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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
September 25, 2007 - February 20, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 422, GLP followed

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Slight yellow crystal
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated:3.5-5.9ºC, range 1-10ºC, dark place
- Stability under test conditions: Yes

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Reason for selecting lineage, It is widely used for toxicity tests using rodents, has abundant background data.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River Japan Co., Ltd. (Atsugi Production Center)
- Age at study initiation: 9 weeks old
- Weight at study initiation:Male:315-366 g, Female: 205-234g range of the animals was within ± 20% of the average body weight.
- Fasting period before study: Yes, the day before the planning dissection was fasted (about 18 to 23 hours).
- Housing: Cages, equipment made by Tokiwa Scientific Instruments Co., Ltd. was sterilized by autoclaving used. However, steel stand is excluded.Using a stainless steel hanging metal gauze cage (195 W × 325 D × 180 H mm). During the pregnancy - using a polycarbonate cage (265 W × 426 D × 200 H mm),
- Diet (e.g. ad libitum): Radio-sterilized solid feed for laboratory animals (CRF-1, Oriental Yeast Co., Ltd.. Free intake except for spontaneous momentum measurement and fresh urine collection and exchange at feeder replacement.
- Water (e.g. ad libitum): Drinking water,After filtering by 5 μm filter, ultraviolet irradiated tap water.Free intake was excluded except for spontaneous momentum measurement and fresh urine collection. Drinking water in the water bottle. During the pregancy:Polycarbonate water bottle (700 mL) was used. Water supply device Tokiwa Scientific Instruments Co., Ltd.
- Acclimation period:The animals were in quarantine/acclimation period for 5 days, observed every day for any abnormalities.

DETAILS OF FOOD AND WATER QUALITY:
Water Quality: Mitsubishi Chemical Analytech Co., Ltd. (former company name: Diamond Analysis Center Co., Ltd.) The inspection is carried out periodically (twice a year), and the obtained analysis value becomes the standard operation manual.
Food Quality: Analysis results conducted at the Japan Food Research Laboratories Foundation, analyzed using Oriental Yeast Co., Ltd. The concentrations of pollutants such as residual agricultural chemicals in lots used were obtained from the company,It meets the standards.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):21.0-23.2ºC (tolerance range 19.0 to 25.0 ºC)
- Humidity (%):47.8-61.3% (tolerance range 35.0 to 75.0%)
- Air changes (per hr):6 to 20 times / hour, all fresh air supply
- Photoperiod (hrs dark / hrs light):12 hours / day (7: 00-19: 00)

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Syringe fitted with stomach probe for rat. Once a day between 8: 07 and 11: 21 (tolerance: 8: 00-13: 00).
Males:14 days before mating, and a total of 42 days from the mating period to the day before necropsy.
Female (pregnancy / nursing): 14 days before mating, mating period, gestation period and 4 days of nursing through delivery (day of delivery nursing 0 Day) for a total of 42 to 50 days.
Females (satelite): during 42 days
Vehicle:
water
Remarks:
Purified
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:Dosing liquid volume: Ten mL / kg, and the liquid volume of each individual was calculated based on the body weight measured on the nearest day.Preparation was carried out in a dispensing room under illumination with ultraviolet rays cut off. Frequency of preparation is test facility. Based on the results of the stability analysis carried out in 7 days, test was conducted once.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical method validation of Hydrazinecarbothioamide in preparation liquid:
After preparation, dispense into polypropylene tubes at each administration day and limit the period of time when stability was confirmed.
Refrigerated (measured value: 3.5 to 5.8 ° C, tolerance: 1 to 10 ° C) · Storage in a dark place
1 mg / mL preparation solution:
A predetermined weight of the test substance was weighed, an appropriate amount of the medium was added, stirred, and dissolved. Thereafter, 1 mg / mL was added using a metric glass or a measuring cylinder to make the concentration of female.
0.2 and 0.04 mg / mL preparation:
1 mg / mL solution was prepared by diluting with a medium.

