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Repeated dose toxicity: oral

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Endpoint:
short-term repeated dose toxicity: oral
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 Oct 2011 - 28 Mar 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 Oct 2011 - 28 Mar 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Dams with offspring and pups were terminated on Day 5 post-partum. Litter weights, anogenital distance and nipple retention were not measured. Thyroid hormones of pups were not assessed.
Qualifier:
according to
Guideline:
other: Circular on Test Methods of New Chemical Substances (Japan)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the refrigerator
- Solubility and stability under test conditions: confirmed by IR at the end of the administration period
- Stability of the test substance in the solvent/vehicle: The test substance is stable for 8 days in the refrigerator and 24 hours at room temperature.
Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Atsugi, Japan
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: 371 - 432 g (males), 223 - 274 g (females)
- Housing: individual animal in cage in steel cages before mating. During mating, one female and one male were in the same metal cage. From Day 17 in gestation period to Day 4 in the lactation period, one dam and its pups were in the plastic cage with white wood chip as bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
- Diet: NMF food (Oriental Yeast Co., Ltd., Itabashi-ku, Japan) was provided ad libitum during the acclimation period and throughout the study. Food was removed the afternoon/evening prior to necropsy.
- Water: Quality of tap water was analysed quarterly.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24
- Humidity (%): 40 - 56
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Oct To: 19 Dec 2011
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Each dosing formulation was prepared every 8 days. Stability of formulation was determined and stable for 8 days in the refrigerator and 24 hours at room temperature. Dosing solutions were prepared by adding the appropriate amount of the test article to a tared container and adding the appropriate amount of corn oil to yield the desired dose level. Dosing solutions were administered by oral gavage with a flexible stomach tube. The test article was administered at a dose volume of 5 mL/kg bw and was calculated from the most recent body weight. Until administration, stock solution was stored in a dark bottle and in a refrigerator (4 - 8°C).

VEHICLE
- Justification for use and choice of vehicle (if other than water): According to the preliminary test by using GC and IR due to the physical and chemical properties of the test substance, the test substance was stable in the corn oil.
- Concentration in vehicle: 6, 30 and 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg b.w.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Study Day 15 up to Day 28
- Proof of pregnancy: Females rats were evaluated daily for evidence of copulation, by confirming the presence of either vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy.
- After successful mating each pregnant female was caged (how): Day 0 of gestation was defined as the day where copulation was observed, at which time the female was individually caged.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations were conducted using GC to confirm the concentrations in all dose groups before and at the end of the administration period. The results of dose concentration analyses were determined to be 97.7 - 107.7%. These results were within the acceptable limits (100.0 ± 10.0% of nominal values).

Homogeneity of formulations was not determined.
Duration of treatment / exposure:
Males:
14 days before mating
14 days during mating
14 days after mating

Females:
14 days before mating
27 - 37 days during mating, gestation and lactation period

Males and females for recovery group:
42 days administration and 14 days post-exposure observation period
Frequency of treatment:
once daily, 7 days/week
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Male treatment group:
7 (control and 750 mg/kg bw/day)
12 (30 and 150 mg/kg bw/day)

Male recovery group:
5 (control and 750 mg/kg bw/day)

Female
12 (treatment group)
10 (recovery group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were based on the results of a range-finding study, in which animals were orally exposed to 250, 500 and 1000 mg/kg bw/day for 14 days. No death occurred in all groups and effects on general clinical signs and body weight were not observed. Males exposed to 1000 mg/kg bw/day showed slightly decreased food consumption at the beginning of the study, decreased red blood cell count, haemoglobin and haematocrit, increased platelet and total protein, increased liver and thyroid weight and decreased testis weight. Males exposed to 500 mg/kg bw/day showed increased total protein and liver weight. Male 250 mg/kg bw/day group showed increased liver weight. Females exposed to 1000 mg/kg bw/day group showed decreased food consumption during the administration period, increased total protein, increased liver, thyroid and ovary weight. Therefore, 30, 150 and 750 mg/kg bw/day were selected as the dose levels for the main study.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
Not performed
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: three times a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males in the treatment and recovery group and females in the recovery group were obsereved once prior to exposure, once a week during the administration period and recovery period.
Females in the treatment group were observed once prior to the exposure, once a week during mating period, on gestation Days (GD) 1, 7, 14, and 20, and on Day 4 post-partum.

BODY WEIGHT: Yes
- Time schedule for examinations: Males in the treatment and recovery groups and females in the recover y group were weighed on Day 1, 8, 15, 22, 29, 36 and 42 during administration period, on Day 1, 8 and 14 during recovery period, and before termination.
Parental females were weighed on Day 1, 8 and 15 during administration period (non-pregnant animals on Day 22), on GD 0, 7, 14 and 20, on Day 0 and 4 post-partum, and before termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No



Oestrous cyclicity (parental animals):
Performed dduring the pre-mating period
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight and epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on Day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, presence of gross anomalies, postnatal mortality, weight gain

GROSS EXAMINATION OF DEAD PUPS:
no

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at the end of the administration or at the end of the observational period
- Maternal animals: All surviving animals were euthanised by isoflurane on post-partum Day 4.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues and organ taken for histopathological examination:
Adrenal, bone, bone marrow, femoral, cerebellum (pons), cerebrum, epididymis, eye, heart, duodenum, jejunum, ileum (Peyer's patch), cecum, colon, rectum, kidney, mesenteric and submandibular lymph nodes, liver, parathyroid, pancreas, pituitary, salivary gland submandibular, sciatic nerve, spleen, stomach, skeletal muscle, femoral, seminal vesicle (coagulating gland), spinal cord, thoracic, testis, thymus, trachea, thyroid, urinary bladder, ovary, uterus, vagina

The following organs were weighed:
Brain, pituitary, thyroids, thymus, heart, liver, spleen, kidneys, adrenals, ovaries, uterus, seminal vesicles, prostate, testis, epididymis,
Statistics:
Bartlett test, Dunnett's test, Steel test, F test, Student's t test, Aspin-Welch's t test, Fischer's test
Reproductive indices:
Copulation index, fertility index, gestation index, implantation index, and delivery index
Offspring viability indices:
Live birth index, viability index on postnatal Day 4, sex ration of total number of pups at birth, sex ration of live pups on Day 4
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One male in the 750 mg/kg bw/day group died on Day 12 of administration. Before death, the male showed reddish urine and smudge of lower abdomen on Day 10, decreased spontaneous movement on Day 11, and prone/lateral position and pale skin on day 12. Reddish urine was also observed in one animal in the 750 mg/kg bw/day group on Week 1, 2 and 3.
One female in the 150 mg/kg bw/day treatment group died on Day 1 of the lactation period. However, there was no abnormal general condition reported.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male in the 750 mg/kg bw/day treatment group died on Day 12 of administration.
One female in the 150 mg/kg bw/day treatment group died on Day 1 post-partum.
All animals in the recovery groups survived the duration of the study.
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):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1) At the end of administration period
Males in the 750 mg/kg bw/day treatment group showed increased platelet numbers and fibrinogen value and prolonged prothrombin time and activated partial thromboplastin time (APTT). The mode of action is not clear but this can be caused by the test substance.
Females in the 750 mg/kg bw/day treatment group showed prolonged APTT, decreased relative and a bsolute neutrophil counts, and increased relative lymphocyte and eosinophil counts. The forementioned changes were considered as incidental due to the slight changes.
Females in the 750 mg/kg bw/day recovery group showed prolonged PT and APTT and increased MCV. Although the relative neutrophil counts were decreased, the absolute counts were not changed. This change was considered to be incidental.
No changes were found in the 30 and 150 mg/kg bw/day treatment group in comparison with the control group.

