Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
unknown
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
only summary available in english

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
2,2'-azobis (2,4-dimethylvaleronitrile) (available from Wako Pure Chemical Industries, Lot No. ALJ4132, purity 100.0%) is a white crystalline at room temperature and stored at cool place after availability. Stability of the test substance, the infrared absorption spectrum was measured before and after dosing period, the test substance was confirmed to be stable during the administration period

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
according to Guideline
Sex:
male/female
Details on test animals and environmental conditions:
65 female and 55 male Crl: CD (SD) rat (SPF), breed at Charles River Japan (Hino Care Center) being 8 weeks old were purchased and acclimatised for 8 days including quarantine for 6 days.. By weight stratified random sampling method to the administration after 2 days ago, they were grouped so that the average body weight of each group are substantially equal. Week-old administration at the start of the animals in both sexes 9 weeks of age, body weight range is male 323.8 ~ 359.2g, female was 205.9 ~ 236.6 g.
Animals, throughout the entire rearing period, including during the quarantine, acclimatization period, temperature 21 ~ 25 ° C, relative humidity of 40 to 70%, and ventilation rate for 10 to 15 times / hour, light-dark cycle 12-hour intervals (7 lighting, 19:00 darkness)
The animal received autoclaved diet (chow, MF, Oriental Yeast) and tap water with added chlorine ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
Preparation of dosing solutions are added olive oil (Fudjimi pharmaceutical plants) the test substance was suspended dosing solution of high dose, low dose and medium dose was diluted prepared from higher doses. The uniformity and the 14-day stability of the test substance preparation liquid was confirmed by high performance liquid chromatography. Administration solution, and store it in a cool, dark place until the administration, and used within 14 days after preparation. It confirmed the test substance concentration in the administration solution at the start of administration
Details on mating procedure:
In the afternoon of the administration 15 days, was transferred to female except for the recovery group to the male of the cage of the same dose, at a ratio of one-to-one, were allowed to live together every day until copulation is confirmed between 14 days as the limit.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The uniformity and the 14-day stability of the test substance preparation liquid was confirmed by high performance liquid chromatography.
Duration of treatment / exposure:
Males 42 days, females from 14 days before mating to day 4 of lactation
Frequency of treatment:
daily
Details on study schedule:
not applicable (no F1-parents)
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
based on pre-test
Administered dose, 14-day repeated dose toxicity studies and repeated oral dose toxicity, reproductive and developmental toxicity annexation attempt
The results of the test were determined on the basis of. In other words, the test substance at a dose of 0,10,50,250 and 1,000 mg / kg / day, 1 group of male and female each of the three animals of Crl: CD (SD) 14-day repeated oral administration as a result of the rat, 1,000 mg of male and female / kg locomotor activity decreased in group, respiratory rate reduction, eyelid closure, food poor, feces amount decrease, prone position with the same group of female, after was breathing deep large Nadogami, each male and female until the administration 7 days 2 examples died. In the survivors of 250 mg / kg or more groups of male and female, liver weight high, swelling and centrilobular hepatocellular hypertrophy was observed. In addition, the extension of the APTT was observed at 10 mg / kg or more groups of females. Although these results were carried out repeated oral dose toxicity, reproductive and developmental toxicity Combined study at a dose of 0,2,10 and 50 mg / kg / day, a few changes in the weight highs and stomach of the liver at 50 mg / kg group since was only seen in cases, in this study, a 250 mg / kg / day to organic changes in the liver are expected to be seen as a high dose, the following in common ratio 5 50 and 10 mg / kg / day the three doses has been set.
Positive control:
none

