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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was carried out 15th July 2002 and 24th September. The final report was issued 6th November 2002.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Meets the criteria for classification as Reliable without restriction according to Klimisch et al (1997)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and We lfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eigh t day repeat dose oral toxicity study.
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Slc:Wistar rats (bred by Japan SLC, Inc.) of 5 weeks old purchased from Sankyo Labo-Services Co., Ltd.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Slc:Wistar rats (bred by Japan SLC, Inc.) of 5 weeks old were purchased from Sankyo Labo-Services Co., Ltd. and acclimatised for 6 days during which time quarantine was conducted. Thirty males and thirty females showing satisfactory growth and health status were accepted into the study. At the start of treatment, mean body weight (range) was 146.7(139-159) g in male and 116.0 (107-125) g in female. Each animal was identified by marking the tail with oil paint, such as line with red oil paint during the quarantine period and numerals indicating group and individual with black oil paint during the study period. In addition, identification cards were attached to racks and cages. Sex was not identified.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Dosing solution was administered orally using a steel stomach cannula attached to a 2.5 ml syringe. The volume of administration was set at 10ml per kg of bodyweight , and calculated on the most recent body weights.
Vehicle:
other: Purified water
Details on oral exposure:
The test material was dissolved in purified water used as a vehicle, and the concentrations were adjusted to become as follows , 100/mg/kg: 1%, 300mg/kg:3.0%, 1000mg/kg: 10%. Solutions were stored in the dark at 4°C.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Solutions of the test material were prepared once a week. Concentrations of the test material in the dosing solutions, homogenity and the 7 - day stability were confirmed by HPLC.
Duration of treatment / exposure:
Test duration: 28 days
Frequency of treatment:
Dosing regime:every day (ie. 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:
Male: 10animals at 0 mg/kg/day ( control with recovery group)Male: 5 animals at 100 mg/kg bw/dayMale: 5 animals at 300 mg/kg bw/dayMale: 10 animals at 1000 mg/kg bw/dayFemale: 10animals at 0 mg/kg/day ( control with recovery group)Female: 5 animals at 100 mg/kg bw/dayFemale: 5 animals at 300 mg/kg bw/dayFemale: 10 animals at 1000 mg/kg bw/dayDoses are given as active ingredient.
Control animals:
yes
Details on study design:
Dose selection rationale : in the dose finding study using 5 males and 5 female rats, no toxicological changes were observed at 1000 or 300mg/kg. Therefore 1000mg/kg was selected as the highest dose, and 300 and 100mg/kg were selected with the common ratio of 3. In addition 14-day recovery test groups were provided in the highest dose group and in the control group. Allocation of animals to each work group was done by a random sampling method after grouping by body weight.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
(1) Observation of general findingsAll animals were observed once a day for external appearance and presence of abnormal behavior. In addition, moribund condition and presence of death were observed twice a day.(2) Detailed observation of clinical signsAll animals were observed for 19 items of detail symptoms such as cognition (stereotypy, passivity, alertness), mood (face washing, vocalisation), central nervous system, excitement (tremor, clonic/tonic convulsion), posture (body posture), ataxia abnormal gait), autonomic symptom (piloerection, hypothermia, skin color,respiration rate, pupil, exophthalmos, salivation, lacrimation, urination), and abnormal behavior (for one minute/animal) according to Irwin's multiple observation method1 on the day before administration start (after allocation to groups), onceafter administration, and once a week thereafter.(3) BodyweightAll animals were weighed on the starting day of administration and twice a week thereafter using an electronic balance.(4) Food intakeFood intake per cage (2~3 animals were housed in a cage) was measured using an electronic balance and food intake per capita in a day was calculated.(5) Functional examinationFive animals each from the control group and high dose group were examined.1) Visual examinationOn the 27th day, animals were examined whether they kept a horizontal posture extending the forelimb before the tactile hairs touch a laboratory desk when the animals were approached from a high position. Evaluation criteria were decided as positive reaction: + (kept a horizontal posture before touching) and negative reaction:- (no extending of forelimb by touching the tip of nose).2) AuditoryexaminationOn the 27th day, reaction to the sound of 3KHz and 120 db was examined using an audiometer (Muromachi Kikai Co., Ltd.). Evaluation criteria were decided as positive reaction: +, and negative reaction:3) Algesimetry (Tail pinch method)On the 27th day, a reaction to pinching the tail by a forceps was examined.Evaluation criteria were decided as a positive reaction or a negative reaction.4) Grasp examinationOn the 27th day, grasp responses of forelimb and hindlimb were measured using a rat-grasp response test system (MK-S80/CM/R: Muromachi Kikai Co., Ltd.). the measurement was performed twice and mean value was recorded.5) Motor activity measurementOn the 22nd day in the treatment period motor activities for male and female were measured by automated activity monitors (Supermex: made by Muromachi machine Co., Ltd.) for 22 hours immediately after administration.Clincial Chemistry and HaematologyDay and method of sacrificeThe animals from test groups after dosing period were sacrificed on the next day of final administration, and the recovery group animals were sacrificed on the next day of recovery period completion by exsanguinations (blood sampling for hematology and blood chemistry) under pentobarbital anesthesia (30 mg/kg).Method of blood samplingThe animals were fasted overnight and supplied with water only. Abdominal incision was made in the animals under pentobarbital anesthesia, and a blood-sampling needle with silicone tube was inserted into the abdominal aorta. The blood flown out naturally was collected. A certain amount of blood was sampled first for coagulation test and hematology, and then sampled for blood chemistry. The blood samples were left untreated for about 50 to 60 minutes in room temperature, and then serum was obtained by centrifugation (5000rpm/min, 10 min) after confirming blood coagulation.The blood sampling was conducted on one animal each from the control group, low dose group, intermediate dose group, and high dose group in this order first, and then reverse order from the high dose group to the control group next. This process was repeated on male first and then conducted on female.The anticoagulant, 3.2 % sodium citrate solution was used for the examination of coagulation system, and EDTA-2K for the other hematology.