2) At the end of the recovery period
Males in the 750 mg/kg bw/day recovery group showed increased platelet. No changes were observed in females after recovery period in comparison with the control group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1) At the end of the administration period
Males in the 750 mg/kg bw/day treatment group showed increased γ-GTP, decreased total bilirubin, decreased potassium, increased calcium, increased total protein, and decreased A/G ratio. Decreased AST was not considered as toxicological effect. Total bilirubin was also decreased in male 150 mg/kg bw/day treatment group.
Females in the 750 mg/kg bw/day treatment group showed increased γ-GTP, decreased total bilirubin, increased BUN and decreased A/G ratio.
Females in the 750 mg/kg bw/day recovery group showed increased γ-GTP, increased total protein, and decreased A/G ratio. Decreased AST was not considered as toxicological effect.
No significant changes in clinical biochemistry parameters were observed in the 30 and 150 mg/kg bw/day treatment group compared to the control group.

2) At the end of the recovery period
Males in the 750 mg/kg bw/day recovery group showed decreased total bilirubin. Slight increased albumin was considered as the physiological variation because this change was not observed at the end of the administration period.
Females in the 750 mg/kg bw/day recovery group didn’t show the any differences as compared with the control group.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
1) At the end of administration period
Yellow urine was observed in 5 males and 3 females in the 750 mg/kg bw/day tratment group.
Increased urine volume and decreased osmotic pressure were observed in male in the 150 mg/kg bw/day treatment group. These changes regarding urine volume and osmotic pressure were not dose-depending.

2) At the end of the recovery period
Potassium value was increased in males in the 750 mg/kg treatment group. This change was regarded to be incidental because it was only a slight change and no abnormality was found at the end of the administration.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
1) During administration
Hearing was decreased in 30 mg/kg bw/day treatment group on Week 6 of administration. However, this change was not dose-depending. Since the dead male animal in the 750 mg/kg bw/day treatment group was not able to walk on Day 11 of administration, detailed clinical signs were not tested.
Females in the 750 mg/kg bw/day recovery group showed also decreased hearing in Week 1 of administration. This was regarded to be incidental because no changes were found afterward in this group and in females of the treatment group at the same time.

2) During recovery period
The increased hearing in females in the 750 mg/kg bw/day recovery group in Week 1 of recovery period was also regarded to be incidental because no change was observed at other time point and general condition was not abnormal.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1) At the end of the administration period
Relative and absolute thyroid and liver weight was increased in male and female rats in the 750 mg/kg bw/day treatment group.
Males in the 30 mg/kg bw/day treatment group showed decreased relative and absolute thymus weight and increased absolute heart weight. Males in the 150 mg/kg bw/day treatment group showed decreased relative thymus weight. But these changes were not dose-dependent. Although the relative kidney weight was increased in females in the 750 mg bw/day treatment group, this was regarded to be incidental due to the lack of dose-dependency. Females in the 750 mg/kg bw/day recovery group showed the increased absolute heart weight. This change was also considered to be incidental because the relative weight was not changed.

2) At the end of the recovery period
Males in the 750 mg/kg bw/day recovery group showed increased absolute and relative thyroid and liver weight.
No changes were observed in females of 750 mg/kg bw/day recovery group in comparison with the control group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1) Dead animals
One male in the 750 mg/kg bw/day treatment group showed pale discoloration of skin and all tissues, smudge of lower abdominal fur, enlargement of and dark red foci in the kidney, enlargement of and dark coloration in the liver, dark coloration in the prostate, small thymus, and large urinary bladder. One female in the 150 mg/kg bw/day treatment group showed dark red foci in the kidney and in the forestomach.

2) At the end of the administration period
One male (1/6) in 750 mg/kg bw/day treatment group showed the enlargement of the kidney. Other slight changes were considered to be incidental.
No abnormalites on reproductive organs and tissues were observed.

3) At the end of the recovery period
No changes caused by the test substance were observed in all animals. Other slight changes were considered as incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Exposure of the test material to male and female rats resulted in effects being observed in the following organs: kidney, urinary bladder, liver, and thyroid. In the kidney, there were minimal tubular regeneration (4/6 males in the 750 mg/kg bw/day treatment group), eosinophilic body found in tubular cell (minimal 3/6, mild 1/6 males in the 750 mg/kg bw/day treatment group) and minimal transitional hyperplasia/hypertrophy observed (1/6 males in the 750 mg/kg bw/day treatment group). In the urinary bladder, there was transitional hyperplasia/hypertrophy seen (minimal 2/6 and moderate 1/6 males in the 750 mg/kg bw/day treatment group, minimal 3/12 females in the 750 mg/kg bw/day treatment group). The observed transitional hyperplasia/hypertrophy in male rats in the 30 mg/kg bw/day treatment group was concluded to be not treatment-related effects but instead caused by incidental inflammation of the urinary bladder. In the liver, there was centrilobular hepatocytic hypertrophy observed (minimal 3/6 males in the 750 mg/kg bw/day treatment group, minimal 9/11 and mild 2/12 females in the 750 mg/kg bw/day treatment group, minimal 4/9 females in the 150 mg/kg bw/day treatment group, and minimal 4/6 and mild 1/6 females in the 750 mg/kg bw/day recovery group). In the thyroids, there was hypertrophy in follicular cell (minimal 2/12 males in the 150 mg/kg bw/day treatment group, mild 6/6 males in the 750 mg/kg bw/day treatment group, minimal 10/12 and mild 1/12 females in the 750 mg/kg bw/day treatment group, and minimal 1/5 and mild 4/5 females in the 750 mg/kg bw/day recovery group). The effect observed in the liver is concluded to be associated with induction of drug metabolising enzyme. It is well known that a substance which induces drug metabolising enzyme also promotes metabolism of thyroid hormone and causes hypertrophy of follicular cell through the hypothalamus-hypophysis system as a secondary effect. Effects observed in the transitional epithelium of the kidneys were also noted in the urinary bladder. The mode of action causing this effect was not clear according to this test result. However, the effects on the transitional epithelium in the kidneys and urinary bladder were reversed by the end of the recovery period; these effects were therefore concluded to be reversible.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no statistically significant differences between controls and treatment groups in the mean body weights on any of the test groups.
There were no differences in the average daily food consumption between controls and the males groups for any of the measured time periods. In the reproductive group females, at 30 and 750 mg/kg bw/day showed a significant increase (p< 0.05 and p<0.01, respectively) in food consumption compared to control at day 2 during the lactation period. But this change was not regarded as treatment related due to the lack of dose response.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There was no statistically significant difference across treatment groups for sperm measures.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no statistically significant difference across treatment groups for estrous cycle, mean days until copulation for males and females, copulation index, insemination index, fertility index, delivery index, gestation length in days, number of corpora lutea, number of implantation sites, implantation index, live birth index, and viability index on postnatal day 4.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No remarkable changes were found in organ weights of reproductive organs in all treatment groups compared to the control group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No remarkable changes were found in reproductive organs in all treatment groups compared to the control group.
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive toxicity observed
Critical effects observed:
not specified
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no statistically significant differences between controls and treatment groups in live birth index, viability index on postnatal Day 4, sex ratio of delivered pups and liveborns, and sex ratio of live pups on Day 4.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant difference was found in any treated groups from control group.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no abnormal findings in male and female pups.
Although one pup had a vestigial tail in 30 mg/kg bw/day, this was considered as effect not associated with the test item.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)
There were no statistically significant differences between controls and treatment groups in live birth index, viability index on postnatal Day 4, sex ratio of delivered pups and liveborns, and sex ratio of live pups on Day 4.