Confirmation of stability
The stability of the test substance in the administration solution under refrigeration, in the dark, and under stoppered condition is 0.01 and 5 mg / mL conducted at the test facility for 8 days.

Confirmation of Concentration
The administration solution at the time of initial preparation was analyzed by Spectrophotometer: Model HITACHI U-3310, Cell: 10 mm quartz cell (n = 2), and the average value of the concentration (actually measured value: 99.4 ~ 106.0%, setting concentration within ± 10%)

Standard and sample solution were prepared.

Measurement of standard solution
Absorbance of standard solutions (ST-1, ST-2 and ST-3) at 235 nm was measured by spectrophotometry with water as a control and a calibration curve was prepared from the concentration of the standard solution.

Measurement of sample solution
The absorbance of the sample solution at 235 nm was measured by spectrophotometry using water as a control, Calculate the HCTA concentration in the sample solution from the calibration curve and the dilution factor, calculate the average value.

Recovery period
In the control group and 5 males and 5 females in the 10 mg / kg group, a recovery period of 14 days after the administration period was administered. However, females were set as satellites without mating.
Duration of treatment / exposure:
MALES: 42 days
FEMALES:from 14 days before mating to day 4 of lactation
FEMALES (satellite) : 42 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
0.4 mg/kg bw/day (nominal)
Dose / conc.:
2 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
RECOVERY
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
RECOVERY
No. of animals per sex per dose:
MALES: 12 (5 for recovery)
FEMALES: 12
SATELLITE FEMALES: 5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of range finding study.

Three doses of 0.4, 2 and 10 mg / kg. It was set with reference to the result of the dose setting test conducted at the test facility. "14-day repeated dose toxicity by oral administration of Hydrazinecarbothioamide in rats Test (test number: B070687) "
FRIST TRIAL: Dose: 0, 0.4, 2 and 10 mg / kg,
Number of animals: each group: 1female, Three male,
The low value of the thymus weight and the low value of the body weight are not significant although there is no significant difference in the males at 10 mg / kg. Slightly high heart weight was found in females of 10 mg / kg group. Because clear expression of toxicity could not be confirmed in this study, 50 mg / kg was used for the highest dose.

Dose setting test (test number: B070903) was performed again. "14-day repeated dose toxicity by oral administration of Hydrazinecarbothioamide in rats
Test (test number: B070903) "
SECOND TRIAL: Dose: 0, 20, 30 and 50 mg / kg,
Number of animals: each group: 3 males and 3 females
All sexes of 20 mg / kg or more died on the 1st day or moribund. It was a state. These animals showed irritability and tonic convulsions, the breathing irregularity. As a result of necropsy, pulmonary edema of 20 mg / kg or more. Pancreatic fluid reserves in males and females in the 20 and 30 mg / kg group and males in the 50 mg / kg group was observed. As a result, although death was observed in the 20 mg / kg group, death occurred in the 10 mg / kg group, No moribund was observed, and changes that could be attributed to the test substance could be confirmed.
Therefore, 10 mg / kg at which some toxicity is expected to occur is set to a high dose, and three doses were set for the lower common ratio 5, medium dose 2 mg / kg, low dose 0.4 mg / kg. Also, a control group to which only body (purified water) was administered was provided.
First trial was selected as a tested doses.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, in the males and females (Tremor, clonic convulsion, breathing)

DETAILED CLINICAL OBSERVATIONS: Yes, observation of reacting from removal from the cage, reactivity to handling, aggression, trauma, color of skin, soiled fur, exophthalmos, palpebral closure, color of conjumctuiva, secretion,lacrimation, salivation, piloerection, pupil size, arousal, urination, defecation, posture, body position, gait, bizzarre behaviour, tremor, clonic convulsion,tonic convulsion, breathing, stereotypy.
- Time schedule: 9 weeks (from week -1 to week 8)

