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EC number: 692-731-2 | CAS number: 76950-43-1
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
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- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Repeated dose toxicity: oral
Administrative data
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 Jan - 6 Jul 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Cross-reference
- Reason / purpose for cross-reference:
- reference to other study
Reference
- Endpoint:
- short-term repeated dose toxicity: oral
- Remarks:
- combined repeated dose and reproduction / developmental screening
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 12 Mar - 03 Jun 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP-Guideline 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)
- Version / remarks:
- 22 Mar 1996
- Deviations:
- yes
- Remarks:
- no data, if special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure was performed.
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Crl:CD(SD)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 10 weeks
- Weight at study initiation: males: 350-436 g (mean: 391.9 g); females: 211-262 g (mean: 234.7 g)
- Housing: single housing in metal bracket-type cages (260 W x 380 D x 180 H, mm) with wire mesh floors; for mated females (GD 17 - lactation day 4) the wire mesh floors of cages were replaced with small trays with bedding
- Diet: pellet diet, CRF-1 (Oriental Yeast Co., Ltd.), ad libitum
- Water: Sapporo City tap water, ad libitum
- Acclimation period: 13 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- Japanese Pharmacopoeia water for injection
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test substance was accurately weighed out. Water was added to obtain the specified concentration. A stirrer was used for dissolving the substance in the vehicle. The dosing solutions were prepared once every 2 to 8 days and stored refrigerated in an airtight and light protected container.
VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no.: 3C90 - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The concentration of the test substance in the first and last dosing solution preparations were analysed. The accuracies (% of the analysed values relative to the theoretical value defined as 100%) of the 10, 30 and 100 mg/L dosing solutions were 97.3, 93.5 and 99.3% for the first preparations and 103, 110 and 112% for the final preparations, respectively. Thus, the acceptance criteria (nominal concentration ± 15%) was met.
Stability of 8-day storage under refrigeration followed by 5 h storage at room temperature after preparation was analysed in the 1 and 100 mg/mL preparations. In the analysis results, the remaining rates of the 1 and 100 mg/mL preparations were 92.2% and 98.3%, respectively, which met the acceptance criteria (100% ± 10%). - Duration of treatment / exposure:
- males: for 42 days, starting 14 days before mating
mated females: for 14 days before mating and during mating period until successful copulation, and during gestation through day 4 after parturition
non-mated females: for 42 days - Frequency of treatment:
- once daily, 7 days/week
- Remarks:
- Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- 12 (main study)
5 males (control and high dose satellite group, selected from the main study group)
5 additional females (control and high dose non-mating group)
5 additional females (control and high dose non-mating satellite group) - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: A preliminary 14-day study (Fujii, 2014) was performed with dose levels of 100, 300 and 1000 mg/kg bw/day using 4 animals/sex/dose. No mortality, clinical findings, body weight changes or changes in food consumption were observed. In urinalysis, the number of females with protein-positive urine increased in the 1000 mg/kg bw/day group. The hemoglobin concentration and hematocrit value were low in males and triglyceride and renal weight were high in females in the 1000 mg/kg bw/day group. No changes related to the test substance administration were noted in the 100 or 300 mg/kg bw/day group. During necropsy no abnormal findings in males or females in any treated group were observed.
- Rationale for selecting satellite groups: five males with body weight around the central value of the population were selected from control and high dose main study group based on the study weight on administration day 28 to approximate the overall mean, after the end of mating period
- Post-exposure recovery period in satellite groups: 14 days - Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily before and after administration during the administration period, twice daily in the morning and afternoon during the recovery period, and once in the morning on the day of necropsy
- Cage side observations included: observations for mortality, external appearance and behaviour.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the start of administration and on administration days 7, 14, 21, 28, 35 and 42 and on recovery days 7 and 14
- Observations included:
1) from outside home cage: body position/posture, respiratory pattern, tremor/convulsion, stereotypes (rolling/repetitive circling) and bizarre behaviour (self-biting)
2) from out of cage: ease of removal, ease of handling, muscle tone, piloerection, fur conditions, appearance of skin, eyes, eyeballs, and mucous membranes, pupil size, lacrimation, salivation, and other secretions or excretions
3) from an open field: gait, co-ordination of movement, reactivity to environmental stimuli, searching (sniffing and standing), excretions (urination and defecation), stereotypes (excessive grooming and unusual head movement), bizarre behaviour (walking backwards and vocalization) and aggression
BODY WEIGHT: Yes
- Time schedule for examinations: all males and females in the satellite groups: before administration on administration days 1, 4, 7, 14, 21, 28, 35 and 42 and on recovery days 1, 7, and 14, and the day of necropsy; females in the main study group: before administration on administration days 1, 4, 7, and 14, and before administration on gestation days 0, 7, 14, and 20, and before administration on lactation days 0 and 4, and the day of necropsy (on the 5th day after parturition, except females without parturition which were necropsied on gestation day 26 (day of necropsy)).
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
OPHTHALMOSCOPIC EXAMINATION: No
HEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (pentobarbital sodium)
- Animals fasted: Yes
- How many animals: 5 animals per group (males in the main study groups: selected in ascending order of animal No. from those not used in the functional observations; females in the main study groups: the same females as those used in the functional observations; animals in the satellite groups: all animals)
- Parameters examined: red blood cell count (RBC), hematocrit (HCT), hemoglobin concentration (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), platelet count, white blood cell count (WBC), reticulocyte count, differential count of WBC (neutrophil, eosinophil, basophil, monocyte, lymphocyte), prothrombin time (PT), activated partial thromboplastin time (APTT)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes
- How many animals: the same 5 animals per group as those used in the hematological examination
- Parameters examined: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GTP), glucose, total cholesterol, triglyceride, total bilirubin, total bile acid, urea nitrogen, creatinine, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein, albumin/globulin ration, albumin, protein fraction (albumin, α1-globulin, α2-globulin, β-globulin, γ-globulin)
URINALYSIS: Yes
- Time schedule for collection of urine: in administration week 6 (on administration days 37 to 38) and recovery week 2 (on recovery days 9 to 10)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- How many animals: the same 5 animals as those used in the functional observations and females to be necropsied at the end of recovery in the satelite group
- Parameters examined: pH, protein, glucose, ketone body, urobilinogen, bilirubin, occult blood, color, urinary sediments, urine volume and specific gravimetry
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in administration week 6 (administration day 36) and recovery week 2 (recovery day 8), on lactation day 4 for femaeles in the main study groups
- Dose groups that were examined: 5 males per group (including the satellite group) selected to approximate the mean body weight on administration day 28, females (necropsied at the end of recovery) in the satellite groups, and 5 females selected in the order of parturition dates in the main study groups
- Battery of functions tested: sensory and motory reactivity to visual, touch, auditory, pain and proprioceptive stimuli and righting reflex; grip strength; motor activity - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
Organs examined: brain (cerebrum, cerebellum, pons), spinal cord, pituitary gland, thymus, thyroids, parathyroids, adrenals, spleen, heart, esophagus, stomach, liver, pancreas, submandibular glands, duodenum, jejunum, ileum (with Peyer's patches), cecum, colon, rectum, trachea, lungs, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicles (with coagulating glands), ovaries, uterus (horn and cervix), vagina, eyeballs and harderian glands, mammary gland (female only, right abdominal region), femur (with bone marrow, right), mesenteric lymph nodes, mandibular lymph nodes, skeletal muscle (biceps femoris), sciatic nerve, and gross lesions (with border to the normal tissue)
Organ weight was determined from the following tissues/organs: brain, heart, liver, kidneys, testes, epididymides, seminal vesicles, pituitary glands, thyroids (with parathyroids), spleen, thymus, adrenals, prostate, ovaries and uterus.
Vaginal smear was collected from all females in the satellite groups before necropsy on the day of necropsy to determine the stage of estrous cycle. These data were used to support the evaluation of organ weights or histopathology.
HISTOPATHOLOGY: Yes
From all the organs and tissues of all animals fixed and preserved at necropsy, paraffin block specimens were prepared and those of all animals in the control and high dose groups were microscopically examined. Stomach was examined in the low and mid dose group due to microscopic findings in the high dose group. - Statistics:
- Statistical analysis were performed using the toxicological data processing system (MiTOX): group means and standard deviations, Bartlett test, one-way analysis of variance, Kruskal-Wallis test, Dunett's test, Steel's test, Fisher's exact probability test, F-test, Student's t-test, Welch test, Wilcoxon rank-sum test; p=0.05
- 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:
- no effects observed
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- 1000 mg/kg bw/day, males (main study group, end of administration): hyperplasia of squamous cells at limiting ridge of the stomach (slight) in all 7 males with significantly high incidence, indicating slight irritability to the stomach mucosa.
- Histopathological findings: neoplastic:
- no effects observed
- Details on results:
- CLINICAL SIGNS AND MORTALITY
Subcutaneous mass was noted in one female each in the control and 1000 mg/kg bw/day group during the gestation and lactation periods. This observation was considered a spontaneous change. No other abnormalities were noted in any main study group or satellite group, respectively during clincal observations. The detailed clinical observations revealed no significant differences between the test substance groups and the control group.
BODY WEIGHT AND WEIGHT GAIN
No significant differences were noted in body weight or body weight gain between the test substance groups and the control group (main study groups and satellite groups).
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No significant differences were noted in food consumption between the test substance groups and the control group (main study groups and satellite groups).
HAEMATOLOGY
Males, main study group [at the end of administration period]:
In the 100 mg/kg bw/day group, reticulocyte count was significantly lower than in the control group, which was considered an incidental change because it was not dose-related. No significant differences were noted in any examined parameter in the 300 or 1000 mg/kg bw/day group compared to the control.
Females, main study group [at the end of administration period]:
In the 100 mg/kg bw/day group, no significant differences were noted in any examined parameter compared to the control group. In the 300 mg/kg bw/day group, platelet count was significantly low, which was considered an incidental change because it was not dose-related. In the 1000 mg/kg bw/day group, mean corpuscular hemoglobin (MCH) and prothrombin time (PT) were significantly low. These changes were considered incidental, because no changes were detected in related parameters, whose values were within the range of historical control data.
Females, satellite group (non-mating group) [at the end of administration period]:
In the 1000 mg/kg bw/day group, no significant differences were noted in any test item compared to the control.
Males, satellite group [at the end of recovery period]:
In the 1000 mg/kg bw/day group, platelet count was significantly higher than in the control group, which was considered an incidental change because it was not detected at the end of administration period and was within the range of historical control data.
Females, satellite group (non-mating group) [at the end of recovery period]:
In the 1000 mg/kg bw/day group, no significant differences were noted in any examined parameter compared to the control group.