BODY WEIGHT (OFFSPRING)
No significant difference was found in any treated groups from control group.

GROSS PATHOLOGICAL FINDINGS
There were no abnormal findings in male and female pups. Although one pup had a vestigial tail in 30 mg/kg bw/day, this was considered as effect not associated with the test item.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no reproductive toxicity observed
Critical effects observed:
no
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Reproductive effects observed:
no

Table 2. Summary of mean reproductive parameters for reproductive group female rats

 

 

Control (0 mg/kg bw/day)

30 mg/ kg bw/day

150 mg/ kg bw/day

750 mg/ kg bw/day

Corpora lutea counts

mean

16.6

16.8

16.2

16.8

S.D.

1.8

2.0

2.4

2.7

N

12

12

10

12

Total implants

mean

15.3

15.6

15.2

14.9

S.D.

1.9

1.4

1.8

2.2

N

12

12

10

12

Days gestation

mean

22.5

22.0

22.3

22.0

S.D

0.5

0.3

0.5

0.3

N

12

12

10

12

Total live pups day 0

mean

13.4

14.3

14.4

13.3

S.D.

1.7

1.8

1.7

2.4

N

12

12

10

12

Male pups

mean

6.8

7.0

7.2

6.6

S.D.

2.4

1.5

2.1

2.2

N

12

12

10

12

Female pups

mean

6.6

7.3

7.2

6.7

S.D

2.1

1.9

2.1

2.8

N

12

12

10

12

Delivered males/Delivered pups (%)

mean

0.50

0.49

0.51

0.51

S.D

0.16

0.10

0.13

0.16

N

12

12

10

12

Day 4 viable pups

mean

13.2

14.1

14.4

13.2

S.D

1.7

1.6

1.7

2.4

N

12

12

9 a)

12

Viable/total liveborns (%)

mean

98.2

99.1

99.3

99.4

S.D.

3.3

3.2

2.2

1.9

N

12

12

10

12

Initial Male Average Pup Weight (g) [Live Pups only]

mean

6.7

6.6

6.7

6.7

S.D.

0.3

0.4

0.5

0.6

N

12

12

10

12

Initial Female Average Pup Weight (g) [Live Pups only]

mean

6.3

6.2

6.3

6.4

S.D.

0.6

0.4

0.4

0.5

N

12

12

10

12

Final Average Male Pup (g)

mean

10.3

10.2

10.0

10.2

S.D.

0.9

0.9

0.9

1.5

N

12

12

9 a)

12

Final Average Female Pup (g)

mean

9.6

9.8

9.4

10.0

S.D.

1.1

0.7

0.9

1.5

N

12

12

9 a)

12

 

a)     One dam died on lactation Day 1

Conclusions:
Based on the results of this study, the NOAEL for reproductive toxicity was set at 750 mg/kg bw/day, the highest dose tested. No adverse effects on development of offspring were observed up to and including 750 mg/kg bw/day.
Reason / purpose:
reference to same study
Reference
Endpoint:
developmental toxicity
Remarks:
screening study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 Oct 2011 - 28 Mar 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Dams with offspring and pups were terminated on Day 5 post-partum. Litter weights, anogenital distance and nipple retention were not measured. Thyroid hormones of pups were not assessed.
Qualifier:
according to
Guideline:
other: Circular on Test Methods of New Chemical Substances (Japan)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the refrigerator
- Solubility and stability under test conditions: confirmed by IR at the end of the administration period
- Stability of the test substance in the solvent/vehicle: The test substance is stable for 8 days in the refrigerator and 24 hours at room temperature.
Species:
rat
Strain:
other: Crl:CD (SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Atsugi, Japan
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: 371 - 432 g (males), 223 - 274 g (females)
- Housing: individual animal in cage in steel cages bevor mating. During mating, one female and one male were in the same metal cage. From Day 17 in gestation period to Day 4 in the lactation period, one dam and its pups were in the plastic cage with white wood chip as bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
- Diet: NMF food (Oriental Yeast Co., Ltd., Itabashi-ku, Japan) was provided ad libitum during the acclimation period and throughout the study. Food was removed the afternoon/evening prior to necropsy.
- Water: Quality of tap water was analysed quarterly.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24
- Humidity (%): 40 - 56
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Oct To: 19 Dec 2011
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Each dosing formulation was prepared every 8 days. Stability of formulation was determined and stable for 8 days in the refrigerator and 24 hours at room temperature. Dosing solutions were prepared by adding the appropriate amount of the test substance to a tared container and adding the appropriate amount of corn oil to yield the desired dose level. Dosing solutions were administered by oral gavage with a flexible stomach tube. The test article was administered at a dose volume of 5 mL/kg bw and was calculated from the most recent body weight. Until administration, stock solution was stored in a dark bottle and in a refridgerator (4 - 8°C).

VEHICLE
- Justification for use and choice of vehicle (if other than water): According to the preliminary test by using GC and IR due to the physical and chemical properties of the test substance, the test substance was stable in the corn oil.
- Concentration in vehicle: 6, 30 and 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg b.w.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations were conducted using GC to confirm the concentrations in all dose groups before and at the end of the administration period. The results of dose concentration analyses were determined to be 97.7 - 107.7%. These results were within the acceptable limits (100.0 ± 10.0% of nominal values).

Stability of formulatios was determined and stable for 8 days in the refrigerator and 24 hours at room temperature.
Homogeneity of formulations was not determined.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Study day 15 up to study day 28
- Proof of pregnancy: Females rats were evaluated daily for evidence of copulation, by confirming the presence of either vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy.
- After successful mating each pregnant female was caged (how): Day 0 of gestation was defined as the day evidence of copulation was observed, at which time the female was individually caged.
Duration of treatment / exposure:
Males:
14 days before mating
14 days during mating
14 days after mating

Females:
14 days before mating
27 - 37 days during mating, gestation and lactation period

Males and females for recovery group:
42 days administration and 14 days post-exposure observation period
Frequency of treatment:
once daily, 7 days/week
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Male treatment group:
7 (control and 750 mg/kg bw/day)
12 (30 and 150 mg/kg bw/day)

Male recovery group:
5 (control and 750 mg/kg bw/day)

Female
12 (treatment group)
10 (recovery group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were based on the results of a range-finding study, in which animals were orally exposed to 250, 500 and 1000 mg/kg bw/day for 14 days. No death occurred in all groups and effects on general clinical signs and body weight were not observed. Males in the 1000 mg/kg bw/day showed slightly decreased food consumption at the beginning of the study, decreased red blood cell, haemoglobin and haematocrit, increased platelet and total protein, increased liver and thyroid weight and decreased testis weight. Males in 500 mg/kg showed increased total protein and liver weight. Male 250 mg/kg group showed increased liver weight. Females in the 1000 mg/kg bw/day group showed decreased food consumption during the administration period, increased total protein, increased liver, thyroid and ovary weight. Therefore, 30, 150 and 750 mg/kg bw/day were selected as the dose levels for the main study.
- Post-exposure recovery period in satellite groups: 14 days
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: three times a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males in the treatment and recovery group and females in the recovery group were obsereved once prior to the exposure, once a week during the administration period and recovery period.
Females in the treatment group were observed once prior to the exposure, once a week during mating period, on gestation days (GD) 1, 7, 14, and 20, and on Day 4 post-partum.