Sacrifice and pathology:
The following items were examined after sacrifice by exsanguinations.1) NecropsyAll animals were observed macroscopically for body surface, orifices, cranial cavity,organs in thoracic and abdominal cavities, and lymph nodes.2) Organ weightThe brain, pituitary, heart, liver, kidneys, thymus, spleen, adrenals, testes, epididymides, prostate, seminal vesicles, ovaries and uterus from all animals were weighed (absolute) with electronic balance (AEX-200B: Shimadzu Corporation), and organ/bodyweight ratio (relative organ weight) was calculated based on the body weight at necropsy. The kidneys, adrenals, testes or ovaries, and epididymides were weighed right and left organs together. The prostate, seminal vesicle and uterus were weighed on the next day.3) Histopathological examinationThe heart, liver, kidneys, adrenals, spleen, testes, epididymides, prostate, seminal vesicles, ovaries and uterus from the control group and high dose group at the end of administration period and the liver and kidneys from the recovery groups were routinely prepared as paraffin sections and stained with H • E staining for microscopic examination. In addition, the kidneys, testes, and epididymides were stained with PAS reaction and examined microscopically. Evaluation criteria for pathological lesions were indicated as minimal: ±, slight: +, moderate: ++ and severe: +++.
Other examinations:
In addition, the organs remained after supplying for the examination described above and the brain, pituitary, eyeball and Harderian gland, salivary gland (submaxillary gland, parotid gland), esophagus, trachea, thyroid (including parathyroid), lung, thymus, heart, stomach and duodenum, small intestine, large intestine, pancreas, adrenals, kidneys, urinary bladder, testes, prostate,epididymides, seminal vesicle, coagulation gland, ovaries, uterus, bone marrow(femur), peripheral nerves, lymph node (mesenteric lymph node) and skin were fixed and preserved in neutral phosphate buffered 10 % formalin. In addition, the testes and epididymides were fixed in Bouin's solution and preserved in neutral buffered 10% formalin
Statistics:
Data obtained by each examination were assessed by Bartlet test for homogeneity of variance with multiple comparison method. Where variances were shown to be homogenous one-way analysis of variance was conducted and followed by Dunnet multiple comparison test when significant differences were recognised. Where the test showed unequal variances Kruskal-Wallis rank sum test was conducted and followed by Dunnett's rank test when significant differences were found. However, statistical analysis was not conducted on the data of general condition, detail observation of signs, function test, necropsy and histopathology.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No abnormalities were seen in the 4-week dosing groups or the recovery test groups.
Mortality:
no mortality observed
Description (incidence):
No mortality was seen either in male or female and no abnormalities were observed in behaviour. In all animals of dosing groups, blue colouration which was similar to test material solution was seen in urine and feces throughout the administration period.However, urine colour returned to normal after 24 hours.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Female of the high dose group showed statistically significant increase from the 7th day to the 27th day compared with the control group.Recovery groupMale and female of the dose group showed normal increase similarly to the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no adverse effect on food consumption throughout the study.
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):
Hematology 4-week dosing groupFemale of the high dose group showed APTT prolongation.Recovery groupDecrease in platelet count was seen in male ofthe dosed group.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Recovery groupDecrease in platelet count was seen in male of the dosed group.In the high dose group, male showed lowered BUN and female showed lowered total protein.Recovery groupIn the high dose group, male showed lowered A/G ratio.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No abnormalities were seen in male or female of each group.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Male and female of the dosing groups showed similar results to that of the control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
4-week dosing groupNo changes were seen in each dose group.Recovery groupNo changes were seen in male or female.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
[4-week dosing group]Liver: Small granulation foci (+) were seen in a male and 2 females of the control group, and a male and 2 females ofthe high dose group.Kidney: Eosinophilic bodies in the proximal tubular epithelium (+) were observed in all males of the control group and the dose groups. In addition, no remarkable changes were seen in the heart, spleen, adrenals, testes, epididymides, seminal vesicles, prostate, ovaries, male liver or female kidneys.[Recoverygroup]Liver: Small granulation fociwere observed in 1 animal of the control group and 1animal ofthe dosed group.Kidney: Eosinophilic bodies in the proximal tubular epithelium (+) were observedin all males of the control group and the dosed group.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
Clinical observations:There were no deaths in either the males or females of each dose group. Colouration of the urine and faeces with the test material solution (blue colour) were seen in all animals of the dose groups throughout the dosing period, however, urine colour returned to normal 24 hours after the administration and no influences were observed in urinalysis. No abnormalities were detected in the detailed observation of the general condition, behaviour, functional test or food intake. Males and females showed normal bodyweight gain and no toxic influences were detected in the animals. Females of the high dose group showed increased bodyweight gain compared with the conrol group. these increased weight gains were thought to be incidental.Laboratory findings:hematology:Females of the high dose group males showed APTT prolongation. Males in the recovery group showed a decreased platelet count.Blood chemistry:In the high dose group males showed a lowered BUN and females showed a lowred count of total protein. In the high dose recovery group, a male showed lowered A/G ratio.Effects in organs:Organ Weight:No changes in organ weight were seen in any of the groups in either the dosing or recovery groups.Histopathological examination:Liver: Sall granulation foci (+) were seen in one male and two females of the control group and one male and two females of the high dose group.Kidney: Eosinophilic bodies in the proximal tubular epithelium (+) were observed in all males of the control group and the dose groups.In addition, no remarkable changes were seen in the heart, spleen, adrenals, testes, epididymides, seminal vesicles, prostate, ovaries, male liver or female kidneys.