CLINICAL CHEMISTRY
Males, main study group [at the end of administration period]:
In the 100 mg/kg bw/day group, γ-GTP was significantly low compared to the control group, which was considered an incidental change because it was not dose-related. In the 300 and 1000 mg/kg bw/day groups, no significant differences were noted in any test parameter compared to the control group.
Females, main study group [at the end of administration period]:
Total protein and potassium in the 100 mg/kg bw/day group were significantly lower than in the control group. Potassium in the 300 mg/kg bw/day group was significantly low. In the 1000 mg/kg bw/day group total bilirubin, total bile acid, total protein, potassium and calcium were significantly low. These changes were considered unrelated to the test substance administration because they were not dose-related or no changes were noted in their related parameters, and the values were within the range of historical control data.
Females, satellite group (non-mating group) [at the end of administration period]:
In the 1000 mg/kg bw/day group, total bilirubin was significantly lower then in the control group, which was considered unrelated to the test substance administration because the values were within the range of historical control data.
Males, satellite group [at the end of the recovery period]:
Total bile acid and creatinine in the 1000 mg/kg bw/day group were significantly lower than in the control group. These changes were considered incidental because they were not detected at the end of administration period and were within the ranges of historical control data.
Females, satellite group (non-mating group) [at the end of recovery period]:
γ-globulin fraction in the 1000 mg/kg bw/day group was significantly higher than in the control group. This was considered an incidental change because it was not detected at the end of administration period and were within the ranges of historical control data.
URINALYSIS
Males, main study group [administration week 6]:
In the 300 and 1000 mg/kg bw/day groups, urine pH was significantly high and urinary protein significantly increased compared to the control group. The high urine pH was considered an incidental change due to the fact, that a large part of rats in the historical control data showed urine pH 8.5 (among 90 rats, pH was 6.5 in 2 rats, 7.0 in 1 rat, 8.0 in 16 rats, and 8.5 in 70 rats), and that many rats in the control group in the present study showed pH 8.0. The urine protein changes (the number of animals indicating + or 2+ increased) were within the range of historical control data (among 90 male rats, - in 4 males, ± in 35 males, + in 42 males, 2+ in 9 males). No damage in the kidneys or no systemic abnormalities were noted in males at the doses up to 1000 mg/kg bw/day. Therefore, these changes were considered incidental and unrelated to test substance administration.
Females, satellite group (non-mating grop) [administration week 6]:
Urinary protein significantly increased (the number of animals indicating + increased) in the 1000 mg/kg bw/day group. This change, however, was within the range of historical control data (among 90 female rats, - in 23 females, ± in 36 females, + in 29 females, 2+ in 2 females). No significant differences wer noted in other examined parameters compared to the control group.
In male and female satellite recovery groups no significant differences were noted in any examined parameter between the test substance groups and the control group.
NEUROBEHAVIOUR
Males, main study group [administration week 6]:
Motor activity count was significantly high (30 to 40 min) in males of the 100 mg/kg bw/day group, which was considered incidental, because the change was not dose-related.
In female main study groups and in male and female satellite groups, no significant differences were noted in any parameter compared to the control group.
ORGAN WEIGHTS
Males, main study group [at the end of administration period]:
The relative kidney weight in all dose groups were significantly higher than in the control group. The absolute kidney weight was also significantly high in the 300 mg/kg bw/day group. These changes were not dose-related, the absolute weight was not changed in the 100 and 1000 mg/kg bw/day groups, and no effects of the test substance administration were found histopathologically; thus, these findings were considered incidental changes unrelated to the test substance administration. In the 1000 mg/kg bw/day group, the relative liver weight was significantly increased, which was considered an incidental range because the absolute weight was not changed and histopathological examination revealed no effects of the test substance administration.
Females, main study group [at the end of administration period]:
No significant differences were noted in organ weight measured in the 100 mg/kg bw/day group compared to the control group. In the 300 mg/kg bw/day group, the relative uterus weight was significantly increased, which was considered an incidental change because it was not dose-related. The absolute and relative liver and kidney weight was significantly increased in the 1000 mg/kg bw/day group.
Females, satellite group (non-mating group) [at the end of the administration period]:
The absolute heart weight was significantly low in the 1000 mg/kg bw/day group, which was considered an incidental change because no significant differences were noted in the relative weight.
Males, satellite group [at the end of the recovery period]:
In the 1000 mg/kg bw/day group, the absolute and relative kidney and adrenal weight was significantly increased and absolute and relative seminal vesicle weight was significantly decreased. These changes were not observed at the end of the administration period and no effects of the test substance administration were found histopathologically, therefore these changes were considered incidental.
Females, satellite group (non-mating group) [at the end of the recovery period]:
The absolute and relative ovary weight was significantly decreased in the 1000 mg/kg bw/day group, which was considered incidental because this was not observed at the end of administration period and histopathological examination revealed no abnormalities.
GROSS PATHOLOGY
Males, main study group [at the end of administration period]:
No abnormal findings were noted in the control or 100 mg/kg bw/day group. Large size of the thymus was noted in one male in the 300 mg/kg bw/day group, which was considered incidental because no abnormal findings were noted in the 1000 mg/kg bw/day group.
Females, main study group [at the end of administration period]:
Grayish white subcutaneous mass was noted in one female in the 1000 mg/kg bw/day group, whtih was considered a spontaneous change because this was also observed in the control group. No abnormal findings were found in the 100 or 300 mg/kg bw/day groups.
Females, satellite group (non-mating group) [at the end of administration period]:
No abnormal finding were noted in the control group. In the 1000 mg/kg bw/day group, multifocal black focus in the glandular stomach mucosa was noted in one female, which was considered unrelated to test substance administration because this occurred in only one animal.
Males, satellite group [at the end of recovery period]:
No abnormal findings were noted in then control or 1000 mg/kg bw/day group.
Females, satellite group (non-mating group) [at the end of recovery period]:
No abnormal findings were noted in then control or 1000 mg/kg bw/day group.
HISTOPATHOLOGY: NON-NEOPLASTIC
Males, main study group [at the end of the administration period]:
Hyperplasia of squamous cells at limiting ridge of the stomach (slight) was noted in all 7 males in the 1000 mg/kg bw/day group with significantly high incidence, indicating slight irritability to the stomach mucosa. This effect was considered an effect of the test substance administration. No abnormal findings were noted in the stomach in the 100 or 300 mg/kg bw/day group. Other findings were aggregation of alveolar macrophage in the lung, focal atrophy of the acinar cells in the pancreas, microgranuloma in the liver, atrophy of the seminiferous tubulus in the testis, interstitial inflammation in the prostate, ultimobranchial body in the thyroid, and focal atrophy of retina sporadically observed in the 1000 mg/kg bw/day group. These findings were observed also in the control group or historical control data, and there were no differences in their incidences or grades compared the control group. Therefore these changes were considered unrelated to the test substance administration. The male with macroscopic findings in the 300 mg/kg bw/day group showed also lymphoma in the thymus.
Females, main study group [at the end of the administration period]:
In the 1000 mg/kg bw/day group, aggregation of alveolar macrophage in the lung, microgranuloma in the liver, cyst, basophilic change of renal tubule and hyaline cast in the kidney, ultimobranchial body in the thyroid, retinal rosette, and adenocarcinoma in the mammary gland were sporadically observed. These findings were observed also in the control group or historical control data and there were no differences in their incidences or grades compared to the control group; therefore, they were considered unrelated to the test substance administration.
Females, satellite group (non-mating group) [at the end of the administration period]:
In the 1000 mg/kg bw/day group, glandular stomach erosion, cyst in pars nervosa in the pituitary gland, ultimobronachial body in the thyroid, and retinal rosette were sporadically observed. These findings were observed also in the control group or historical control data, and there were no differences in their incidences or grades compared to the control group; therefore these findings were considered unrelated to the test substance administration.
Males, satellite group [at the end of recovery period]:
In the 1000 mg/kg bw/day group, aggregation of alveolar macrophage in the lung, focal atrophy of the acinar cells in the pancreas, hyline cast, and basophilic change of renal tubule in the kidney, interstitial inflammation in the prostate, ultimobranchial body in the thyroid, and cyst of zona reticularis in the adrenal were sporadically observed. These findings were observed also in the control group or historical control data, and there were no differences in their incidences or grades compared to the control group; therefore, they were considered unrelated to the test substance administration.
Females, satellite group (non-mating group) [at the end of recovery period]:
In the 1000 mg/kg bw/day group, microgranuloma in the liver, cyst in pars distalis in the pituitary gland, and ultimobranchial body in the thyroid were sporadically observed. These findings were observed also in the control group or historical control data, and there were no differences in their incidences or grades compared to the control group; therefore, they were considered unrelated to the test substance administration. - Dose descriptor:
- NOAEL
- Remarks:
- systemic
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects upto and including the highest dose
- Dose descriptor:
- NOAEL
- Remarks:
- local
- Effect level:
- 300 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male
- Basis for effect level:
- other: at 1000 mg/kg bw/day: Hyperplasia of squamous cells at limiting ridge of the stomach (slight), indicating slight irritability to the stomach mucosa
- Dose descriptor:
- NOAEL
- Remarks:
- local
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- other: no adverse effects upto and including the highest dose
- Critical effects observed:
- not specified
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Version / remarks:
- adopted in 1998
- Deviations:
- yes
- Remarks:
- some additional endpoints were considered, including e.g., estrous cyclicity and sperm analysis
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Version / remarks:
- adopted in 2008
- Deviations:
- yes
- Remarks:
- some additional endpoints were considered, including e.g., estrous cyclicity and sperm analysis
- GLP compliance:
- yes
- Limit test:
- no
Test material
- Reference substance name:
- {1,4-diazabicyclo[2.2.2]octan-2-yl}methanol
- EC Number:
- 692-731-2
- Cas Number:
- 76950-43-1
- Molecular formula:
- C7H14N2O
- IUPAC Name:
- {1,4-diazabicyclo[2.2.2]octan-2-yl}methanol
- Test material form:
- solid
Constituent 1
- Specific details on test material used for the study:
- Name: RZETA (A mixture composed of (1,4-diazabicyclo[2.2.2]octane-2- yl)methanol (89.8%) and 1,5-diazabicyclo[3.2.2]nonan-3-ol (9.8%)
Supplier: Tosoh Corporation, Japan
Lot No.: 6Z02RZETACR
Analytical purity for 76950-43-1 (1,4-diazabicyclo[2.2.2]octane-2-yl)methanol): 89.8%
Appearance: pale yellow solid
Expiration date: December 2, 2018
Storage conditions: at room temperature in an airtight, light-protected, and N2 sealed container.