BODY WEIGHT: Yes
- Time schedule for examinations: Males in the treatment and recovery groups and females in the recovery group were weighed on Day 1, 8, 15, 22, 29, 36 and 42 during administration period, on Day 1, 8 and 14 during recovery period, and before termination.
Females in the treatment group were weighed on Day 1, 8 and 15 during administration period (non-pregnant animals on Day 22), on GD 0, 7, 14 and 20, on Day 0 and 4 post-partum, and before termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 5
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
Statistics:
Barlett test, Dunnett test, Steel test, F test, Student's t test, Aspin-Welch's t test, Fischer's test
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One male in the 750 mg/kg bw/day group died on Day 12 of administration. Before death, the male showed reddish urine and smudge of lower abdomen on day 10, decreased spontaneous movement on Day 11, and prone/lateral position and pale skin on day 12. Reddish urine was also observed in one male in the 750 mg/kg bw/day treatment group on Week 1, 2 and 3.
One female in the 150 mg/kg bw/day treatment group died on Day 1 of the lactation period. However, there was no abnormal general condition reported.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Treatment groups:
One male in the 750 mg/kg bw/day treatment group died on Day 12 of administration.
One female in the 150 mg/kg bw/day treatment group died on Day 1 post-partum.
All animals of the recovery groups survived the duration of the study.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1) At the end of administration period
Males in the 750 mg/kg bw/day treatment group showed increased platelet numbers and fibrinogen value and prolonged prothrombin time and activated partial thromboplastin time (APTT). The mode of action is not clear but this can be caused by the test substance.
Females in the 750 mg/kg bw/day treatment group showed prolonged APTT, decreased relative and absolute neutrophil counts, and increased relative lymphocyte and eosinophil counts. The forementioned changes were considered as incidental due to the slight changes.
Females in 750 mg/kg bw/day recovery group showed prolonged PT and APTT and increased MCV. Although the relative neutrophil counts were decreased, the absolute counts were not changes. This change was considered to be incidental.
No changes were found in 30 and 150 mg/kg bw/day treatment group in comparison with the control group.
2) At the end of the recovery period
Males in 750 mg/kg bw/day recovery group showed increased platelet. No changes were observed in females after recovery period in comparison with the control group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1) At the end of the administration period
Males in the 750 mg/kg bw/day tratment group showed increased γ-GTP, decreased total bilirubin, decreased potassium, increased calcium, increased total protein, and decreased A/G ratio. Decreased AST was not considered as toxicological effect. Total bilirubin was also decreased in male 150 mg/kg bw/day treatment group.
Females in the 750 mg/kg bw/day treatment group showed increased γ-GTP, decreased total bilirubin, increased BUN and decreased A/G ratio.
Females in the 750 mg/kg bw/day recovery group showed increased γ-GTP, increased total protein, and decreased A/G ratio. Decreased AST was not considered as toxicological effect.
No significant changes in clinical biochemistry parameters were observed in the 30 and 150 mg/kg bw/day treatment group compared to the control group.
2) At the end of the recovery period
Males in the 750 mg/kg bw/day recovery group showed decreased total bilirubin. Slight increased albumin was considered as the physiological variation because this change was not observed at the end of the administration period.
Females in the 750 mg/kg bw/day recovery group didn’t show the any differences as compared with the control group.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
1) At the end of administration period
Yellow urine was observed in 5 males and 3 females in 750 mg/kg bw/day tratment group. Increased urine volume and decreased osmotic pressure were observed in male 150 mg/kg bw/day. These changes regarding urine volume and osmotic pressure were not dose-depending.
2) At the end of the recovery period
Potassium value was increased in male 750 mg/kg bw/day. This change was regarded as an incidental issue because it was a slight change and no abnormality was found at the end of the administration.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
1) During administration
Hearing was decreased in 30 mg/kg bw/day treatment group on week 6 of administration. However, this change was not dose-depending. Since the dead male animal in the 750 mg/kg bw/day treatment group was not able to walk on Day 11 of administration, detailed clinical signs were not tested.
Females in the 750 mg/kg bw/day recovery group showed also decreased hearing in Week 1 of administration. This was regarded to be incidental because no changes were found afterward in this group and in females of treatment group at the same time.

2) During recovery period
The increased rearing in female 750 mg/kg bw/day recovery group in Week 1 of recovery period was also regarded to be incidental because no change was observed at other time point and general condition was not abnormal.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1) At the end of the administration period
Relative and absolute thyroid and liver weight was increased in male and female rats in the 750 mg/kg bw/day treatment group.
Males in the 30 mg/kg bw/day treatment group showed decreased relative and absolute thymus weight and increased absolute heart weight. Males in the 150 mg/kg bw/day treatment group showed decreased relative thymus weight. But these changes were not dose-dependent. Although the relative kidney weight was increased in females in the 750 mg bw/day treatment group, this was regarded to be incidental due to the lack of dose-dependency. Females in the 750 mg/kg bw/day recovery group showed the increased absolute heart weight. This change was also considered to be incidental because the relative weight was not changed.