BODY WEIGHT: Yes,
Males animals were treated on days 1, 8, 15, 22, 29, 36, 42 and 43, In addition, in male-recovered animals it was measured on days 50 and 56. Female satellite animals (the same as males) are recovered from male,measured at the same frequency. Female test animals - 0, 7 and 14 days before mating, during gestation - 0, 7, 14 and 20 days, lactation - 0 and 4 days.
Measurement - an electronic balance weight (EB - 3200S: Shimadzu Corporation, PB 3002 - S: Metlitore Co., Ltd.) was used.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Males animals were treated on days 8, 15, 29, 36, 40. In addition, in male-recovered animals it was measured on days 50 and 54.
Female satellite animals were treated on days 8, 15, 22, 29, 36, 42, recovered 50 and 56 days.
Female test animals - 7 and 14 days before mating, during gestation - 7, 14, 20 days, and during lactation - 4 days.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Measurement - an electronic balance weight (EB-3200S: Shimadzu Corporation, PB 3002-S : METTLER TOLEDO CO., LTD.)

FOOD EFFICIENCY:No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (Red/White blood cell count, hemoglobin concentration, hematocrit, MCV, MCH, MCHC, platelet count, reticulocyte ratio, PT, APTT, Lymphocute, neutrophil, eosinphil, basophil, monocyte )
Males, Females:day 43 and 57

CLINICAL CHEMISTRY: Yes (ASAT(GOT), ALAT(GPT), GAMA-GT, ALP, Total bilirubin, urea nitrigen, creatinine, glucose, total cholesterol, tryglyceride, total protein, albumin, A/G ratio, calcium, Inorganic phosphorus, Na, K, Cl)
Males, Females:day 43 and 57

URINALYSIS: Yes, only in males on day 40: (pH, protein, glucose, ketones, bilirubin, occult blood, urobirinogen)

NEUROBEHAVIOURAL EXAMINATION: Yes,the day before the administration, once a week until the 6 th week both after 13: 00
Touch response, auditory respione, tail pinch response, aerial righting reaction, forelimb, hindlimb

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY:
Organ weight:
Males:Day 43 and 57,Organ weight and relative organ weight :(Final body weight, brain, thymus, heart, liver, spleen, kidneys, adrenals, testes, epicidymides)
Satelite females: on day 57, Organ weight and relative organ weigh(Final body weight, brain, thymus, heart, liver, spleen, kidneys, adrenals)
For the measurement, an electronic balance (AW 120: stock Shimadzu Corporation) was used.

Pathological anatomical examination:After the blood sampling, the animals to be examined are collected under anesthesia.After artery was cut / bleeding, it was euthanized and necropsied. Females who did not deliver were confirmed after copulation.After euthanasia by the above method on the day, necropsy was done in the same way. The dead animals were necropsied promptly.

HISTOPATHOLOGY: Yes The following organs and tissues of all animals were collected and fixed with 10 vol% neutral phosphate buffered formalin solution.The testes and epididymis of male were fixed with Bouin's solution, then 10 vol% and stored in neutral phosphate buffered formalin solution.
Brain, pituitary gland, thymus, lymph node (mandible / mesenteric), trachea, lung, stomach, intestinal tract (twelve fingers Intestine, jejunum, ileum, cecum, colon, rectum), thyroid / parathyroid (bilateral), heart, liver, Spleen, kidney (both sides), adrenal gland (both sides), bladder, testis (both sides), epididymis (both sides), semen Bulla (including coagulated gland), prostate ventral lobe, ovary (both sides), uterus, vagina, bone marrow (right side femur) Sciatic nerve (right side), spinal cord, grossly abnormal site