Certificate of Analysis of the test article provided: yes, attached to this report (Annex 1)
Test animals
- Species:
- rat
- Strain:
- other: Crl:CD(SD)
- Details on species / strain selection:
- as recommended by the OECD Guideline 408
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Females nulliparous and non-pregnant: not stated
- Age at study initiation: 5 weeks (males) and 6 weeks (females)
- Weight at study initiation: 170 - 192 g (males) and 150 - 172 g (females)
- Fasting period before study: no
- Housing: animals were housed in metal bracket cages (260 W × 380 D × 180 H, mm) with wire mesh floors; three or less animals per cage during the quarantine and acclimation period, and one after group assignment
- Diet: Pellet diet, CRF-1, Oriental Yeast Co., Ltd., ad libitum
- Water: Sapporo-City tap water, ad libitum
- Acclimation period: at least 6 days
DETAILS OF FOOD AND WATER QUALITY:
According to the study report, each lot of the diet used was analyzed for contaminants; the contamination analyses were performed by Eurofins Food and Product Testing Japan, K.K. (Analysis report: Nos. AR-16-JP-110873-01-JA, AR-16-JP-113015-01-JA and AR-17-JP-001203-01-JA) and the diet manufacturer (Analytical test report Nos. 16G03-103, 16G03-117 and 17G03-013). The analysis data of each lot was obtained from the diet manufacturer. The analysis items and the acceptable values were those specified in the Standard Operating Procedures of Safety Research Institute for Chemical Compounds Co., Ltd., and all values were within the acceptable ranges. No further details are available from the study report.
According to the study report, a sample of drinking water was collected at the animal room No. 301 used in this study on January 5, 2017, and July 4, 2017, which was analyzed for contaminants. The analysis was performed by Nihon Eisei Co., Ltd. (Results of water quality test: No. A285508 and A291241) and the analysis data were obtained. The analysis items and the acceptable values were those specified in the Standard Operating Procedures of Safety Research Institute for Chemical Compounds Co., Ltd., and all values were within the acceptable ranges. No further details are available from the study report.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 24 Jan To: 10 May 2017
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- Otsuka distilled water
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test article was accurately weighed out, to which the vehicle was added to achieve the prescribed concentration; the test article was dissolved using a stirrer. Dosing solution was used within the verified stability period (within 8 days). Dosing solutions were refrigerated (actual range 1.7 ºC to 7.1 ºC) in an air-tight and light-protected container.
VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL, respectively
- Amount of vehicle: 10 mL/kg bw
- Lot/batch nos. : 6I83 and 6G74 - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Stability of the dosing solutions:
According to the study report, the stability of dosing solutions was investigated prior to the conduct of the present study (Report NCAS 14-012). Within this study, the stability of 1 and 100 mg/mL preparations of the test item during 8-day storage under refrigeration followed by 5-hr storage at room temperature after preparation was confirmed. With respect to the dose levels considered within the present study, the concentrations of test item in the dosing solutions were analysed at the first preparation, at mid-term of administration period and at the final preparation; in fact, all prepared concentrations were subjected to analysis. The analytic study report (study No. 16-182) is provided within the 90-Day study report in Annex 2. In brief, the analytical results confirmed adequate concentrations of the test item in the dosing solutions: the nominal concentrations of 10, 30 and 100 mg/L which were analysed showed final dose concentrations of 9.98, 31.1 and 101 mg/mL, respectively, indicating recoveries >= 99.8%. - Duration of treatment / exposure:
- 90 or 91 days, followed by a 14 days post-exposure (recovery) observation period
- Frequency of treatment:
- once daily, 7 days/week
Doses / concentrationsopen allclose all
- 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:
- 10 (main study)
5 (recovery) - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The dosage levels were selected based on the results of a previous screening test according to OECD Guideline 422 conducted with the test item (Fuji, 2014). In this study, the test item was administered at 0, 100, 300, and 1000 mg/kg bw/day by gavage to male and female Crl:CD rats. Squamous cell hyperplasia at the limiting ridge of the stomach with high incidence in males at 1000 mg/kg bw/day and increased liver and kidney weight in females at 1000 mg/kg bw/day were reported. Therefore, 1000 mg/kg bw/day was selected as the highest test dose for the present study, and 300 and 100 mg/kg bw/day were selected by dividing the highest dose by a common ratio of 3.
- Post-exposure recovery: 14 days
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily during acclimation; at least twice daily, before and after dosing, during administration period; at least twice daily, in the morning and afternoon, during recovery period
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the start of administration, on administration day 7, and once a week thereafter
BODY WEIGHT: Yes
- Time schedule for examinations: on administration days 1, 4, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 90, and necropsy day; before dosing on each measurement day; during recovery period: on recovery days 1, 7, and 14
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of administration (during the acclimation period) and in administration week 12 and recovery week 2
- Dose groups that were examined: all animals before the start of administration and during recovery period; all animals in the control- and high dose-toxicity study groups and recovery groups during administration period
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: red blood cell count (RBC), hematocrit (HCT), hemoglobin concentration (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (Platelet), white blood cell count (WBC), reticulocyte count (Reticulocyte), differential count of WBC (neutrophils, eosinophils, basophils, monocytes, lymphocytes), prothrombin time (PT), activated partial thromboplastin time (APTT)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters examined: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GTP), glucose, total cholesterol (T-Cho), triglyceride (TG), total bilirubin (T-Bil), urea nitrogen (UN), creatinine (Crea), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), inorganic phosphorus (IP), total protein (TP), albumin/globulin ratio (A/G ratio), protein fraction (albumin, α1-globulin, α2-globulin, β-globulin, γ-globulin)
URINALYSIS: Yes
- Time schedule for collection of urine: in administration week 13 and recovery week 2
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters examined: pH, protein, glucose, ketone body, urobilinogen, bilirubin, occult blood, urine volume, specific gravity
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in administration week 13 and recovery week 2
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
ORGAN WEIGHTS: Yes
- the following organ weights were recorded for all animals (except the one that died): brain, heart, liver, kidneys, lung, testes, epididymides, seminal vesicles (with coagulating glands), spleen, thymus, adrenals, pituitary gland, and thyroids (with parathyroids), prostate, ovaries, uterus
HISTOPATHOLOGY: Yes
- The following organs and tissues were preserved and examined: brain, pituitary gland, spinal cord (cervical, mid thoracic, and lumber regions), thyroids, parathyroids, thymus, heart, lung, larynx, trachea, bronchus, aorta, liver, spleen, kidneys, adrenals, tongue, submandibular lymph nodes, salivary glands (submandibular glands, sublingual glands), esophagus, stomach, duodenum, jejunum, ileum (with Peyer’s patches), cecum, colon, rectum, mesenteric lymph nodes, pancreas, mammary gland (right abdomen), skin (abdomen), urinary bladder, testes (left side), epididymides, prostate, seminal vesicles (with coagulating glands), ovaries, uterus, vagina, eyeballs, harderian glands, sternum (with bone marrow), femur (with bone marrow, right), skeletal muscle (biceps femoris) with sciatic nerve, and gross lesions (with a border to the normal tissue)
- Microscopic specimens of all the organs and tissues fixed and preserved at necropsy were prepared in the control and high dose-toxicity study groups and microscopically examined. Histopathological examination of the high dose group revealed effects of the test article administration in the stomach of males and females. Consequently, microscopic specimens were prepared from all animals in the middle- and low-dose groups and recovery groups and microscopically examined.
- For the animal that died, microscopic specimens were prepared from all the organs and tissues fixed and preserved and microscopically examined. - Other examinations:
- ESTROUS CYCLICITY:
- Time schedule for examination: main test, from administration day 77 to 90; recovery, from recovery day 1 to 14
- Dose groups that were examined: main test, all groups; recovery, control and 1000 mg/kg bw/day group
SPERM ANALYSIS:
- Time schedule for examination: main test, at necropsy; recovery, at necropsy
- Dose groups that were examined: main test, all groups; recovery, control and 1000 mg/kg bw/day group - Statistics:
- please refer to section "Any other information on materials and methods"
Results and discussion
Results of examinations
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- 13-Week treatment:
No clinical symptoms of toxicity were noted during clinical and detailed observations of all treated male and female rats; the control animals were inconspicuous.
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group):
No clinical symptoms of toxicity were noted during clinical and detailed observations of the male and female rats of the treated group during the recovery period. The control animals were inconspicuous. - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One male in the mid-dose group died within the course of day 87 of treatment; this case of death was rather incidental and not related to the treatment; no clear cause of death could however be assessed.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 13-Week treatment:
Except for the female rats of the mid-dose group (i.e., 300 mg/kg bw/day), no effects on body weight and body weight gain were noticed. In fact, for the females of the mid-dose group, body weight on treatment days 28 to 56 and on day 70, was statistically significantly reduced, compared to untreated females of the control group. The following mean body weight values were reported:
For day 28, at 300 mg/kg bw/day, the mean body weight for female rats was 230 ± 14.2 g versus 250.2 ± 21 g for control (p<0.05); for purpose of comparison, the mean body weight was 240.4 ± 15.3 and 241.8 ± 22.3 g for the 100 and the 1000 mg/kg bw/day group, respectively.
For day 56, at 300 mg/kg bw/day, the mean body weight for female rats was 263.3 ± 17.3 g versus 290.5 ± 21.7 g for control (p<0.05); for purpose of comparison, the mean body weight was 275.1 ± 21.6 and 280.7 ± 29.1 g for the 100 and the 1000 mg/kg bw/day group, respectively.
For day 70, at 300 mg/kg bw/day, the mean body weight for female rats was 281.1 ± 19.0 g versus 305.7 ± 21.6 g for control (p<0.05); for purpose of comparison, the mean body weight was 294.8 ± 23.8 and 300.8 ± 28.1 g for the 100 and the 1000 mg/kg bw/day group, respectively.
With respect to the body weight gain, this also was statistically significantly reduced for the females of the same test group, i.e., 300 mg/kg bw/day, on treatment days 4 to 77 and on day 90, compared to untreated control females. The following mean body weight gain values were reported:
For day 4, at 300 mg/kg bw/day, the mean body weight gain for female rats was 11.6 ± 3.8 g versus 15.9 ± 4.2 g for control (p<0.05); for purpose of comparison, the mean body weight gain was 12.6 ± 3.2 and 15.7 ± 3.2 g for the 100 and the 1000 mg/kg bw/day group, respectively.
For day 14, at 300 mg/kg bw/day, the mean body weight gain for female rats was 40.7 ± 10.2 g versus 52.7 ± 9.2 g for control (p<0.01); for purpose of comparison, the mean body weight gain was 45.2 ± 10.2 and 46.5 ± 8.6 g for the 100 and the 1000 mg/kg bw/day group, respectively.