2) At the end of the recovery period
Males in the 750 mg/kg bw/day recovery group showed increased absolute and relative thyroid and liver weight.
No changes were observed in females of 750 mg/kg bw/day recovery group in comparison with the control group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1) Dead animals
One male in the 750 mg/kg bw/day treatment group showed pale discoloration of skin and all tissues, smudge of lower abdominal fur, enlargement of and dark red foci in the kidney, enlargement of and dark coloration in the liver, dark coloration in the prostate, small thymus, and large urinary bladder. One female in the 150 mg/kg bw/day treatment group showed dark red foci in the kidney and in the forestomach.
2) At the end of the administration period
One male (1/6) in the 750 mg/kg bw/day treatment group showed the enlargement of the kidney. Other slight changes were considered to be incidental.
3) At the end of the recovery period
No changes caused by the test substance was not observed in all animals. Other slight changes were considered as incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Exposure of the test material to male and female rats resulted in effects being observed in the following organs: kidney, urinary bladder, liver, and thyroid. In the kidney, there were minimal tubular regeneration (4/6 males in the 750 mg/kg bw/day treatment group), eosinophilic body found in tubular cell (minimal 3/6, mild 1/6 males in the 750 mg/kg bw/day treatment group) and minimal transitional hyperplasia/hypertrophy observed (1/6 males in the 750 mg/kg bw/day treatment group). In the urinary bladder, there was transitional hyperplasia/hypertrophy seen (minimal 2/6 and moderate 1/6 males in the 750 mg/kg bw/day treatment group, minimal 3/12 females in the 750 mg/kg bw/day treatment group). The observed transitional hyperplasia/hypertrophy in male rats in the 30 mg/kg bw/day treatment group was concluded to be not treatment-related effects but instead caused by incidental inflammation of the urinary bladder. In the liver, there was centrilobular hepatocytic hypertrophy observed (minimal 3/6 males in the 750 mg/kg bw/day treatment group, minimal 9/11 and mild 2/12 females in the 750 mg/kg bw/day treatment group, minimal 4/9 females in the 150 mg/kg bw/day treatment group, and minimal 4/6 and mild 1/6 females in the 750 mg/kg bw/day recovery group). In the thyroids, there was hypertrophy in follicular cell (minimal 2/12 males in the 150 mg/kg bw/day treatment group, mild 6/6 males in the 750 mg/kg bw/day treatment group, minimal 10/12 and mild 1/12 females in the 750 mg/kg bw/day treatment group, and minimal 1/5 and mild 4/5 females in the 750 mg/kg bw/day recovery group). The effect observed in the liver is concluded to be associated with induction of drug metabolising enzyme. It is well known that a substance which induces drug metabolising enzyme also promotes metabolism of thyroid hormone and causes hypertrophy of follicular cell through the hypothalamus-hypophysis system as a secondary effect. Effects observed in the transitional epithelium of the kidneys were also noted in the urinary bladder. The mode of action causing this effect was not clear according to this test result. However, the effects on the transitional epithelium in the kidneys and urinary bladder were reversed by the end of the recovery period; these effects were therefore concluded to be reversible.
Histopathological findings: neoplastic:
no effects observed
Details on results:
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There were no statistically significant differences between controls and treatment groups in the mean body weights on any of the test groups.
There were no differences in the average daily food consumption between controls and the males groups for any of the measured time periods. In the reproductive group females, at 30 and 750 mg/kg bw/day, there was a significant increase (p< 0.05 and p<0.01, respectively) in food consumption from control at day 2 during the lactation period. But this cahnge was not regarded effect associated with the test item due to the lack of dose response.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
In the testes there was no statistically significant difference across treatment groups for sperm measures.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no statistically significant difference across treatment groups for estrous cycle, mean days until copulation for males and females, copulation index, insemination index, fertility index, delivery index, gestation length in days, number of corpora lutea, number of implantation sites, implantation index, live birth index, and viability index on postnatal day 4.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No remarkable changes were found in organ weights of reproductive organs in all treatment groups compared to the control group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No remarkable changes were found in reproductive organs in all treatment groups compared to the control group.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
There were no statistically significant differences between controls and treatment groups in live birth index, viability index on postnatal Day 4, sex ratio of delivered pups and liveborns, and sex ratio of live pups on Day 4.

BODY WEIGHT (OFFSPRING)
No significant difference was found in any treated groups from control group.

GROSS PATHOLOGICAL FINDINGS
There were no abnormal findings in male and female pups. Although one pup had a vestigial tail in 30 mg/kg bw/day, this was considered as effect not associated with the test item.
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
Based on the results of this study, no adverse effects on development of offspring were observed up to and including 750 mg/kg bw/day, the highest dose tested.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Dams with offspring and pups were terminated on Day 5 post-partum. Litter weights, anogenital distance and nipple retention were not measured. Thyroid hormones of pups were not assessed.
Qualifier:
according to
Guideline:
other: Circular on Test Methods of New Chemical Substances (Japan)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep the substance in cool and dark place; tightly closed, in refrigerator (1 - 10°C); fill nitrogen after opening of package.
- Solubility and stability under test conditions: The test substance is stable. The stability was confirmed at the end of exposure using IR spectroscopy.
- Stability of the test substance in the solvent/vehicle: The test substance was determined to be stable for 8 days in the refrigerator and 24 hours at room temperature.

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Atsugi, Japan
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: 371 - 432 g (males), 223 - 274 g (females)
- Housing: individual animal in cage in steel cages before mating. During mating: one female and one male were kept in the same metal cage. From Day 17 in gestation period to Day 4 in the lactation period: one dam and its pups were kept in the plastic cage with white wood chip as bedding.
- Diet: ad libitum
- Water: tap water ad libitum
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
- Diet: NMF food (Oriental Yeast Co., Ltd., Itabashi-ku, Japan) was provided ad libitum during the acclimation period and throughout the study. Food was removed afternoon/evening prior to necropsy.
- Water: tap water, the quality of water was analysed quarterly.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24
- Humidity (%): 40 - 56
- Air changes (per hr): 10 - 15 times
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Oct to 19 Dec 2011

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Each dosing formulation was prepared every 8 days. The stability of the formulations was determined and concluded to be stable for 8 days in the refrigerator and 24 hours at room temperature. Dosing solutions were prepared by mixing the appropriate amount of the test item and corn oil in a tared container to yield the desired dose levels. Dosing formulations were administered by oral gavage with a flexible stomach tube. The test item was administered at a dose volume of 5 mL/kg bw and was calculated from the most recent body weight. Until administration, stock solution was stored in a dark bottle and in a refrigerator (4 - 8°C).

VEHICLE
- Justification for use and choice of vehicle (if other than water): In a preliminary test, GC and IR was used due to the physical and chemical properties of the test substance, to confirm that the test substance was stable in the corn oil.
- Amount of vehicle (if gavage): 5 mL/kg b.w.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the dosing formulations were conducted using GC to confirm the concentrations in all dose groups before and at the end of the administration period. The results of dose concentration analyses were determined to be 97.7 - 107.7%. These results were within the acceptable limits (100.0 ± 10.0% of nominal values).

Homogeneity of formulations was not determined.
Duration of treatment / exposure:
Males:
14 days before mating
14 days during mating
14 days after mating

Females:
14 days before mating
27 - 37 days during mating, gestation and lactation period

Males and females of recovery groups:
42 days administration and 14 days post-exposure recovery period
Frequency of treatment:
once daily, 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Male treatment group:
7 (control and 750 mg/kg bw/day)
12 (30 and 150 mg/kg bw/day)

Male recovery group:
5 (control and 750 mg/kg bw/day)

Female
12 (treatment group)
10 (recovery group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were based on the results of a dose range-finding study, in which animals were orally exposed to 250, 500 and 1000 mg/kg bw/day for 14 days. No deaths occurred in all groups and effects on general clinical signs and body weight were not observed. Males in the 1000 mg/kg bw/day treatment group showed slightly decreased food consumption at the beginning of the study, decreased red blood cell concentration, haemoglobin and haematocrit, increased platelet and total protein, increased liver and thyroid weight and decreased testis weight. Males in 500 mg/kg bw/day showed increased total protein and liver weight. Male in the 250 mg/kg bw/day treatment group showed increased liver weight. Females in the 1000 mg/kg bw/day treatment group showed decreased food consumption during the administration period, increased total protein, increased liver, thyroid and ovary weight. Therefore, 30, 150 and 750 mg/kg bw/day were selected as the dose levels for the main study.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: three times a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males in the treatment and recovery groups and females in the recovery group were obsereved once prior to exposure, once a week during the administration period and recovery period.
Parental females were observed once prior to exposure, once a week during mating period, on gestation days (GD) 1, 7, 14, and 20, and on Day 4 post-partum.