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical abnormalities throughout the administration and recovery periods, tonic convulsion was observed in 2 males and 1 female in the 10 mg/kg group at detailed clinical observation.
One male in the 10 mg/kg group showed a hypersensitive reaction in touch response and tail pinch response
Observation within home cage, observation at handling and within open field: No abnormalities were found in any animals at the time of observation.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female in the group of dosing 10 mg/kg was found dead before dosing on day 2 of administration. She had pulmonary edema.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males at 10 mg/kg, body weight decreased throughout the administration and recovery periods from day 8 onward.
The body weight gain was comparable to that of the control group from day 15 onwards. It only occurred in the early stage of administration and recovered thereafter. In the 0.4 and 2 mg / kg groups, there was no significant difference between the sexes and the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Throughout administration and recovery period in both males and females, there was a significant difference between the control group and the test substance administered group.
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):
No change due to the test substance was observed.
At the end of administration period
A low tendency of white blood cell count was observed in males of 10 mg / kg group,as a result, all cases were within the range of the average value of the background data ± 2 S. D. (accidental change)
In addition, the low value of MCHC and shortening tendency of PT were observed in males of the 10 mg / kg group. Only for MCHC, it is slight change compared with the control group, and hemoglobin concentration and the hematocrit value was almost the same as that of the control group, and for PT, the control group one case of high value, it is a change opposite to extension which is usually toxicologically problematic. As a result, it was judged that any changes were not toxicologically meaningful. Also, in the 0.4 mg / kg group extension of PT in females, prolongation of APTT in females at 0.4 and 2 mg / kg group, white in females at 2 mg / kg group. A low value of blood cell count was observed. However, neither correlation with the dose is recognized. (accidental change)

At the end of the recovery period
There was no significant difference between the test items in both sexes and the control group.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of administration period
High levels of total cholesterol were observed in males in the 10 mg / kg group. In addition, low or low trends of ASAT and ALAT were observed in males of the 10 mg / kg group. Since it is the opposite change from the high value which is usually toxicologically problematic, it is taken as a emergent change. Also, ALP and creatinine were elevated in males of the 0.4 mg / kg group. Although it was not observed correlation with the dose, it was determined to be an accidental change.

At the end of the recovery period
A low value of total protein and a high value of inorganic phosphorus were observed in males of the 10 mg / kg group. No change is recognized at the end, background data of the test facility. As a result of comparison with the total protein, all cases were within the average value of background data ± 2 S. D. Inorganic phosphorus showed a high value in 1 case, but in other test items since no relevant changes are noticed, it is judged that there is no toxicological significance
Urinalysis findings:
no effects observed
Description (incidence and severity):
In the urine qualitative test, in any of the test items, between the control group and the test substance administered group, there was no significant difference between them.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
One male in the 10 mg / kg group, at the time of examination of contact reaction and tail pinch reaction behind the act of crying was observed. Tactile reaction: Violent reaction, tail pinch reaction: jump, reaction to exaggeration in the 0.4 and 2 mg / kg groups, in both sexes. In the grip force measurement, no significant difference was observed between the control group and the test substance-administered group. In the examination during the recovery period, these changes were not observed and abnormalities were found in any animals

In the 10 mg / kg group two males and one female showed tonic convulsions in the 6th week.It was observed at the time of maintenance, at the time of function test execution or after returning to the cage after the examination was ended. In the 0.4 and 2 mg / kg group, no abnormality was observed in any animals in both males and females. These changes were not observed in the examination during the recovery period, and abnormalities were observed in any animals.

Measurement of locomotor activity
There was no significant difference between the control group and the test substance administration group in both males and females.


Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At the end of administration period
Absolute and relative weight, low and low thymus in males in 2 and 10 mg / kg group. Value trend, high or high trend of liver and kidney was observed in males of 10 mg / kg group.
At the end of the recovery period
In the males of the 10 mg / kg group, the relative weight of the brain was high. The absolute weight is a similar value and a low value of the final body weight is seen, it is attributed to a low value of body weight.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No change due to the test substance was observed. Testicular and epididymal compacts were bilateral in one case of 2 mg / kg group, one in 10 mg / kg group It is taken as change not related to the test substance.
Besides, dark red spots or red spots of the lungs are administered or after the recovery period finishes in the animal, 2, 1, and 1 males in the 0.4, 2 and 10 mg / kg group there was a deformity of the unilateral adrenal glands in the group of 0.4 mg / kg. It was found in one female group. All of these changes are sporadic emergence without correlation with dose. Since it is present, it is judged that there is no toxicological significance.