For day 35, at 300 mg/kg bw/day, the mean body weight gain for female rats was 80.9 ± 12.3 g versus 104.0 ± 15.1 g for control (p<0.01); for purpose of comparison, the mean body weight gain was 88.9 ± 17.3 and 92.4 ± 21.6 g for the 100 and the 1000 mg/kg bw/day group, respectively.
For day 77, at 300 mg/kg bw/day, the mean body weight gain for female rats was 125.1 ± 17.9 g versus 149.5 ± 16.4 g for control (p<0.05); for purpose of comparison, the mean body weight gain was 138.5 ± 26.0 and 143.9 ± 29.8 g for the 100 and the 1000 mg/kg bw/day group, respectively.
For day 90, at 300 mg/kg bw/day, the mean body weight gain for female rats was 128.7 ± 18.6 g versus 154.1 ± 15.1 g for control (p<0.05); for purpose of comparison, the mean body weight gain was 142.2 ± 25.2 and 146.0 ± 29.9 g for the 100 and the 1000 mg/kg bw/day group, respectively.
No such findings were reported for the male rats of all treated groups. Thus, these findings were not considered to be due to the treatment.
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group):
No statistically significant differences were noted with respect to body weight and body weight gain of the male and female rats of the high-dose group (i,e., 1000 mg/kg bw/d) compared with those in the control group. - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- 13-Week treatment:
Except for the female rats of the mid-dose group (i.e., 300 mg/kg bw/day), no effects on food consumption were noticed. The findings as reported for the mid-dose females were not considered to be treatment-related. In fact, food consumption for female rats in the mid-dose group was statistically significantly reduced on day 21 of treatment, when compared to the females of the control group (18.18 ± 1.37 g versus 20.54 ± 1.95 g). No such findings were reported for the females of the low- and high-dose groups (i.e., 100 and 1000 mg/kg bw/day); no such findings were reported for the male rats of all treated groups.
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group):
No statistically significant differences were noted with respect to food consumption for male or female rats in the 1000 mg/kg bw/day group compared with those in the control 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):
- Ophthalmological examinations revealed no abnormalities in any of the test animals.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 13-Week treatment, male rats (Table 1A):
Hemoglobin concentration (HGB) was statistically significantly decreased in the male rats of the high-dose group (.i.e., 1000 mg/kg bw/day) compared to the control value (14.85 ± 0.72 g/dL versus 16.11 ± 0.28 g/dL for control; p<0.01).
Hematocrit (HCT) was statistically significantly decreased in the male rats of the high-dose group compared to the control value (41.36 ± 1.85% versus 45.10 ± 0.73% for control; p<0.01).
Mean corpuscular volume (MCV) was statistically significantly decreased in the male rats of the high-dose group compared to the control value (47.32 ± 1.29 fL versus 50.77 ± 2.00 fL for control; p<0.01).
Mean corpuscular hemoglobin (MCH) was statistically significantly decreased in the male rats of the high-dose group compared to the control value (16.99 ± 0.47 pg versus 18.13 ± 0.68 pg for control; p<0.01).
Platelet count was statistically significantly increased in the male rats of the high-dose group compared to the control value (125.31 ± 17.80 104/μL versus 102.62 ± 7.55 104/μL for control; p<0.01).
Prothrombin time (PT) was statistically significantly increased in the male rats of the high-dose group compared to the control value (23.86 ± 4.05 sec versus 18.49 ± 2.83 sec for control; p<0.05).
Activated partial thromboplastin time (APTT) was statistically significantly increased in the male rats of the high-dose group compared to the control value (21.27 ± 3.30 sec versus 17.73 ± 2.22 sec for control; p<0.01).
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), male rats (Table 1B):
Hematological examinations in those males of the high-dose group which were subjected to an additional treatment-free 14 day-recovery period, revealed no statistically significant differences between treated and control animals.
13-Week treatment, female rats (Table 1C):
Activated partial thromboplastin time (APTT) was statistically significantly increased in the female rats of the high-dose group compared to the control value (13.47 ± 1.45 sec versus 11.54 ± 1.30 sec for control; p<0.01).
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), female rats (Table 1D):
Hematological examinations in those female rats of the high-dose group which were subjected to an additional treatment-free 14 day-recovery period, revealed no statistically significant differences between treated and control animals.
In fact, regarding the treated males, the decreased platelets count and PT value as reported were statistically significantly different from the control values; however they remained with the respective historical control ranges of the testing facility (64 to 155.8 × 104/μL for PLT and 15 to 31.9 sec for PT; Historical control data on hematology (2012 to 2017). Data on file. Safety Research Institute for Chemical Compounds Co., Ltd.). With respect to the decreased MCV, MCH, HGB and HCT values, the latter was reported being slightly below the historical control range of the testing facility in only 2/10 males; no information regarding historical control range was reported with respect to MCV, MCH, HGB. Nevertheless, since no supporting changes in red blood cell count (RBC), mean corpuscular hemoglobin concentration (MCHC) and/or reticulocytes count were noticed and since at necropsy, histopathological examination provided no evidence for bleeding, the effects as described above are neither considered adverse nor to be of toxicological relevance. Regarding the APTT value, this also slightly exceeded the historical control range of the testing facility in only 2/10 males (i.e > 28.4 sec; according to historical control data on hematology (2012 to 2017). Data on file. Safety Research Institute for Chemical Compounds Co., Ltd.).
For the females, the APTT value remained within the historical control range of the testing facility for this gender (9.9 – 18.8 sec for females, according to historical control data on hematology (2012 to 2017). Data on file. Safety Research Institute for Chemical Compounds Co., Ltd.).
No such effects were reported for the low- and mid-dose levels (i.e., 100 and 300 mg/kg bw/day) for both, male and female rats.
Thus, with respect to hematology, effects almost were noticed at the highest dose level of 1000 mg/kg bw/day. Taking into account both the results after treatment and after recovery, the effects are neither considered to be adverse nor of particular toxicological relevance, and none of them is considered treatment-related. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 13-Week treatment, male rats (Table 2A):
The alkaline phosphatase (ALP) level was statistically significantly increased in the male rats of the high-dose group (i.e., 1000 mg/kg bw/day) compared to the control value (477.7 ± 114.5 IU/L versus 304.0 ± 63.4 IU/L for control; p<0.01).
The Gamma- glutamyl transpeptidase (γ-GTP) level was statistically significantly increased in the male rats of the high-dose group (i.e., 1000 mg/kg bw/day) compared to the control value (0.53 ± 0.18 IU/L versus 0.24 ± 0.23 IU/L for control; p<0.05).
Calcium (Ca) also was statistically significantly increased in the male rats of the high-dose group (i.e., 1000 mg/kg bw/day) compared to the control value (10.01 ± 0.30 mg/dL versus 9.59 ± 0.26 mg/dL for control; p<0.05).
Total cholesterol was statistically significantly decreased in the male rats of the high-dose group (i.e., 1000 mg/kg bw/day) compared to the control value (38.6 ± 9.4 mg/dL versus 53.9 ± 11.1 mg/dL for control; p<0.01).
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), male rats (Table 2B):
No relevant effects were noticed.
13-Week treatment, female rats (Table 2C):
Alanine aminotransferase (ALT) was statistically significantly decreased in mid- and high-dose females (i.e., 300 and 1000 mg/kg bw/day) compared to the control values (27.2 ± 6.4 IU/L for the mid dose, p<0.05 and 23.4 ± 7.6 IU/L for the high dose, p<0.01, versus 49.3 ± 28.6 IU/L for control).
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), female rats (Table 2D):
The only remarkable effect noticed at the end of the recovery period, was a statistically significant increase in inorganic phosphorus reported for the females of the high-dose group; no such finding was observed at the end of administration period. Thus, the increased inorganic phosphorus level was not given more importance, since no further concomitant findings were noticed.
Regarding the males, for the increased γ-GTP and Ca levels, the values in 9/10 male rats were statistically significantly different from control but remained within the respective historical control ranges of the testing facility (0.0 – 0.8 IU/L for γ-GTP and 8.8 – 10.4 mg/dL for Ca; Historical control data on biochemistry (2012 to 2017). Data on file. Safety Research Institute for Chemical Compounds Co., Ltd.). Only in one male each, the γ-GTP and the Ca level was slightly above the upper historical control limit, respectively.
With respect to the total cholesterol, only in 3/10 animals the values were found below the lower limit of the historical control range of the testing facility (36 – 100 mg/dL; Historical control data on biochemistry (2012 to 2017). Data on file. Safety Research Institute for Chemical Compounds Co., Ltd.
In conclusion, since the reported changes were neither accompanied nor supported by any relevant histopathological findings in organs of concern, i.e., liver and/or thyroid, and in the absence of any relevant changes affecting other particular parameters such as e.g. the triglyceride (TG) level, the clinical-chemical findings in the male rats were considered to be neither adverse nor toxicologically relevant.
With respect to the decreased ALT levels reported for mid- and high-dose females, these are considered of no toxicological relevance, since, according to the study report, low values are clinically not important. Further, according to the study report, individual values at both dose levels were within the historical control range of the testing facility. Hereby it is to be noticed that the unit for ALT values as reported in the tables of the study report was given as IU/L whereas the historical control range refers to the values in mg/dL (18 – 119 mg/dL).
Additional findings such as a statistically significant decrease in total bilirubin seen in males of the low-dose group only, or a significant decrease in aspartate aminotransferase (AST) as reported for the females of the mid-dose group only, are considered incidental and not related to treatment.
Thus, with respect to clinical-chemistry, effects were noticed at the highest dose level of 1000 mg/kg bw/day. Taking into account both the results after treatment and after recovery, the effects are neither considered to be adverse nor of particular toxicological relevance, and none of them is considered treatment-related. - Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 13-Week treatment, male rats:
Urinalysis revealed statistically significantly increased protein content and specific gravity for the males of the high-dose group (i.e., 1000 mg/kg bw/day) when compared to control.
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), male rats:
All parameters considered within urinalysis were inconspicuous for the males.
13-Week treatment, female rats:
Urinalysis revealed statistically significant increases in protein content and increased pH for the females of the high-dose group (i.e., 1000 mg/kg bw/day): a statistically significant increase in protein content was also reported for the females of the mid-dose group (i.e., 300 mg/kg bw/day).
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), female rats:
Except for a statistically significantly decreased specific gravity, all parameters considered within urinalysis were inconspicuous for the females. No particular toxicological meaning can be associated to this finding.