Examinations

Parental animals: Observations and examinations:
1) the general state
Male, were observed in twice and dissection day every day until the day before dissection from the start date of administration. Female, twice daily until recovery group administered the last day of the period, the animals to be subjected to mating was observed in the labor situation from the start date of administration, twice daily until delivery four days, including the nursing state and delivery 5 days (anatomy date) . During the recovery period, it was observed once a day both to dissection Date male and femal
2) detailed the general state observation
For male and female all cases, once before the start of the administration, after the start of the administration were observed in the weekly-blind after administration, we were scoring. Blinded rearranges all of the animals by a random number, using a test label, was a state in which the administration group can not be determined. However, in the case of the inspection target animal is one example at the time of inspection of labor four days did not blinded.
Reaction when extracting from the cage was observed near or hands to hold the animal, the ease and utterance out of the animal when the animal is given an external stimulus, etc. hold. Detailed observation for the hands, muscle tone, decreased body temperature and the presence or absence of a nap, of the hair condition (dirt, SoTsuyoshi), of the skin and mucous membrane color (pallor, redness, cyanosis), abnormalities of the eye (lacrimation, eye protruding, pupil diameter), to observe the presence or absence of salivation and secretions. Observation of the action in the arena, attitude at 1 minute animal in the arena, activity, breathing, eyelid closure, walking state, tremors, spasms, convulsions, were observed for the presence of stereotyped behavior and abnormal behavior. In addition, it was recorded Haikuso number of times (the number of feces) and frequency of urination of one minute (the number of pool of urine).
3) function testing
Male, was examined after administration of the administration last week for five which were selected in order of small animal number for each dose. Female, before fasting for delivery four days for five close of delivery date of each dose of the calving animals, recovery group were examined after administration of the administration last week. Inspection of during the recovery period, in order to change to doubt the effect of the test substance administered in the inspection of during the administration period was not observed, it was not carried out.
Reactivity test, observing the animal's reactions when given the appropriate external stimulus to a sensory organ to be inspected, subjected to scoring. Inspection was carried out in continued blind to detailed observation. Visual (approach and touch), the closer the ball-point pen of the sheath to the face before 3 cm, was recorded the reaction of when held as it is 4 seconds. Hearing, it was recorded the reaction of when you play a finger in the overhead. Pain was recorded reaction when sandwiching the third ridge portion of the tail with clothespin. Pupillary, after blocking the light it was recorded and the reaction of the pupil when exposed to light. Aerial SeiMuko reflections were recorded reaction when dropped animals abdominal from a height of approximately 30 cm in a state of facing upward.
Grip strength measurements, grip strength meter (FGC-2, Meitisu) was measured in the blind using. Forelimbs, hind limb both measured twice, and the average value was used as the grip strength value of an individual's forelimb and hind limb.
Locomotor activity measurement, rats for momentum measurement device (ACTIMO-10, Shintekuno) was used to measure the animal's momentum. The number of crossed an infrared beam as a measurement value was measured (6 times at 10 minute intervals) for 1 hour.
4) body weight
Male was measured 1,3,7,14,21,28,35,42 and 43 day administration (carry-out at the time, fasted state).
Female, 1, 3, 7 and 14 days administration, pregnancy 0,7,14,17 and 20 days, delivery 0 (delivery date), was measured in the 4 and 5 days (carry-out at the time, fasted state). Female of the satellite was measured in male and the same day. During the recovery period, it was measured in both sexes 1,3,7,14 and 15 days (carry-out at the time, fasted state).
5) food consumption
Male administration 1,3,7,14,28,35 and 42 days, female, administered 1, 3, 7 and 14 days, pregnancy 0,7,14,17 and 20 days, delivery 0 (calving day) and was measured 4 days. Female of the satellite was measured in male and the same day. During the recovery period, it was measured in both the 1, 3, 7 and 14 days male and female. For both sexes each period, in search of food consumption from the measurement date to the next measurement date, to calculate the average daily food consumption.
) hematology
After overnight fasting at the free drinking water from the afternoon to the anatomy of the administration the last day of the period or the recovery period the last day (16 to 20 hours), 5 males in the ascending order of the animal number out of each dose of the surviving animals, females each for male and female all cases of five and recovery group close to the dose with calving date, it was bled EDTA-2K as an anti-coagulant, fully automated comprehensive hematology analyzer (CELL-DYN3500, Abbott Laboratories) by the number of red blood cells (RBC) , white blood cell count (WBC), hemoglobin concentration (Hb), hematocrit value (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), to measure the mean corpuscular hemoglobin concentration (MCHC) and the number of platelets (platelet) . In addition, comprehensive hematology testing equipment (ADVIA 120, Siemens) by, to measure the reticulocyte count ratio (Reticulo) and white blood cell percentage (Differentiation of leukocyte). Furthermore, blood was collected 3.2% aqueous solution of sodium citrate as an anticoagulant, using the obtained isolated from blood plasma, the blood coagulation automatic measuring apparatus (KC-10A, Amerungu), prothrombin time (PT) and activated It was measured partial thromboplastin time (APTT).
7) Blood biochemical tests
For animals was performed hematology, with serum, the automatic biochemical analyzer (7180 Automatic Analyzer, Hitachi, Ltd.), aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein (T- Protein), albumin (albumin), total bilirubin (T-Bil), alkaline phosphatase (ALP), cholinesterase (ChE), γ- guru Tamil trans peptidase (γ-GTP), total cholesterol (T-Cho), triglyceride (TG ), glucose (glucose), urea nitrogen (BUN), was measured creatinine (Creatinine), calcium (Ca) and inorganic phosphorus (IP). Furthermore the electrolyte analyzing apparatus (PVA-EXII, A & T) was thus measured sodium (Na), potassium (K) and chlorine (Cl). A / G ratio (A / G ratio) was calculated from the total protein and albumin.