Effect levels

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Study top dose
Remarks on result:
other:
Remarks:
No adverse effects were observered in the study at any dose level.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test substance NOEL was 1000 mg/kg/bw/day in both male and female rats.
Executive summary:

Introduction.

The study was designed to investigate the systemic toxicity of the test material and was performed in compliance with the standards stipulated in the GLP of Japanese Chemical Substances Control Law "Testing facilities in conducting tests stipulated in article 4 of the Order Prescribing Those Items of the Test Relating to the New Chemical Substances and Study on Harmful Effects of Designated Chemical Substances". (Kanpoan No.41, March 1, 2000, Japanese Ministry of the Environment, Integrated Environmental Policy Bureau, Seieihatu No. 268, the Japanese Ministry of the Health, Labour and Welfare, Medical Safety Bureau, Kikyoku No.1, the Japanese Ministry of Economy,Trade and Industry, Manufacturing Industries Bureau Joint Notification) and Guideline of Japanese Chemical Substance Control Law " Methods for the Test on New Chemical Substances" Partial Amendment and Others (Kanpogyo No. 700, December 5, 1986, Japanese Ministry of the Environment, Integrated Environmental Policy Bureau, Yakuhatsu No. 1039, the Japanese Ministry of the Health, Labour and Welfare, Medical Safety Bureau, Kikyoku No. 1014, 1986, the Japanese Ministry of Economy, Trade and Industry, Manufacturing Industries Bureau, Notification).

Method

Test material at 1000 mg/kg bw/day (high dose group), 300 mg/kg bw/day (intermediate dose group) or 100 mg/kg bw /day (low dose group) or purified water used as vehicle was administered. In addition, 5 males and 5 females each from the high dose group and the control group were allocated to recovery test groups.

Results

There were no deaths on the study and no changes attributable to the influence of test material were detected in any dose group. Blue staining similar in colour to the test material was seen in feces and urine as well as alimentary tract contents and renal cortex at necropsy. This change was judged not to be toxicity effect.

Conclusion

The NOEL was concluded to be 1000mg/kg/day in both male and female rats