BODY WEIGHT: Yes
- Time schedule for examinations: Males in the treatment and recovery groups and females in the recovery group were weighed on Day 1, 8, 15, 22, 29, 36 and 42 during administration period, on Day 1, 8 and 14 during recovery period, and before termination.
Parental females were weighed on Day 1, 8 and 15 during administration period (non-pregnant animals on Day 22), on GD 0, 7, 14 and 20, on Day 0 and 4 post-partum, and before termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal was determined and the mean daily diet consumption was calculated as g food/kg body weight/day: No
- Compound intake was calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 was calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isofulfuran)
- Animals fasted: Yes, between 16 to 21 hours before termination
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Yes between 16 to 21 hours before termination
- How many animals: all animals

URINALYSIS: Yes
- Time schedule for collection of urine: 24-hour urine was collected on Day 36 and 37 of the administration period for treatment groups, and during Day 8 and Day 9 in recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Animals were fasted for the first 4 hours. The 4-hour fasted urine samples were obtained and analysed for pH, protein, ketone body, glucose, occult blood, bilirubin, urobilinogen, color, and urine sediment. Urine samples of 20-hour were analysed for osmotic pressure, sodium, potassium and chloride.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Males in the treatment and recovery groups and females in the recovery group were obsereved once prior to exposure, once a week during the administration and recovery period
Parental females were observed once prior to exposure, once a week during mating period, on GD 1, 7, 14, and 20, and on Day 4 post-partum.
- Dose groups that were examined: 30, 150 and 750 mg/kg bw/day
- Battery of functions tested: Auditory response, approach response, touch response, pupillary reflex, aerial righting reflex, and landing foot splay

IMMUNOLOGY: No

OTHER:
Blood hormone measuring for T3, T4 and TSH at termination. Blood samples taken for clinical chemistry investigations were used to analyze hormone values.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Brain, pituitary, thyroid, thymus, heart, liver ,spleen, kidney, adrenal, seminal vesicle, prostate, testis, epididymis, ovary, uterus

HISTOPATHOLOGY: Yes
Adrenal, bone, bone marrow, femoral, cerebellum (pons), cerebrum, epididymis, eye, heart, duodenum, jejunum, ileum (Peyer's patch), cecum, colon, rectum, kidney, mesenteric and submandibular lymph nodes, liver, parathyroid, pancreas, pituitary, salivary gland submandibular, sciatic nerve, spleen, stomach, skeletal muscle, femoral, seminal vesicle (coagulating gland), spinal cord, thoracic, testis, thymus, trachea, thyroid, urinary bladder, ovary, uterus, vagina
Statistics:
Barlett test, Dunnett's test, Steel test, F test, Student's t test, Aspin-Welch's t test, Fischer's test

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One male in the 750 mg/kg bw/day treatment group died on Day 12 of administration. Before death, the male showed reddish urine and smudge of lower abdomen on Day 10, decreased spontaneous movement on Day 11, and prone/lateral position and pale skin on Day 12. Reddish urine was also observed in one male in the 750 mg/kg bw/day treatment group on Week 1, 2 and 3.
One female in the 150 mg/kg bw/day treatment group died on Day 1 of the lactation period. However, there was no abnormal general condition reported.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Treatment groups:
One male in the 750 mg/kg bw/day treatment group died on Day 12 of administration.
One female in the 150 mg/kg bw/day treatment group died on Day 1 post-partum.
All animals in the recovery groups survived the duration of the study.
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:
effects observed, treatment-related
Description (incidence and severity):
1) At the end of administration period
Males in the 750 mg/kg bw/day treatment group showed increased platelet numbers and fibrinogen value and prolonged prothrombin time and activated partial thromboplastin time (APTT). The mode of action is not clear but this could have been caused by the test substance.
Females in the 750 mg/kg bw/day treatment group showed prolonged APTT, decreased relative and absolute neutrophil counts, and increased relative lymphocyte and eosinophil counts. All forementioned changes were considered to be incidental as only a slight change was observed.
Females in the 750 mg/kg bw/day recovery group showed prolonged PT and APTT and increased MCV. Although the relative neutrophil counts were decreased, the absolute counts were not changed. This change was considered to be incidental.
No changes were found in 30 and 150 mg/kg bw/day treatment group in comparison with the control group.
2) At the end of the recovery period
Males in the 750 mg/kg bw/day recovery group showed increased platelet. No changes were observed in females after recovery period in comparison with the control group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1) At the end of the administration period
Males in the 750 mg/kg bw/day treatment group showed increased γ-GTP, decreased total bilirubin, decreased potassium, increased calcium, increased total protein, and decreased A/G ratio. Decreased AST was not considered as a toxicological effect. Total bilirubin was also decreased in males in the 150 mg/kg bw/day treatment group.
Females in the 750 mg/kg bw/day treatment group showed increased γ-GTP, decreased total bilirubin, increased BUN and decreased A/G ratio.
Females in the 750 mg/kg bw/day recovery group showed increased γ-GTP, increased total protein, and decreased A/G ratio. Decreased AST was not considered as a toxicological effect.
No significant changes in clinical biochemistry parameters were observed in the 30 and 150 mg/kg bw/day treatment group compared to the control group.
2) At the end of the recovery period
Males in the 750 mg/kg bw/day recovery group showed decreased total bilirubin. Slight increased albumin was considered as a physiological variation because this change was not observed at the end of the administration period.
Females in the 750 mg/kg bw/day recovery group did not show the any differences when compared with the control group.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
1) At the end of administration period
Yellow urine was observed in 5 males and 3 females in the 750 mg/kg bw/day treatment group. Increased urine volume and decreased osmotic pressure were observed in males in the 150 mg/kg bw/day treatment group. These changes were not dose-depending.
2) At the end of the recovery period
Potassium value was increased in males in the 750 mg/kg bw/day treatment group. This change was regarded to be incidental because it was a slight change and no abnormality was found at the end of the administration.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
1) During administration
Hearing was decreased in the 30 mg/kg bw/day treatment group on week 6 of administration. However, this change was not dose-depending. Since the dead male animal in the 750 mg/kg treatment group was not able to walk on Day 11 of administration, detailed clinical signs were not tested.
Females in the 750 mg/kg bw/day recovery group showed also decreased hearing in Week 1 of administration. This was regarded to be incidental because no changes were found afterward in this group and in females in the treatment group at the same time.

2) During recovery period
The increased hearing in females in the 750 mg/kg bw/day recovery group in Week 1 of recovery period was also regarded to be incidental because no change was observed at other time point and general condition was not abnormal.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1) At the end of the administration period
Relative and absolute thyroid and liver weight was increased in male and female rats in the 750 mg/kg bw/day treatment group.
Males in the 30 mg/kg bw/day treatment group showed decreased relative and absolute thymus weight and increased absolute heart weight. Males in the 150 mg/kg bw/day treatment group showed decreased relative thymus weight. But these changes were not dose-dependent. Although the relative kidney weight was increased in females in the 750 mg bw/day treatment group, this was regarded to be incidental due to the lack of dose-dependency. Females in the 750 mg/kg bw/day recovery group showed the increased absolute heart weight. This change was also considered to be incidental because the relative weight was not changed.