Oestrous cyclicity (parental animals):
1) of the periodic inspection
For female all cases, until copulation verification day from 1 day administration, vaginal plaque from the females was microscopy with Giemsa staining in the morning every day. The sexual cycle of up to administration day 15, (in the case of a multi-day consecutive, the first day of estrus) each estrus was calculated average of the period the number of days in search of sexual cycle number of days from up to the next estrus. In addition, to calculate the incidence of individuals (not shown a regular sexual cycle individual) indicating the abnormal estrous cycle.
Sperm parameters (parental animals):
not determined
Litter observations:
To the date of birth (reckoned with 0-day-old), birth number (stillbirth number of children + birth live number of children), stillbirth number of children, birth live number of children, birth children gender, 0-day survival number of children, 0-day survival babies gender, birth It was observed rashly outside table. Later, the general state up to four days after birth, the survival number of children, to observe the death number of children and gender, birth rate :( birth live number of children / implantation marks number) × 100,0 day survival :( birth live number of children / birth number) × 100, birth child sex ratio: male birth number of children / birth number, 0-day survival child sex ratio: of postnatal day 0 male survival number of children / survival number of children of postnatal day 0, the outer table abnormal rate: external surface of the anomaly birth live number of children with / of birth raw number of children) × 100,4 day survival :( old 4-day survival number of children / birth rashly
Number) × 100,4 day survival child sex ratio: was calculated the survival number of children of male survival number of children / age 4 days old 4 days. In addition, body weight was measured every male and female in the belly unit to the day of birth and age four days.
Postmortem examinations (parental animals):
8) autopsy
For all cases, the day after the administration the last day of the period (excluding the recovery group) and recover the last day of the period, body surface, openings, subcutaneous, cranial cavity, chest cavity, abdominal cavity, the naked eye observation of the pelvic cavity and its contents went It was. Ovary is the number of pregnancy corpus luteum, uterus was recorded the number of implantation marks after the incision.
9) organ weight and tissue collection
For all cases, (including the duodenum - the rectum, the Peyer's patches), trachea and lungs, stomach, intestines, liver, heart, kidney, bladder, testis, epididymis, prostate, seminal vesicles, ovary, uterus, vagina, brain (cerebrum , including the cerebellum and pons), spinal cord, sciatic nerve, bone marrow (femur), lymph nodes (axillary and mesenteric lymph nodes), spleen, thymus, pituitary gland, thyroid (including parathyroid) and the adrenal glands taken and, liver, heart, kidney, testis, epididymis, brain, spleen, the weight of thymus and adrenal gland were measured. Moreover, to calculate the relative weight of the organs based on the weight at unloading. That was well taken gross lesions. Trachea, lung and bladder, was taken after the injection of a 10% neutral buffered formalin solution. Stomach and intestines, after injecting 10% neutral buffered formalin, and immersed in the same solution, and then the contents removed by washing with water. Harvested organs and tissues were fixed in 10% neutral buffered formalin. However, the testis and epididymis were fixed in Bouin's solution
10) histopathological examination
Each dose, five in the order male small control group and 250 mg / kg group of animal numbers, female for five close to that of one calving date of birth animal, trachea, lung, stomach, intestines, liver, heart, kidney , bladder, testis, epididymis, prostate, seminal vesicles, ovary, uterus, vagina, brain, spinal cord, sciatic nerve, bone marrow, axillary lymph nodes, mesenteric lymph nodes, spleen, thymus, pituitary gland, thyroid and adrenal glands It was examined. Tests, to prepare a paraffin-embedded thin sections, was subjected to hematoxylin-eosin staining was performed on the optical microscopic. Other 250 mg / kg organs and tissues change doubt the impact of the administration of the test substance was observed in the group, was also tested for all of the other dose groups and recovery group. In addition, it was examined gross lesions of the histopathological examination subject animal. Female of this addition to infertility were examined ovaries, the uterus and vagina in order to investigate the cause of the infertility. Female all Haraji died were examined stomach before in order to examine the condition of the mother animal, glandular stomach, liver, heart, kidney, spleen, thymus and adrenal glands.
In HE staining samples of 250 mg / kg group of male, eosinophilic bodies increase in kidney was observed, in order to confirm the association between alpha 2u- globulin, for one each example of the control group and 250 mg / kg group, by anti-alpha2u- globulin antibody was performed immunohistochemistry. In addition, small leaves around hepatocellular vacuolization was seen in one patient of liver of female in the control group, for the accumulation of fat in the liver cells was suspected, were oil red O staining of the liver.
Postmortem examinations (offspring):
non described
Statistics:
Of the parent animal body weight (except when carried out), food consumption, grip strength and motor activity, hematology item, blood biochemical test items, organ weight, the average of the period the number of days, number of days required mating, pregnancy period, pregnancy corpus luteum the number and implantation marks the number, birth number of births, stillbirths number of children, birth live number of children, birth children ratios, 0-day survival children __ number, 0-day survival child sex ratio, 4 Nissei number of children, 4-day survival child sex ratio and body weight, performs a test by Bartlett method, was one-way analysis of variance when the equal variance was observed in 5% significance level. If the significant differences in variance analysis, between the control group and each dosage group were assayed by Dunnett method. If the equal variance is not observed, to test the significance of all groups in the rank sum test method of the Kruskal-Wallis, the case was significant difference, non-parametric and the control group in a multiple comparison assay of Dunnett the significant difference between went.
Haikuso number of times and frequency of urination conducted a test of Kruskal-Wallis.
Abnormalities cycle incidence of parental animals, mating rate, insemination rate, conception rate and fertility rate, birth child sex ratio of offspring, 0-day survival child ratios and 4-day survival child
sex ratio is, in Fisher's exact test and significant difference tests were performed between the control group.
Of the parent animal implantation rate, pre-implantation embryo loss rate, post-implantation embryo loss rate and birth rate, fertility rate of birth, 0-day survival, the outer table abnormal rate and the 4-day survival rate, rank sum of the Kruskal-Wallis It was tested for significance of all groups in assays.
Body weight of the offspring were treated to every male and female of the litter as a sample unit.
Reproductive indices:
Pregnant animals were natural birth the whole example. And nesting mother animal by 3:00 pm, it is assumed that labor in case you start breast-feeding to the child has been completed, and that day delivery date and (delivery day 0). Animal birth to 3 pm or later completed was the day after the delivery date. The number of days until the delivery is complete the day before from the date of admitted copulation was the pregnancy period. From the results obtained, to calculate the birth rate :( live births female number / female number) × 100, calving rate :( birth number / implantation marks number) × 100.
Offspring viability indices:
To the date of birth (reckoned with 0-day-old), birth number (stillbirth number of children + birth live number of children), stillbirth number of children, birth live number of children, birth children gender, 0-day survival number of children, 0-day survival babies gender, birth It was observed rashly outside table. Later, the general state up to four days after birth, the survival number of children, to observe the death number of children and gender, birth rate :( birth live number of children / implantation marks number) × 100,0 day survival :( birth live number of children / birth number) × 100, birth child sex ratio: male birth number of children / birth number, 0-day survival child sex ratio: of postnatal day 0 male survival number of children / survival number of children of postnatal day 0, the outer table abnormal rate: external surface of the anomaly birth live number of children with / of birth raw number of children) × 100,4 day survival :( old 4-day survival number of children / birth rashly
Number) × 100,4 day survival child sex ratio: was calculated the survival number of children of male survival number of children / age 4 days old 4 days. In addition, body weight was measured every male and female in the belly unit to the day of birth and age four days.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
During the administration period, in males, in the group of 50 mg / kg or more, immediately after administration, transient salivation was found in all 12 subjects at each dose. Besides this, upper incisor teeth defects were found in one of the 10 mg / kg group on administration 35 to 41 days, but the relationship with the dose was not clear change. In females, transient salivation immediately after administration was observed in 3 of 12 cases during pregnancy in the 50 mg / kg group, all in pregnancy and pregnancy in 250 mg / kg group, 7 For example, in all 5 cases of 250 mg / kg group of satellites. In addition, one patient in the control group showed scarification on 3 to 5 days of nursing. No abnormality was found in the 10 mg / kg group.
No abnormality was observed in both males and females during the recovery period
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
During the administration period, both during the recovery period, significant difference in the male and female of the test substance administered group was not observed.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
During the administration period, in the males, the 10 mg / kg group showed a low value on the 28 th to 42 th day of administration, but the relationship with the dose was not clear change. There was no significant difference in the 50 and 250 mg / kg groups.
In females, high levels were observed on the 4th day of nursing in the 10 and 250 mg / kg group, but there was no significant difference in the 50 mg / kg group and the dose relationship was unclear and it was judged to be an accidental change.
