192.45 mg/kg bw/day). Under electron microscopy, a series of ultrastructural changes were observed. At the subcellular level the influence of test substance was found on the mitochondria, endoplasmic reticulum and other membrane type structure of liver and kidney. Based on these results, the NOAEL of the test substance was determined to be 64.15 mg/kg bw/day.
cineole (50 days, rats, oral):
1,8-cineole was daily administered to Wistar rats (male and female) for 50 days at doses of 0, 100, 500 and 1000 mg / kg bw/day by gavage. Test method was according to OECD guideline 407. In all experimental groups, animals were observed for signs of toxicity, such as piloerection, diarrhea, changes in locomotor activity or mortality. Body weight, food and water consumption were determined once a week. Haematological and clinical tests 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. Histological preparations 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. Occasional alterations in rats of both sexes were observed, as evidenced in hematological and biochemical parameters and histopathological analysis. However, these changes showed low clinically relevant, since they occurred in a non-generalized manner between animals of the treated groups. Since the lower dose (100 mg/kg) induced reduction of water consumption and mean platelet volume and alkaline phosphatase, it was not possible to establish the NOAEL.
Camphene (14 days, rats, oral):
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.
Cumene (90 days, rats, inhalation):
A 13-week repeated dose toxicity study (inhalation route) was performed on cumene following a method similar to OECD guideline 413. Groups male and female rats were exposed to the test item by whole body inhalation at concentrations of 0, 50, 100, 500 and 1200 ppm, 6 hours per day, 5 days per week for 13 weeks. Exposure to cumene vapor resulted in mild toxicity at 1,200 ppm, minimal effects at 500 ppm, and no observable effects at 50 and 100 ppm.
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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