2) At the end of the recovery period
Males in the 750 mg/kg bw/day recovery group showed increased absolute and relative thyroid and liver weight.
No changes were observed in females of 750 mg/kg bw/day recovery group in comparison with the control group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1) Dead animals
One male in the 750 mg/kg bw/day treatment group showed pale discoloration of skin and all tissues, smudge of lower abdominal fur, enlargement of and dark red foci in the kidney, enlargement of and dark coloration in the liver, dark coloration in the prostate, small thymus, and large urinary bladder. One female in the 150 mg/kg bw/day treatment group showed dark red foci in the kidney and in the forestomach.
2) At the end of the administration period
One male (1/6) in the 750 mg/kg bw/day treatment group showed the enlargement of the kidney. Other slight changes were considered to be incidental.
3) At the end of the recovery period
No changes caused by the test substance were observed in all animals. Other slight changes were considered as incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Exposure of the test material to male and female rats resulted in effects being observed in the following organs: kidney, urinary bladder, liver, and thyroid. In the kidney, there were minimal tubular regeneration (4/6 males in the 750 mg/kg bw/day treatment group), eosinophilic body found in tubular cell (minimal 3/6, mild 1/6 males in the 750 mg/kg bw/day treatment group) and minimal transitional hyperplasia/hypertrophy observed (1/6 males in the 750 mg/kg bw/day treatment group). In the urinary bladder, there was transitional hyperplasia/hypertrophy seen (minimal 2/6 and moderate 1/6 males in the 750 mg/kg bw/day treatment group, minimal 3/12 females in the 750 mg/kg bw/day treatment group). The observed transitional hyperplasia/hypertrophy in male rats in the 30 mg/kg bw/day treatment group was concluded to be not treatment-related effects but instead caused by incidental inflammation of the urinary bladder. In the liver, there was centrilobular hepatocytic hypertrophy observed (minimal 3/6 males in the 750 mg/kg bw/day treatment group, minimal 9/11 and mild 2/12 females in the 750 mg/kg bw/day treatment group, minimal 4/9 females in the 150 mg/kg bw/day treatment group, and minimal 4/6 and mild 1/6 females in the 750 mg/kg bw/day recovery group). In the thyroids, there was hypertrophy in follicular cell (minimal 2/12 males in the 150 mg/kg bw/day treatment group, mild 6/6 males in the 750 mg/kg bw/day treatment group, minimal 10/12 and mild 1/12 females in the 750 mg/kg bw/day treatment group, and minimal 1/5 and mild 4/5 females in the 750 mg/kg bw/day recovery group). The effect observed in the liver is concluded to be associated with induction of drug metabolising enzyme. It is well known that a substance which induces drug metabolising enzyme also promotes metabolism of thyroid hormone and causes hypertrophy of follicular cell through the hypothalamus-hypophysis system as a secondary effect. Effects observed in the transitional epithelium of the kidneys were also noted in the urinary bladder. The mode of action causing this effect was not clear according to this test result. However, the effects on the transitional epithelium in the kidneys and urinary bladder were reversed by the end of the recovery period; these effects were therefore concluded to be reversible.
Histopathological findings: neoplastic:
no effects observed
Details on results:
BODY WEIGHT
There were no statistically significant differences in the mean body weights between controls and test groups.

FOOD CONSUMPTION
There were no statistically significant differences in the mean amount of food consumption between controls and test groups.

HAEMATOLOGY
Decreased ratio and number of neutrophil count in females in the 750 mg/kg bw/day treatment group and decreased ratio of neutrophil count in females in the 750 mg/kg bw/day recovery group was considered to be the toxicological effect of the test substance, but the mode of action is not clear. Increased platelet and fibrinogen, prolonged pro-thrombin time and APTT in males in the 750 mg/kg bw/day treatment group and prolonged pro-thrombin time and APTT in females in the 750 mg/kg bw/day treatment group were also considered as treatment-related effects.

CLINICAL CHEMISTRY
Decreased potassium levels was considered to be in the physiological range as changes in urine volume and diarrhea were not observed. BUN was also not considered to be treatment-related because no impairment was found in the kidneys. Increased total protein was correlated to induction of drug metabolising enzyme in theliver. Increased calcium levels was regarded as a secondary effect of increased total protein, but the mode of action was not clear. Due to the lack of impairment in the livers, increased gamma GTP was considered to be not toxicological significant. Changes in the total bilirubin levels was also considered to be not toxicological significant, because these changes were only slight.

URIANALYST
Yellow urine was considered to be a treatment related effect, because there were histopathological changes in the kidneys and bladder.

ORGAN WEIGHTS
There were statistically significant differences observed in absolute organ weights in males compared to controls. Thyroids (increase at 750 mg/kg bw/day treatment group), thymus (decrease at 30 mg/kg bw/day treatment group), heart (increase at 30 mg/kg bw/day treatment group), and liver (increase at 750 mg/kg bw/day treatment group).
There were statistically significant differences in the mean percentage of organ weights relative to body weights for males: thyroids (increase at 750 mg/kg bw/day treatment group), thymus (decrease at 30 and 150 mg/kg bw/day treatment group), and liver (increase at 750 mg/kg bw/day treatment group).
There were statistically significant differences noted for absolute organ weights in females compared to controls; thyroids (increase at treatment and recovery 750 mg/kg bw/day group), heart (increase at 750 mg/kg bw/day recovery group), and liver (increase at 750 mg/kg bw/day treatment and recovery groups).
There were statistically significant differences for the mean percentage of organ weights relative to body weights for thyroids (increase at 750 mg/kg bw/day treatment and recovery groups), liver (increase at 750 mg/kg bw/day treatment and recovery groups), and kidneys (increase at 750 mg/kg bw/day treatment group) in females.
However, the changes were reversed by the end of the recovery period. These effects could be considered to be reversible.

GROSS PATHOLOGY
Effects attributable to test article administration in males occurred in the liver, kidney, prostate, thymus, bladder, and skin. In females test material-related effects were observed in the kidney and forestomach.

HISTOPATHOLOGY: NON-NEOPLASTIC
Exposure of the rats (male and female) to the test material resulted in effects being observed in the following organs: kidney, urinary bladder, liver, and thyroid. In the kidney, there were minimal tubular regeneration (4/6 males in the 750 mg/kg bw/day treatment group), eosinophilic body found in tubular cell (minimal 3/6, mild 1/6 males in the 750 mg/kg bw/day treatment group) and minimal transitional hyperplasia/hypertrophy observed (1/6 males in the 750 mg/kg bw/day treatment group). In the urinary bladder, there was transitional hyperplasia/hypertrophy seen (minimal 2/6 and moderate 1/6 males in the 750 mg/kg bw/day treatment group, 3/12 females in the 750 mg/kg bw/day treatment group). The observed transitional hyperplasia/hypertrophy in male rats in the 30 mg/kg bw/day treatment group was concluded not to be a treatment-related effects but instead caused by incidental inflammation of the urinary bladder. In the liver, there was centrilobular hepatocytic hypertrophy observed (minimal 3/6 males in the 750 mg/kg bw/day treatment group, minimal 9/11 and mild 2/12 females in the 750 mg/kg bw/day treatment group, minimal 4/9 females in the 150 mg/kg bw/day treatment group, and minimal 4/6 and mild 1/6 females in the 750 mg/kg bw/day recovery group). In the thyroids, there was hypertrophy in follicular cell (minimal 2/12 males in the 150 mg/kg bw/day treatment group, mild 6/6 males in the 750 mg/kg bw/day treatment group, minimal 10/12 and mild 1/12 females in the 750 mg/kg bw/day treatment group, and minimal 1/5 and mild 4/5 females in the 750 mg/kg bw/day recovery group). The effect observed in the liver is concluded to be associated with induction of drug metabolising enzyme. It is well known that a substance which induces drug metabolising enzyme also promotes metabolism of thyroid hormone and causes hypertrophy of follicular cell through the hypothalamus-hypophysis system as a secondary effect. Effects observed in the transitional epithelium of the kidneys were also noted in the urinary bladder. The mode of action causing this effect was not clear according to this test result. However, the effects on the transitional epithelium in the kidneys and urinary bladder were reversed by the end of the recovery period; these effects were therefore concluded to be reversible.