During the recovery period, there was no significant difference in the test substance-administered group in both males and females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in the male, but the low value of the hemoglobin concentration and hematocrit value in the group of more than 50 mg / kg were found, none of which are differed on the value of the 50 and 250 mg / kg group, dose and it related is not clear, since the abnormality in red blood cell count is not seen, it is determined that the accidental changes.
Significant differences in 10 mg / kg group was observed. In females, significant difference in all of the test substance administered group was not observed.
During the recovery period, in the male, MCHC at 250 mg / kg group, height of the platelet count and lymphocyte ratio, but low value of neutrophil ratio was observed, that the same change is not observed at the time of the end of the administration period , from the fact that no abnormality was observed in the number of red blood cells and white blood cell count, it is determined that the accidental change. In females, significant difference in the 250mg / kg group were observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in the male, 250 mg / kg high of γ-GTP in the group, the low value of triglycerides was observed. Low value of total bilirubin that is dependent on the dose in this addition to 50 mg / kg or more groups, calcium higher in the 250 mg / kg group, but the low value of the chlorine was observed, that a change to any relevant is not seen from, it is determined that the accidental changes. Significant differences in 10 mg / kg group was observed. In females, although low levels of potassium were observed at 10 mg / kg group, for association with dose is not clear, it is determined that the accidental changes. Significant differences in 50 and 250 mg / kg group was observed.
During the recovery period, in the male, but the low value of AST at 250 mg / kg group was seen, from the fact that the relevant change is not observed, it is determined that the accidental change. In addition, although high calcium in the same group was observed, as compared with at the end of the administration period, the degree had been reduced. In females, but the low value of albumin in the 250 mg / kg group was seen, from the fact that the relevant change is not observed, it is determined that the accidental change.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No abnormality was observed in the test substance-administered group in males during the administration period. In females, the high value of locomotor activity was observed in 0 to 10 minutes in the 250 mg / kg group, but there was no abnormality in the total value of 0 to 60 minutes due to transient change, general condition observation, arena Since there was no change related to behavior within the judge, it was determined to be an accidental change. No abnormality was found in the 10 and 50 mg / kg groups.
The examination during the recovery period was not conducted because there was no doubt about the influence of administration of the test substance in the examination during the administration period 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 the administration period, in the male, height of the absolute and relative weight of the liver was observed in the 250 mg / kg group.
Significant differences in 10 and 50 mg / kg group was observed. In females, the peak value of the absolute and relative weight of the liver that is dependent on the dose was observed in the group of more than 50 mg / kg. Absolute and relative weight of the high price of the thymus in this addition to 250 mg / kg group, but the low value of the absolute and relative weight of the adrenal glands were observed, because the changes that any relevant is not seen, toxicologically significant changes It was determined not to be. Significant differences in 10 mg / kg group was observed.
During the recovery period, in the male, but high in the absolute and relative weight of the liver was observed in the 250 mg / kg group, as compared with at the end of the administration period, the degree was reduced. The absolute weight of the low value of the adrenal gland in this addition to 250 mg / kg group was observed, since the change of the same at the end of the administration period is not observed, it is determined that the accidental change. In females, but high relative weight of the liver was observed in the 250 mg / kg group, as compared with at the end of the administration period, the degree was reduced.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in the male, enlargement of the liver is one case in 12 cases of 50 mg / kg group, was seen in seven patients all patients of 250 mg / kg group. Nodule and the white part of the epididymis was observed in one case each in this addition to 250 mg / kg group. No abnormalities were observed in the 10 mg / kg group. In females, enlargement of the liver was observed in 12 patients all patients in the 250 mg / kg group. Mucosa recessed portion of the front stomach in this other control group, mucosa black part and the size of the thymus is one example of the glandular stomach, mucous membranes black part is one example of the glandular stomach at 10 mg / kg group, the glands in the 50 mg / kg group mucosa the black part of the stomach was observed in two patients.
During the recovery period, no abnormalities were observed in male and female with 250 mg / kg group. In addition, the white part is an example of the epididymis in male control group, dark red of the thymus was observed in another example.
No abnormalities were observed in each example of the female in the control group and 10 mg / kg group of infertility.