Thus, with respect to urinalysis, the main finding for both male and female rats refers to statistically significantly elevated urinary protein content noticed at the high-dose level of 1000 mg/kg bw/day. In this group, specific gravity was also elevated, however in males only, whereas high pH was reported for females only. In those animals of the high-dose group subjected to an additional 14-day recovery period (i.e., without treatment), all parameters considered within urinalysis were inconspicuous for males whereas for the females, a statistically significantly decreased specific gravity was the only effect noticed. Taking into account the fact that necropsy revealed no concomitant findings affecting the kidneys, and that no clinical-chemical changes were found that may reflect any impairment of the renal function, and in the view of the results following recovery, the effects described above are considered neither as adverse nor as toxicologically relevant. Thus no treatment-related effect could be evidenced within urinalysis. - Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- Significant differences in functional observations in treated males or females (any dose level) compared with the control group were neither seen within the treatment nor during recovery.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 13-Week treatment, male rats (Table 3A):
At necropsy, weighing of organs revealed that the mean absolute liver weight was statistically significantly increased in male rats of the high-dose group (1000 mg/kg bw/day), as compared to control (16.29 ± 2.024 g versus 14.06 ± 1.412 g; p<0.05); the mean relative liver weight also was statistically significantly increased (2.902 ± 0.204 versus 2.545 ± 0.236 g/100 g bw; p<0.01). Also, for these males, the mean absolute kidney weight was statistically significantly increased as compared to control (3.742 ± 0.367 g versus 3.258 ± 0.330 g; p<0.05), as well as the mean relative kidney weight (0.669 ± 0.054 versus 0.590 ± 0.045 g/100 g bw; p<0.01). For the males of the low- and mid-dose group, values with respect to liver and kidneys were comparable to control and thus, inconspicuous.
Further statistically significant but incidental differences were noticed for the mean absolute and relative thymus weights which were significantly increased compared to control values in males of the low- and high-dose groups (100 and 1000 mg/kg bw/day). No such findings were noticed at 300 mg/kg bw/day. Further, histopathological examination for the high dose group revealed no concomitant abnormalities. A further incidental finding in males refers to the mean absolute testis weight which was found to be statistically significantly increased as compared to control value, but only in the low-dose group; testis weight in the other two test groups was inconspicuous.
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), male rats (Table 3B):
At the end of the recovery period, the mean relative liver weight of the high-dose male rats was reported to be statistically significantly increased as compared to control (2.620 ± 0.131 versus 2.362 ± 0.158 g/100 g; p<0.05); however, the extent of the increase was lowered as compared to the value after as reported for the 13 –Week treatment (2.902 ± 0.204 g/100 g bw), indicating reversibility. The mean absolute and relative kidney weights of the high-dose male rats both were comparable to those of the control animals.
13-Week treatment, female rats (Table 3C):
At necropsy, weighing of organs revealed that the mean relative kidney weight was statistically significantly increased in female rats of the high-dose group (1000 mg/kg bw/day), as compared to control (0.728 ± 0.060 versus 0.661 ± 0.060 g/100 g bw; p<0.05).The mean absolute kidney weight was comparable to control. The mean absolute and relative kidney weights of the low- and mid-dose female rats were comparable to those of the control animals. Since no concomitant histopathological findings could be evidenced, the increase in relative kidney weight as reported above is not considered as toxicologically relevant.
As statistically significant but incidental differences, the mean absolute heart weight and the mean absolute and relative pituitary gland weight were reported to be statistically significantly decreased for the females of the mid-dose group (i.e., 300 mg/kg bw/day); for the low- and high-dose group, the values were similar to control and thus inconspicuous.
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), female rats (Table 3D):
At the end of the recovery period, the mean relative spleen weight was reported to be statistically significantly increased in the high-dose females as compared to control (180.608 ± 17.691 versus 154.032 ± 13.916 mg/100 g bw; p<0.05). The mean absolute spleen weight was slightly but not statistically significantly increased as compare to control. Since no such finding was evidenced at the end of the treatment period, and since the finding was only seen in females, this was not considered a relevant toxicological finding.
Thus, the only changes in organ weight for which a relationship to treatment cannot be excluded refer to the statistically significant increases in mean absolute and relative liver weight and in mean absolute and relative kidney weight as reported for the male rats of the high-dose group (1000 mg/kg bw/day) at the end of the 13-Week treatment period. After recovery, the mean absolute liver weight still was slightly but no more statistically significantly increased as compared to control, whereas the mean relative liver weight of the high-dose male rats still was statistically significantly increased. However, with respect to the relative liver weight, the extent of the increase was lowered as compared to the value as reported for the 13–Week treatment. Thus the findings at the end of the recovery period are clearly indicative of reversibility. With respect to the kidney weights, the mean absolute and relative kidney weights of the high dose male rats after recovery both were comparable to those of the control animals, thus also indicating reversibility. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- 13-Week treatment, male rats:
At necropsy gross pathology revealed no abnormalities which could be associated to the treatment. In fact, only spontaneous findings including e.g., small sized testes and epididymis in one control animal and an unilateral yellowish white focus in the cauda of the epididymis in one high-dose animal, were noticed.
With respect to the one animal of the mid-dose group which died on day 87 of treatment, gross pathology was inconspicuous.
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), male rats:
Gross pathology was inconspicuous.
13-Week treatment, female rats:
At necropsy gross pathology revealed no abnormalities which could be associated to the treatment. In fact, only spontaneous findings including e.g., adhesion of the spleen to fat tissue and to the pancreas in one control animal, were noticed.
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), female rats:
Gross pathology was inconspicuous. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- 13-Week treatment, male rats (Table 4):
The main findings as revealed by histopathology for male rats were focused on the stomach of the animals of the high-dose group (1000 mg/kg bw/day). In fact, infiltration of inflammatory cells in the lamina propria at the limiting ridge of the stomach was noticed in 4 animals, hyperkeratosis at the limiting ridge of the stomach was noticed in 4 animals, and infiltration of inflammatory cells in the submucosa of the glandular stomach was noticed in 5 animals of this test group. All findings were graded +1 on a severity scale of 4. No such findings were detected in males of the control, the low- and the mid-dose groups. Thus a relationship to the treatment and the dose level cannot be excluded.
Further slight changes were occasionally noted in the lung, liver, kidneys, and other organs in males in the high-dose group. These were also noted in the control group, and were known to occur spontaneously, and thus were considered unrelated to the treatment.
With respect to the one male that died in the mid dose group, histopathology revealed slight aggregation of alveolar macrophages in the lung and slight focal inflammation in the heart.
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), male rats (Table 4):
At the end of the recovery period, histopathology revealed no abnormal findings; especially regarding the stomach no such findings as described above could be evidenced.
13-Week treatment, female rats (Table 4):
The main finding as revealed by histopathology for female rats was focused on the stomach of the animals of the high-dose group (1000 mg/kg bw/day). In fact, hyperkeratosis at the limiting ridge of the stomach was reported for 2 animals in this group; the finding was graded +1 on a severity scale of 4. No such finding was detected in females of the control, the low- and the mid-dose groups. Thus a relationship to the treatment and the dose level cannot be excluded, especially in the view of the findings reported above for the male rats of the high-dose group.
Further slight changes were occasionally noted in the lung, liver, kidneys, and other organs in females in the high dose group. These were also noted in the control group, and were known to occur spontaneously, and thus were considered unrelated to the treatment.
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), female rats (Table 4):
At the end of the recovery period, histopathology revealed no abnormal findings; especially regarding the stomach no such findings as described above could be evidenced. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- ESTROUS CYCLICITY
13-Week treatment, female rats:
Estrous cyclicity was not affected by the treatment; in fact no statistically significant differences were noticed when comparing all test groups including the control to each other.
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), female rats:
No significant differences in estrous cyclicity were noticed when comparing the high-dose group with the control.
SPERM ANALYSIS
13-Week treatment, male rats:
The only reported finding refers to the straight line velocity and straightness of epididymal sperm which were reported as statistically significantly high for the males of the high-dose group; however no differences in motility between this group and the control group were noticed. Further, there were no significant differences between the treated and control groups regarding all other parameters examined. Thus, the finding is considered incidental and not related to the treatment.
13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), male rats:
No significant differences were noticed between the high-ose and the control group with respect to all sperm parameters considered.