In the death animal:Pulmonary edema accompanied by increase of pleural effusion was observed as a change caused by the test substance. In addition, hepatic diaphragmatic surface nodules were observed, but this is a spontaneous change and contingent.
In the non-delivering animal: No change.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
One female in the group of dosing 10 mg/kg had pulmonary edema (dead)
The pulmonary edema was seen in all dead or moribund animals on day 1 of administration at 20 mg/kg and above in a preliminary study.

Planned anatomy
Changes due to the test substance were observed in the male thymus group of 10 mg / kg group. Accepted change.
In males in the 10 mg / kg group, atrophy of the thymus was observed in 3 cases. After the recovery period the dissected animals, this change was not recognized.
In the female spleen of the 10 mg / kg group, 3 cases showed a slight erythroidic extramedullary hematopoiesis enhanced. This change was seen in 1, 3, 1 cases in the control group, 0.4 and 2 mg / kg group, respectively. It was not correlated with the apparent frequency and frequency of expression. In addition,
Increased hematopoiesis is also known to develop with pregnancy. Therefore, it was determined not to be caused by the pregnancy, but to be expressed with pregnancy. In addition, 0.4 mg / kg in one female group, focal fibrosis was observed in the unilateral adrenal glands which had deformed at autopsy.
Dark red spots of the lung were seen in 2, 1, 1 cases of males in the 0.4, 2 and 10 mg / kg groups, respectively. The red plaques are localized bleeding, and from the number of examples of expression it is related to the test substance. It is admited as no change. Also, in the testes, the diffuse atrophy of seminiferous tubules was 2 mg / kg group, one case (mild, bilateral), 1 case of 10 mg / kg group (moderate, one side). Localized degeneration (minor) of seminiferous tubules was observed in the remaining unilateral testis of the group. However, there was no change in the testes in the other 10 mg / kg group, and these atrophy / degenerative findings were raren the testis but also in the background, it is judged that it is not related to the test substance.
In addition, various tissue changes were observed. However, they occur nonspecifically in rats, it is a present change, and it is not correlated with an obvious dose in the number of cases of expression, it was judged that the change was not related to the test substance.

Dead Animal: Moderate pulmonary edema was observed as a possible change in the cause of death. In addition, spleen white- A slight increase in the nuclear fracture image was observed in the splenic cord, mandibular lymph node and mesenteric lymph node. At the necropsy, hepatic diaphragmatic nodules found in the liver showed minor fibrosis at the base.
Histopathological findings: neoplastic:
not specified
Other effects:
not examined

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 2 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Key result
Dose descriptor:
NOEL
Effect level:
ca. 0.4 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOEL
Effect level:
ca. 2 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: No substance related changes.

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Thiosemicarbazide by oral route to rats produced some changes in body weight and clinical signs of tested animals. The NOAEL was judged to be 2 mg/kg/day for males and females, NOEL of 0.4 mg/kg/day for males and NOEL of 2 mg/kg/bw for females.
Executive summary:

Thiosemicarbazide was tested by oral route to rats in the doses 0, 0.4, 2 and 10 mg/kg/bw. Dosing period was 42 days far males, for satellite females 42 days and for females 14 days before mating to day 4 of lactation. There were used 12 male animals per group (5 for recovery), 12 females animals and 5 satellite females. Recovery period was also applied and it was 14 days for males and satellite females. Animals were killed after treatment and examinated fro clinical signs, body weight and weight gain, histopathological findings. Offspring were sacrificied on day 4 after birth. According to the data from National Institute of Technology and Evaluation, high level (10 mg/kg/bw) of thiosemicarbazide by oral route to rats produced  decreases in bodyweight (males), convulsion and excessive response to stimuli in both sexes and one death of female animal. Therefore NOAEL was judged to be 2 mg/kg/day for males and females, NOEL of 0.4 mg/kg/day for males and NOEL of 2 mg/kg/bw for females.