One thing in the 10 mg / kg group of females in which the wholeborn died on the first day of nursing showed miniaturization of the thymus.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in the male, liver of diffuse liver cell hypertrophy at 250 mg / kg group 5 cases all cases, is eosinophilic bodies increase in the kidney was observed in one patient. 50 mg / kg 1 of 5 patients are solitary cyst of the medulla of the kidney in the group as this addition to naturally occurring lesions, 1 case localized myocarditis of heart at 250 mg / kg group, bilateral in another example away and out inhibition and basolateral stagnation of the sperm cells of mature sperm cells of the testes, sperm granuloma of one side of the epididymis was observed in two patients. In addition, although lymphocyte infiltration of the prostate was observed in 4 cases of three patients and 250 mg / kg group in 5 patients in the control group, the extent of 250 mg / kg group is weaker than the control group, revealed to be associated with dose because not, it is determined that the accidental change. No abnormalities were observed in the 10 mg / kg group. Eosinophilic bodies increase in the kidney that was observed in 250 mg / kg group, as a result of the immunohistochemistry with an anti-alpha 2u- globulin antibodies for one each example of the control group and 250 mg / kg group, the anti-alpha that 2u- globulin antibody-positive substances have accumulated it has been confirmed. In females, 250 mg / kg small centrilobular liver cell hypertrophy of the liver in the group were seen in 5 cases all cases. Besides this erosion of previous stomach in the control group as a natural occurrence lesions, stomach bottom gland mucosal necrosis and lobular peripheral liver cell fat of the liver is one example of the glandular stomach, small leaves surrounding liver cells good of the liver to another example basified, basophilic tubules of the kidney, the renal tubular epithelial degeneration and necrosis, atrophy in 2 cases of thymus, cell infiltration is one example of the cortex of both sides of the adrenal glands, stomach glandular stomach at 50 mg / kg group one example is the bottom gland mucosal necrosis, cell infiltration of the cortex of the mineral deposits and both sides of the adrenal glands of Peyer's patches of the jejunum was observed in one patient in the 250 mg / kg group. For small leaves around hepatocellular vacuolization of the liver was observed in one patient in the control group, as a result of the oil red O staining, placed in liver cells
Le Red O-positive material was observed in a large amount, that the fat is accumulated was confirmed in the hepatocytes.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
In one case of the 50 mg / kg group, the resting period was sustained from 1 to 15 days after administration, but the relationship with the dose was unclear. No abnormality was found in the mating performance of this animal. No animals showing abnormal sexual cycle were found in the control group, 10 and 250 mg / kg group, and no significant difference was observed in the group to which the test substance was administered even in the average cycle period.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mate is satisfied in all animals in the control group and the test substance administered group, the control group, 10, 50 and 250 mg / kg number of pregnant animals of the group, were respectively 11,11,12 and 12 cases. Insemination rate and conception rate is one case each of the control group and 10 mg / kg group became 91.7% for infertility, 50 and 250 mg / kg group showed 100% is not clear association between doses was the change. There was no significant difference seen in the Copulation number of days required of the test substance administration group, gestation period, pregnancy corpus luteum number, implantation marks the number, implantation rate, pre-implantation embryo loss rate, embryo loss rate after implantation, and birth rate

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
> 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no significant difference in the body weight of the test substance-administered group in both males and females.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Examination of the birth date and nursing period of pediatric animals
Infant animals were not affected by administration of the test substance. Seven deaths were seen in the control group, and 1 litter of all the belly died in the 10 mg / kg group. Breast milk was not found in the stomach of this belly, and body temperature was falling. Three cases of mortality in the 50 mg / kg group, three cases of death in the 250 mg / kg group, and one case of tail defect were observed. However, because the association with the dose was not clear, the number of cases of expression was small, it was judged to be an accidental change. There was no significant difference in the number of births, birth rate, 0 day survival rate, sex ratio, external table and 4-day survival rate in the test substance-administered group.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL and NOEL for the reproductive/developmental toxicity were 250
mg/kg/day for parental animals and offspring.
Executive summary:

There were no changes attributable to the test substance on estrous cvclicitv:司

copulation index, fertilization index, conception index, implantation index, preimplantation

loss, post implantation loss, or delivery dam index of the reproductive ability of the parental

animals, or the number of pups born, live birth index, external appearance, viability index,

sex ratio or body weights of the pups.