Effect levels
open allclose all
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- local
- Effect level:
- 300 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- systemic
- 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
Target system / organ toxicity
- Critical effects observed:
- no
Any other information on results incl. tables
Table 1A_ 13-Week treatment_male rats_Hematological parameters (mean value ± SD of n animals):
Parameters |
Dose level (mg/kg bw/day) |
|||
0 (n=10) |
100 (n=10) |
300 (n=9) |
1000 (n=10) |
|
RBC (104/μL) |
889.3 ± 29.4 |
907.5 ± 48.4 |
891.6 ± 30.1 |
874.3 ± 44.7 |
HGB (g/dL) |
16.11 ± 0.28 |
16.21 ± 0.43 |
15.69 ± 0.50 |
14.85 ± 0.72** |
HCT (%) |
45.10 ± 0.73 |
44.76 ± 1.24 |
43.78 ± 1.55 |
41.36 ± 1.85** |
MCV(fL) |
50.77 ± 2.00 |
49.42 ± 2.14 |
49.14 ± 2.31 |
47.32 ± 1.29** |
MCH (pg) |
18.13 ± 0.68 |
17.88 ± 0.72 |
17.61 ± 0.73 |
16.99 ± 0.47** |
MCHC (g/dL) |
35.74 ± 0.36 |
36.21 ± 0.39 |
35.84 ± 0.61 |
35.90 ± 0.35 |
Reticulocyte (%) |
2.877 ± 0.586 |
2.878 ± 0.180 |
2.863 ± 0.318 |
2.939 ± 0.345 |
Platelet (104/μL) |
102.62 ± 7.55 |
113.00 ± 13.23 |
112.84 ± 13.56 |
125.31 ± 17.80** |
WBC (102/μL) |
84.92 ± 19.27 |
79.36 ± 16.59 |
82.58 ± 16.31 |
101.72 ± 33.69 |
Neutrophil (102/μL) |
11.60 ± 2.86 |
10.64 ± 3.31 |
10.86 ± 3.00 |
14.34 ± 4.37 |
Lymphocyte(102/μL) |
68.76 ± 16.16 |
64.18 ± 14.11 |
67.27 ± 16.26 |
81.95 ± 29.59 |
Monocyte(102/μL) |
3.07 ± 0.94 |
3.03 ± 1.10 |
3.08 ± 1.10 |
3.30 ± 1.00 |
Eosinophil(102/μL) |
1.46 ± 0.77 |
1.49 ± 0.46 |
1.34 ± 0.40 |
2.10 ± 0.63 |
Basophil(102/μL) |
0.03 ± 0.05 |
0.02 ± 0.04 |
0.03 ± 0.05 |
0.03 ± 0.05 |
PT (sec) |
18.49 ± 2.83 |
20.30 ± 1.25 |
20.24 ± 2.24 |
23.86 ± 4.05* |
APTT (sec) |
17.73 ± 2.22 |
19.36 ± 1.46 |
19.64 ± 1.69 |
21.27 ± 3.30** |
* : p<0.05; **: p< 0.01
Table 1B_ 13-Week + 14-Day recovery_male rats_Hematological parameters (mean value ± SD of n animals):
Parameters |
Dose level (mg/kg bw/day) |
|
0 (n=5) |
1000 (n=5) |
|
RBC (104/μL) |
916.2 ± 57.2 |
894.6 ± 30.0 |
HGB (g/dL) |
15.80 ± 0.28 |
15.64 ± 0.34 |
HCT (%) |
44.54 ± 1.62 |
44.06 ± 1.33 |
MCV(fL) |
48.76 ± 3.70 |
49.28 ± 1.91 |
MCH (pg) |
17.30 ± 0.93 |
17.50 ± 0.55 |
MCHC (g/dL) |
35.48 ± 0.79 |
35.48 ± 0.41 |
Reticulocyte (%) |
3.792 ± 1.775 |
3.448 ± 0.737 |
Platelet (104/μL) |
105.32 ± 7.94 |
104.56 ± 7.34 |
WBC (102/μL) |
88.56 ± 22.32 |
79.94 ± 18.70 |
Neutrophil (102/μL) |
12.74 ± 5.31 |
17.70 ± 6.46 |
Lymphocyte(102/μL) |
70.68 ± 16.60 |
57.60 ± 14.33 |
Monocyte(102/μL) |
4.04 ± 1.37 |
3.36 ± 1.00 |
Eosinophil(102/μL) |
1.06 ± 0.32 |
1.26 ± 0.55 |
Basophil(102/μL) |
0.04 ± 0.05 |
0.02 ± 0.04 |
PT (sec) |
19.82 ± 4.12 |
21.64 ± 3.66 |
APTT (sec) |
18.20 ± 3.43 |
19.14 ± 2.23 |
Table 1C_ 13-Week treatment_female rats_Hematological parameters (mean value ± SD of n animals):
Parameters |
Dose level (mg/kg bw/day) |
|||
0 (n=10) |
100 (n=10) |
300 (n=10) |
1000 (n=10) |
|
RBC (104/μL) |
817.5 ± 44.6 |
823.0 ± 46.4 |
853.2 ± 38.1 |
801.6 ± 44.6 |
HGB (g/dL) |
15.14 ± 1.16 |
15.28 ± 0.58 |
15.82 ± 0.32 |
14.77 ± 0.64 |
HCT (%) |
42.34 ± 2.64 |
42.62 ± 1.54 |
43.74 ± 1.02 |
40.87 ± 1.77 |
MCV (fL) |
51.78 ± 1.41 |
51.87 ± 2.28 |
51.33 ± 1.96 |
51.04 ± 1.82 |
MCH (pg) |
18.51 ± 0.65 |
18.60 ± 0.80 |
18.58 ± 0.60 |
18.46 ± 0.57 |
MCHC (g/dL) |
35.73 ± 0.82 |
35.84 ± 0.22 |
36.18 ± 0.44 |
36.14 ± 0.34 |
Reticulocyte (%) |
3.113 ± 1.530 |
2.595 ± 0.558 |
2.650 ± 0.500 |
2.515 ± 0.785 |
Platelet (104/μL) |
105.60 ± 20.45 |
91.49 ± 8.88 |
92.25 ± 14.38 |
98.38 ± 14.24 |
WBC (102/μL) |
61.45 ± 32.01 |
47.40 ± 16.76 |
59.40 ± 16.69 |
53.26 ± 20.82 |
Neutrophil (102/μL) |
14.55 ± 22.20 |
6.52 ± 1.85 |
8.06 ± 3.28 |
6.81 ± 3.58 |
Lymphocyte(102/μL) |
43.67 ± 13.33 |
38.41 ± 15.38 |
48.41 ± 15.12 |
43.88 ± 17.95 |
Monocyte(102/μL) |
2.24 ± 1.34 |
1.65 ± 0.72 |
1.68 ± 0.63 |
1.77 ± 0.87 |
Eosinophil(102/μL) |
0.98 ± 0.37 |
0.81 ± 0.49 |
1.23 ± 0.64 |
0.79 ± 0.40 |
Basophil(102/μL) |
0.01 ± 0.03 |
0.01 ± 0.03 |
0.02 ± 0.04 |
0.01 ± 0.03 |
PT (sec) |
15.56 ± 0.79 |
15.69 ± 0.98 |
15.82 ± 0.70 |
15.61 ± 0.76 |
APTT (sec) |
11.54 ± 1.30 |
12.14 ± 1.23 |
12.41 ± 0.82 |
13.47 ± 1.45** |
* : p<0.0; **: p< 0.01
Table 1D_ 13-Week + 14-Day recovery_female rats_Hematological parameters (mean value ± SD of n animals):
Parameters |
Dose level (mg/kg bw/day) |
|
0 (n=5) |
1000 (n=5) |
|
RBC (104/μL) |
796.6 ± 38.9 |
823.8 ± 20.4 |
HGB (g/dL) |
14.86 ± 0.38 |
15.14 ± 0.78 |
HCT (%) |
41.82 ± 0.89 |
42.06 ± 1.69 |
MCV(fL) |
52.58 ± 1.88 |
51.06 ± 1.65 |
MCH (pg) |
18.68 ± 0.67 |
18.38 ± 0.61 |
MCHC (g/dL) |
35.52 ± 0.22 |
35.98 ± 0.70 |
Reticulocyte (%) |
3.280 ± 0,647 |
3.346 ± 0.436 |
Platelet (104/μL) |
94.02 ± 11.54 |
105.18 ± 20.06 |
WBC (102/μL) |
46.16 ± 16.09 |
54.16 ± 22.91 |
Neutrophil (102/μL) |
8.16 ± 2.32 |
7.94 ± 3.00 |
Lymphocyte(102/μL) |
35.40 ± 17.28 |
43.18 ± 19.98 |
Monocyte(102/μL) |
1.64 ± 0.21 |
1.98 ± 0.75 |
Eosinophil(102/μL) |
0.94 ± 0.25 |
1.06 ± 0.26 |
Basophil(102/μL) |
0.02 ± 0.04 |
0.00 ± 0.00 |
PT (sec) |
16.00 ± 0.51 |
16.92 ± 1.37 |
APTT (sec) |
12.38 ± 0.80 |
11.90 ± 1.39 |
Table 2A_ 13-Week treatment_male rats_Clinical chemical parameters (mean value ± SD of n animals):
Parameters |
Dose level (mg/kg bw/day) |
|||
0 (n=10) |
100 (n=10) |
300 (n=9) |
1000 (n=10) |
|
AST (IU/L) |
61 ± 8.0 |
57.8 ± 7.5 |
56.0 ± 6.9 |
66.1 ± 8.7 |
ALT (IU/L) |
29.5 ± 4.5 |
30.7 ± 4.1 |
27.6 ± 2.8 |
32.7 ± 7.5 |
ALP (IU/L) |
304.0 ± 63.4 |
313.1 ± 43.6 |
339.4 ± 79.1 |
477.7 ± 114.5** |
γ-GTP (IU/L) |
0.24 ± 0.23 |
0.45 ± 0.29 |
0.42 ± 0.20 |
0.53 ± 0.18* |
T-Bil (mg/dL) |
0.060 ± 0.013 |
0.047 ± 0.009 |
0.048 ± 0.011 |
0.049 ± 0.010 |
Glucose (mg/dL) |
162.4 ± 11.7 |
163.3 ± 16.2 |
174.9 ± 18.1 |
179.6 ± 16.9 |
T-Cho (mg/dL) |
53.9 ± 11.1 |
52.3 ± 5.2 |
48.0 ± 8.3 |
38.6 ± 9.4** |
TG (mg/dL) |
60.1 ± 30.2 |
72.7 ± 45.7 |
60.9 ± 20.3 |
61.6 ± 24.4 |
TP (g/dL) |
5.60 ± 0.16 |
5.63 ± 0.14 |
5.59 ± 0.15 |
5.57 ± 0.24 |
UN (mg/dL) |
13.72 ± 0.82 |
14.17 ± 1.39 |
14.16 ± 1.92 |
14.41± 2.59 |
Crea (mg/dL) |
0.307± 0.048 |
0.312± 0.019 |
0.290± 0.027 |
0.283± 0.054 |
Na (mEq/L) |
141.2± 1.5 |
142.5± 0.7 |
142.3± 1.3 |
142.5± 1.4 |
K (mEq/L) |
4.737± 0.207 |
4.672± 0.228 |
4.781± 0.178 |
4.835± 0.283 |
Cl (mEq/L) |
104.3± 1.6 |
104.5± 1.1 |
104.9± 1.1 |
104.7± 1.3 |
Ca (mg/dL) |
9.59± 0.26 |
9.88± 0.37 |
9.73± 0.25 |
10.01± 0.30* |
IP (mg/dL) |
6.64± 0.75 |
6.60± 0.67 |
6.58± 0.76 |
7.12± 0.71 |
A/G |
0.877± 0.065 |
0.859± 0.057 |
0.878± 0.060 |
0.894± 0.076 |
Albumin (%) |
46.72± 1.80 |
46.16± 1.72 |
46.70± 1.75 |
47.13± 2.04 |
α1-G (%) |
22.32± 2.60 |
24.03± 2.29 |
23.53± 1.73 |
22.02± 1.74 |
α2-G (%) |
9.52± 1.36 |
9.24± 0.92 |
8.83± 1.15 |
9.27± 1.11 |
β-G (%) |
16.21± 0.94 |
15.75± 0.69 |
15.48± 0.83 |
16.57± 1.31 |
γ-G (%) |
5.23± 0.70 |
4.82± 0.96 |
5.46± 0.95 |
5.01± 1.63 |
* : p<0.05; **: p< 0.01
Table 2B_ 13-Week + 14-Day recovery_male rats_Clinical chemical parameters (mean value ± SD of n animals):
Parameters |
Dose level (mg/kg bw/day) |
|
0 (n=5) |
1000 (n=5) |
|
AST (IU/L) |
62.6 ± 6.3 |
80.8 ± 31.2 |
ALT (IU/L) |
28.4 ± 3.2 |
39.6 ± 21.0 |
ALP (IU/L) |
257.6 ± 53.7 |
300.6 ± 113.4 |
γ-GTP (IU/L) |
0.30 ± 0.14 |
0.46 ± 0.23 |
T-Bil (mg/dL) |
0.072 ± 0.028 |
0.060 ± 0.012 |
Glucose (mg/dL) |
160.8 ± 11.0 |
164.2 ± 36.0 |
T-Cho (mg/dL) |
64.2 ± 9.8 |
55.4 ± 9.0 |
TG (mg/dL) |
63.4 ± 29.2 |
55.2 ± 12.7 |
TP (g/dL) |
5.58 ± 0.22 |
5.50 ± 0.07 |
UN (mg/dL) |
14.50 ± 1.43 |
14.48± 1.51 |
Crea (mg/dL) |
0.334± 0.051 |
0.320± 0.029 |
Na (mEq/L) |
143.2± 0.8 |
143.4± 2.1 |
K (mEq/L) |
4.700± 0.274 |
4.462± 0.315 |
Cl (mEq/L) |
107.4± 1.7 |
107.2 ± 2.6 |
Ca (mg/dL) |
9.70 ± 0.45 |
9.60 ± 0.25 |
IP (mg/dL) |
5.78 ± 0.63 |
6.10 ± 0.62 |
A/G |
0.876 ± 0.065 |
0.864 ± 0.059 |
Albumin (%) |
46.68 ± 1.83 |
46.30 ± 1.62 |
α1-G (%) |
22.32 ±1.68 |
24.10± 2.62 |
α2-G (%) |
9.24± 0.50 |
8.60± 0.57 |
β-G (%) |
16.36± 0.71 |
15.70± 0.73 |
γ-G (%) |
5.40± 1.15 |
5.30± 1.10 |
Table 2C_ 13-Week treatment_female rats_Clinical chemical parameters (mean value ± SD of n animals):
Parameters |
Dose level (mg/kg bw/day) |
|||
0 (n=10) |
100 (n=10) |
300 (n=10) |
1000 (n=10) |
|
AST (IU/L) |
112.3 ± 53.9 |
84.2 ± 25.3 |
64.2 ± 13.8* |
72.2 ± 25.8 |
ALT (IU/L) |
49.3 ± 28.6 |
32.1 ± 14.3 |
27.2 ± 6.4* |
23.4 ± 7.6** |
ALP (IU/L) |
150.0 ± 82.1 |
162.6 ± 70.3 |
178.2 ± 72.2 |
162.2 ± 57.0 |
γ-GTP (IU/L) |
2.01 ± 4.64 |
0.66 ± 0.17 |
1.05 ± 0.37 |
0.70 ± 0.37 |
T-Bil (mg/dL) |
0.082 ± 0.037 |
0.074 ± 0.017 |
0.065± 0.014 |
0.063 ± 0.014 |
Glucose (mg/dL) |
159.2 ± 18.7 |
155.1 ± 19.8 |
149.4 ± 20.8 |
154.6 ± 15.2 |
T-Cho (mg/dL) |
62.3 ± 8.6 |
65.5 ± 13.1 |
60.1 ± 21.9 |
59.4 ± 7.2 |
TG (mg/dL) |
15.4 ± 8.4 |
12.2 ± 4.3 |
10.6 ± 4.7 |
13.3 ± 6.2 |
TP (g/dL) |
6.17 ± 0.47 |
6.15 ± 0.61 |
6.17 ± 0.22 |
6.06 ± 0.25 |
UN (mg/dL) |
15.71 ± 1.58 |
16.18 ± 2.27 |
15.50 ± 2.40 |
16.64 ± 2.41 |
Crea (mg/dL) |
0.368 ± 0.041 |
0.374± 0.033 |
0.353± 0.037 |
0.368± 0.047 |
Na (mEq/L) |
142.4± 1.0 |
142.3± 1.5 |
143.0 ± 0.8 |
141.5 ± 1.8 |
K (mEq/L) |
4.227 ± 0.380 |
4.184 ± 0.297 |
4.248 ± 0.271 |
4.372 ± 0.305 |
Cl (mEq/L) |
105.7 ± 1.2 |
105.7 ± 1.8 |
106.1 ± 1.4 |
105.3 ± 1.8 |
Ca (mg/dL) |
9.81 ± 0.27 |
9.94 ± 0.44 |
9.97 ± 0.20 |
10.09 ± 0.35 |
IP (mg/dL) |
5.42 ± 1.30 |
5.65 ± 1.15 |
5.55± 0.88 |
6.12± 0.74 |
A/G |
1.083± 0.224 |
1.142± 0.095 |
1.101± 0.106 |
1.092± 0.128 |
Albumin (%) |
51.34± 6.88 |
53.24± 2.07 |
52.31± 2.30 |
52.04± 2.75 |
α1-G (%) |
17.94± 2.99 |
17.09± 1.90 |
16.91± 0.91 |
16.79± 1.55 |
α2-G (%) |
9.14± 2.19 |
8.40± 1.63 |
8.29± 1.27 |
8.87± 1.11 |
β-G (%) |
15.05± 2.04 |
14.23± 1.27 |
14.88± 0.85 |
15.21± 1.73 |
γ-G (%) |
6.53 ± 1.07 |
7.04 ± 0.88 |
7.61 ± 1.37 |
7.09 ± 1.45 |
* : p<0.05; **: p< 0.01
Table 2D_ 13-Week + 14-Day recovery_female rats_Clinical chemical parameters (mean value ± SD of n animals):
Parameters |
Dose level (mg/kg bw/day) |
|
0 (n=5) |
1000 (n=5) |
|
AST (IU/L) |
121.0 ± 71.5 |
56.6 ± 12.6 |
ALT (IU/L) |
52.8 ± 30.6 |
25.2 ± 6.2 |
ALP (IU/L) |
108.0 ± 23.9 |
112.0 ± 23.5 |
γ-GTP (IU/L) |
0.78 ± 0.51 |
0.64 ± 0.22 |
T-Bil (mg/dL) |
0.086 ± 0.011 |
0.070± 0.020 |
Glucose (mg/dL) |
155.2 ± 10.7 |
154.8 ± 26.4 |
T-Cho (mg/dL) |
79.6 ± 13.2 |
69.0 ± 8.3 |
TG (mg/dL) |
18.8 ± 11.5 |
21.4 ± 13.6 |
TP (g/dL) |
6.50 ± 0.35 |
6.08 ± 0.22 |
UN (mg/dL) |
15.82 ± 2.18 |
18.54 ± 1.77 |
Crea (mg/dL) |
0.416 ± 0.054 |
0.422 ± 0.030 |
Na (mEq/L) |
141.6 ± 1.1 |
142.4 ± 1.3 |
K (mEq/L) |
4.178 ± 0.283 |
4.120 ± 0.298 |
Cl (mEq/L) |
107.0± 1.4 |
107.2 ± 1.3 |
Ca (mg/dL) |
10.12 ± 0.29 |
9.90 ± 0.07 |
IP (mg/dL) |
3.78 ± 0.19 |
4.38 ± 0.37 |
A/G |
1.204 ± 0.141 |
1.098 ± 0.051 |
Albumin (%) |
54.46 ± 2.94 |
52.36 ± 1.17 |
α1-G (%) |
17.84 ± 0.77 |
18.44± 1.34 |
α2-G (%) |
7.18± 0.58 |
7.34± 0.29 |
β-G (%) |
14.12± 1.22 |
14.96± 0.51 |
γ-G (%) |
6.40± 1.76 |
6.90± 1.54 |
Table 3A_ 13-Week treatment_male rats_Organ weights (values as mean ± SD)
Dose level |
N |
Mean body weight (g) at treatment end |
Liver |
Kidneys |
Heart |
Lung |
Spleen |
Thymus |
Salivary gland |
Adrenals |
||||||||
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
|||
0 mg/kg bw/d |
10 |
552.9± 42.0 |
14.060±1.412 |
2.545± 0.236 |
3.258± 0.330 |
0.590±0.045 |
1.565±0.136 |
0.282±0.02 |
1.731±0.173 |
0.314±0.026 |
861.1±135.2 |
155.928±22.348 |
314.4±78.9 |
56.754±12.917 |
794±69.7 |
143.836±10.755 |
70.6±13.7 |
12.798±2.379 |
100 mg/kg bw/d |
10 |
577.1± 54.9 |
14.849 ±2.068 |
2.567± 0.175 |
3.513± 0.405 |
0.608±0.04 |
1.613±0.152 |
0.281±0.032 |
1.697±0.135 |
0.293±0.019 |
802.9±134.2 |
139.319±20.149 |
506±142.5 |
87.01±20.94 |
888.3±88.2 |
154.135±10.254 |
68±11.6 |
11.743±1.244 |
300 mg/kg bw/d |
9 |
570.0± 44.0 |
14.499 ±1.521 |
2.541± 0.116 |
3.599± 0.337 |
0.631±0.049 |
1.510±0.084 |
0.267±0.018 |
1.719±0.144 |
0.303±0.037 |
827.6±94.4 |
145.274±13.512 |
359.7±62.3 |
63.608±12.72 |
812.2±78.3 |
142.936±14.906 |
65.1±5.5 |
11.492±1.393 |
1000 mg/kg bw/d |
10 |
561.1± 53.9 |
16.290 ±2.024* |
2.902± 0.204** |
3.742± 0.367* |
0.669±0.054** |
1.472±0.128 |
0.263±0.027 |
1.666±0.131 |
0.299±0.027 |
840.4±142.3 |
149.741±18.569 |
464±170.3* |
82.403±28.22* |
843±126.2 |
150.419±19.27 |
61.7±7.8 |
11.111±1.895 |
Dose level |
N |
Mean body weight (g) at treatment end |
Pituitary gland |
Thyroid |
Testis |
Epididymis |
Prostate |
Seminal vesicles |
Brain |
N; number of animals AW; absolute weight (g or mg) RW; relative weight (g/100g body weight or mg/100g) * ; p<0.05 **;p< 0.01
|
||||||||
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
|||||
0 mg/kg bw/d |
10 |
552.9± 42.0 |
11.47± 1.76 |
2.076± 0.281 |
22.49±2.62 |
4.103±0.678 |
3.083±0.800 |
0.565±0.158 |
1.359±0.270 |
0.248±0.057 |
837.9±177.1 |
151.300±27.309 |
2.092±0.178 |
0.381±0.048 |
2.237±0.093 |
0.405±0.025 |
||
100 mg/kg bw/d |
10 |
577.1± 54.9 |
11.49± 2.09 |
1.986 ± 0.296 |
23.45±4.09 |
4.083±0.750 |
3.699±0.270 |
0.646±0.069 |
1.430±0.165 |
0.249±0.026 |
926.8±154.3 |
161.338±25.925 |
2.174±0.253 |
0.378±0.035 |
2.286±0.155 |
0.398±0.027 |
||
300 mg/kg bw/d |
9 |
570.0± 44.0 |
11.36± 1.68 |
1.991 ± 0.241 |
21.97±3.19 |
3.863±0.554 |
3.501±0.327 |
0.618±0.069 |
1.411±0.091 |
0.250±0.023 |
824.6±158.8 |
145.242±29.781 |
2.311±0.325 |
0.408±0.069 |
2,224±0.085 |
0.393±0.035 |
||
1000 mg/kg bw/d |
10 |
561.1± 53.9 |
11.68± 1.54 |
2.094 ± 0.301 |
23.91±4.34 |
4.252±0.612 |
3.588±0.263 |
0.643±0.058 |
1.407±0.101 |
0.252±0.015 |
925.5±167.8 |
165.480±29.833 |
2.221±0.256 |
0.399±0.064 |
2.205±0.102 |
0.396±0.035 |
Table 3B_13-Week + 14-Day recovery_male rats_ Organ weights (values as mean ± SD):
Dose level |
N |
Mean body weight (g) at the end of recovery |
Liver |
Kidneys |
Heart |
Lung |
Spleen |
Thymus |
Adrenals |
Pituitary gland |
||||||||
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
|||
0 mg/kg bw/d |
5 |
615.6±130.9 |
14.626±3.832 |
2.362±0.158 |
3.506±0.531 |
0.578±0.055 |
1.582±0.246 |
0.260±0.025 |
1.722±0.198 |
0.284±0.035 |
872.8±223.4 |
141.558±19.368 |
338.6±84.6 |
54.852±5.162 |
65.2±223.5 |
10.454±2.039 |
11.24±1.40 |
1.862±0.283 |
1000 mg/kg bw/d |
5 |
576.0±39.0 |
15.060±0.823 |
2.620±0.131 |
3.698±0.260 |
0.642±0.046 |
1.578±0.098 |
0.274±0.021 |
1.748±0.139 |
0.304±0.030 |
872.0±67.4 |
151.360±4.828 |
353.6±100.9 |
61.216±16.798 |
70.8±14.4 |
12.428±3.223 |
11.50±1.06 |
2.000±0.180 |
Dose level |
N |
Mean body weight (g) at the end of recovery |
Thyroid |
Testis |
Epididymis |
Prostate |
Seminal vesicles |
Brain |
N; number of animals AW; absolute weight (g or mg) RW; relative weight (g/100g body weight or mg/100g) * ; p<0.05 **;p< 0.01
|
|||||||||
AW (mg) |
RW (mg/100g) |
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (mg) |
RW (mg/100g) |
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
|||||||
0 mg/kg bw/d |
5 |
615.6±130.9 |
21.18±5.25 |
3.522±0.985 |
3.478±0.329 |
0.582±0.110 |
1.430±0.239 |
0.238±0.047 |
852.4±372.8 |
140.624±60.790 |
2.336±0.364 |
0.390±0.082 |
2.306±0.074 |
0.388±0.079 |
||||
1000 mg/kg bw/d |
5 |
576.0±39.0 |
20.28±3.49 |
3.528±0.613 |
3.616±0.254 |
0.628±0.045 |
1.542±0.080 |
0.268±0.022 |
1018.8±75.4 |
177.570±18.205 |
2.636±0.2326 |
0.462±0.044 |
2.300±0.070 |
0.402±0.024 |
Table 3C_ 13-Week treatment_female rats_Organ weights (values as mean ± SD)
Dose level |
N |
Mean body weight (g) at treatment end |
Liver |
Kidneys |
Heart |
Lung |
Spleen |
Thymus |
Salivary gland |
Adrenals |
||||||||
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
|||
0 mg/kg bw/d |
10 |
290.8±14.3 |
7.272±0.852 |
2.498±0.244 |
1.920±0.183 |
0.661±0.060 |
0.9580±0.054 |
0.329±0.019 |
1.280±0.104 |
0.440±0.036 |
523.2±70.7 |
180.134±20.961 |
309.1±78.9 |
106.137±25.854 |
434.4±53.8 |
149.803±20.229 |
73.7±9.7 |
25.428±3.794 |
100 mg/kg bw/d |
10 |
284.6±27.4 |
6.804±0.526 |
2.395±0.108 |
1.818±0.11 |
0.634±0.063 |
0.918±0.089 |
0.324±0.022 |
1.253±0.105 |
0.443±0.041 |
546.5±78 |
192.104±20.889 |
251.1±70.1 |
87.972±21.934 |
469.9±49.0 |
165.845±18.389 |
71.9±11.4 |
25.255±3.324 |
300 mg/kg bw/d |
10 |
273.9±22.3 |
6.687±0.623 |
2.441±0.097 |
1.842±0.237 |
0.673±0.055 |
0.865±0.094* |
0.315±0.026 |
1.197±0.074 |
0.438±0.045 |
496.5±40.2 |
181.910±15.920 |
309.5±103.5 |
114.098±40.765 |
456.2±52.6 |
167.681±24.523 |
65.1±7.1 |
23.927±3.430 |
1000 mg/kg bw/d |
10 |
281.4±21.4 |
7.527±0.620 |
2.674±0.098 |
2.046±0.151 |
0.728±0.060* |
0.876±0.057 |
0.312±0.015 |
1.194±0.088 |
0.425±0.026 |
521.0±62.1 |
184.955±14.893 |
324.8±77.8 |
115.662±28.205 |
461.0±36.8 |
164.419±15.097 |
77.0±10.0 |
27.541±4.482 |
Dose level |
N |
Mean body weight (g) at treatment end |
Pituitary gland |
Thyroid |
Ovary |
Uterus |
Brain |
N; number of animals AW; absolute weight (g or mg) RW; relative weight (g/100g body weight or mg/100g) * ; p<0.05 **;p<0.01
|
||||||||||
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
AW (g) |
RW (g/100g) |
|||||||||
0 mg/kg bw/d |
10 |
290.8±14.3 |
15.17±2.05 |
5.227±0.737 |
16.11±3.46 |
5.569±1.323 |
96.3±10.7 |
33.212±4.190 |
686.8±219.3 |
236.275±74.543 |
2.021±0.065 |
0.696±0.033 |
||||||
100 mg/kg bw/d |
10 |
284.6±27.4 |
13.39±2.03 |
4.714±0.653 |
13.08±2.30 |
4.587±0.605 |
112.0±26.9 |
39.644±10.332 |
897.1±354 |
319.380±137.277 |
2.049±0.092 |
0.724±0.067 |
||||||
300 mg/kg bw/d |
10 |
273.9±22.3 |
11.86±1.99** |
4.349±0.768* |
15.34±3.64 |
5.696±1.670 |
98.9±19.2 |
36.588±8.876 |
743.5±241.9 |
273.838±95.196 |
2.028±0.065 |
0.746±0.076 |
||||||
1000 mg/kg bw/d |
10 |
281.4±21.4 |
13.81±2.15 |
4.922±0.765 |
15.43±3.60 |
5.491±1.235 |
101.4±22.6 |
36.112±7.567 |
676.4±295.3 |
240.593±99.50 |
2.052±0.084 |
0.733±0.053 |
Table 3D_ 13-Week + 14-Day recovery_female rats_ Organ weights (values as mean ± SD):
Dose level |
N |
Mean body weight (g) at the end of recovery |
Liver |
Kidneys |
Heart |
Lung |
Spleen |
Thymus |
Adrenals |
||||||||
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (g) |
RW (g/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
||||
0 mg/kg bw/d |
5 |
310.8±29.9 |
7.538±0.845 |
2.424±0.121 |
1.896±0.199 |
0.612±0.052 |
1.022±0.121 |
0.330±0.023 |
1.238±0.026 |
0.400±0.042 |
480.0±75.1 |
154.032±13.916 |
240.2±381 |
78.018±16.311 |
70.8±8.7 |
22.746±1.082 |
|
1000 mg/kg bw/d |
5 |
310.2±41.2 |
7.198±0.671 |
2.330±0.114 |
1.936±0.091 |
0.632±0.056 |
0.916±0.083 |
0.298±0.038 |
1.186±0.097 |
0.386±0.040 |
554.6±26.3 |
180.608±17.691 |
304.6±104.1 |
97.966±30.921 |
68.0±12.3 |
22.166±4.416 |
|
Dose level |
N |
Mean body weight (g) at treatment end |
Pituitary gland |
Thyroid |
Ovary |
Uterus |
Brain |
N; number of animals AW; absolute weight (g or mg) RW; relative weight (g/100g body weight or mg/100g) * ; p<0.05 **; p<0.01
|
|||||||||
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
AW (mg) |
RW (mg/100g) |
AW (g) |
RW (g/100g) |
||||||||
0 mg/kg bw/d |
5 |
310.8±29.9 |
17.44±2.91 |
5.678±1.232 |
13.84±3.18 |
4.418±0.666 |
93.6±21.9 |
29.982±5.751 |
744.2±205.4 |
238.626±60.913 |
2.112±0.023 |
0.684±0.065 |
|||||
1000 mg/kg bw/d |
5 |
310.2±41.2 |
14.10±3.17 |
4.550±0.860 |
16.06±3.33 |
5.258±1.441 |
96.4±11.5 |
31.254±3.108 |
721.8±193.0 |
227.396±39.058 |
2.068±0.110 |
0.674±0.063 |
Table 4: Incidence and severity of treatment-related stomach changes at end of treatment period
Sex |
Male |
Female |
||||||
Dose (mg/kg bw/day) |
0 |
100 |
300 |
1000 |
0 |
100 |
300 |
1000 |
Number of animals |
10 |
10 |
9 |
10 |
10 |
10 |
10 |
10 |
Infiltration, inflammatory cell, lamina propria, limiting ridge |
||||||||
slight |
0 |
0 |
0 |
4 |
0 |
0 |
0 |
0 |
mild |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
moderate |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
severe |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Total |
0 |
0 |
0 |
4 |
0 |
0 |
0 |
0 |
Hyperkeratosis, limiting ridge |
||||||||
slight |
0 |
0 |
0 |
4 |
0 |
0 |
0 |
2 |
mild |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
moderate |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
severe |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Total |
0 |
0 |
0 |
4 |
0 |
0 |
0 |
2 |
Infiltration, inflammatory cell, submucosa, glandular stomach |
||||||||
slight |
0 |
0 |
0 |
5 |
0 |
0 |
0 |
0 |
mild |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
moderate |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
severe |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
Total |
0 |
0 |
0 |
5 |
0 |
0 |
0 |
0 |
Applicant's summary and conclusion
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