HORMONE VALUES:
In the 750 mg/kg bw/day treatment group, males showed increased TSH. Decreased T4 and increased TSH were observed in females in the 750 mg/kg bw/day treatment and recovery groups, respectively. These changes were considered to be caused by induction of drug metabolism system in the liver. However, the direct effect of the test item on the thyroids is not clear.

Effect levels

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on transitional epithelial cell hyperplasia in the kidney and urinary bladder of male rats and transitional epithelial cell hyperplasia in the urinary bladder in female rats observed in the 750 mg/kg bw dose group.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Haematology

Dose

mg/kg

No.

 

Male (end of administration)

Male (end of recovery)

 

 

 

Platelet

X10000/µL

PT

s

APTT

s

Fibrinogen

mg/dL

Platelet

X10000/µL

0

7

Mean

S.D.

97.0

7.4

15.7

2.4

24.5

4.5

276

43

111.5

4.6

30

5

Mean

S.D.

95.4

12.5

15.2

2.8

23.9

2.6

279

33

-

150

5

Mean

S.D.

105.7

7.6

15.6

2.1

23.4

2.2

283

20

-

750

6

5 a)

Mean

S.D.

121.4**

19.1 D

31.1**

9.1 ST

45.4**

9.9 ST

362**

62 D

129.2**

4.8 T

 

 

 

Female (lactation day 5)

 

 

 

APTT

S

Differential leukocyte counts (%)

Lymph.

Differential leukocyte counts (%)

Neut.

Differential leukocyte counts (%)

Eosino.

Differential leukocyte counts (100/µL)

Neut.

0

12

Mean

S.D.

16.0

2.1

57.9

5.7

38.5

5.5

0.5

0.3

42.1

7.9

30

5

Mean

S.D.

15.6

1.5

58.5

8.0

38.1

7.8

0.7

0.4

44.7

9.9

150

5

Mean

S.D.

16.5

2.5

63.3

4.0

33.0

4.7

0.7

0.2

33.4

6.9

750

12

Mean

S.D.

19.8**

2.4 D

65.9*

9.2 D

30.1

9.4 D

0.9**

0.4D

29.3*

12.8

 

 

 

Female satellite group (end of administration)

 

 

 

MCV

fL

PT

s

APTT

s

Differential leukocyte counts (%)

Neut.

0

5

Mean

S.D.

48.8

1.0

12.2

0.6

17.5

3.5

20.5

2.7

750

5

Mean

S.D.

50.3*

0.8 T

13.8*

1.1 T

25.8*

4.7

14.8*

4.0 T

*: p<0.05; **: p<0.01

D: Dunnett’s test, ST: Steel’s test, T: Student’s t-test,

a)     Number of animals was 5 at the end of recovery

Table 2 Clinical chemistry

Dose

mg/kg

No.

 

Male (end of administration)

 

 

 

AST (GOT)

IU/L

γ-GTP

IU/L

T. bilirubin

mg/dL

K

mmol/L

Ca

mg/L

TP

g/dL

A/G

0

7

Mean

S.D.

74

16

1

1

0.1

0.0

4.6

0.3

9.4

0.4

5.6

0.3

1.24

0.10

30

5

Mean

S.D.

59

2

1

0

0.1

0.0

4.5

0.2

9.4

0.3

5.6

0.2

1.19

0.10

150

5

Mean

S.D.

64

11

1

0

0.0*

0.0 ST

4.7

0.3

9.5

0.3

5.8

0.2

1.18

0.15

750

6

Mean

S.D.

52*

6 ST

2*

1 D

0.0**

0.0 ST

4.2*

0.2 D

10.0*

0.3 D

6.0*

0.2 D

1.06*

0.10 D

 

 

 

Female (lactation day 5)

 

 

 

γ-GTP

IU/L

T. bilirubin

mg/dL

BUN

mg/dL

A/G

0

12

Mean

S.D.

1

1

0.1

0.1

12

2

1.25

0.06

30

5

Mean

S.D.

1

0

0.1

0.1

13

1

1.28

0.12

150

5

Mean

S.D.

1

1

0.1

0.1

12

3

1.26

0.18

750

12

Mean

S.D.

3**

1 ST

0.0*

0.0 ST

17**

3 D

1.12**

0.07 D

 

 

 

Female (Satellite, end of administration)

 

 

 

γ-GTP

IU/L

ALP

IU/L

TP

g/dL

A/G

0

5

Mean

S.D.

1

0

203

33

6.3

0.3

1.40

0.15

750

5

Mean

S.D.

2**

1 T

156*

27 T

6.8*

0.3 T

1.19*

0.08 T

 

 

 

Male (end of recovery)

 

 

 

T. bilirubin

mg/dL

Albumin

g/dL

0

5

Mean

S.D.

0.1

0.0

3.0

0.1

750

5

Mean

S.D.

0.0**

0.0 T

3.2 **

0.1 T

*: p<0.05; **: p<0.01

D: Dunnett’s test, ST: Steel’s test, T: Student’s t-test,

 

Table 3. Hormone

Dose mg/kg

 

Male (end of administration)

Female (lactation day 5)

Female (satellite, end of administration)

Male (end of recovery)

 

 

No.

TSH

ng/mL

No.

T4

µg/dL

No.

TSH

ng/mL

No

T4

µg/dL

0

Mean

S.D.

7

5.61

4.62

12

3.6

1.1

5

3.10

2.13

5

4.0

1.0

30

Mean

S.D.

5

4.04

2.13

5

3.1

0.8

-

-

-

-

150

Mean

S.D.

5

10.62

10.55

5

2.8

0.9

-

-

-

-

750

Mean

S.D.

6

17.03*

14.39 ST

12

2.6*

0.8 D

5

8.32*

3.68 T

5

5.7 *

1.0 T

*: p<0.05

-: not performed

D: Dunnett’s test, ST: Steel’s test, T: Student’s t-test,

 

Table 4. Organ weight

Dose

 

Male (end of administration)

mg/kg

 

Thyroid (R+L)

Thymus

Heart

Liver

 

 

Absolute

mg

Relative

mg/100 g BW

Absolute

mg

Relative

mg/100 g BW

Absolute

mg

Absolute

mg

Relative

mg/100 g BW

0

No.

Mean

S.D.

7

24.2

3.1

7

4.9

0.7

7

341

100

7

67

12

7

1.36

0.14

7

12.81

2.19

7

2.56

0.18

30

No.

Mean

S.D.

12

22.9

3.7

12

4.6

0.7

12

251*

68 D

12

50*

13 D

12

1.50*

0.12 D

12

13.22

1.59

12

2.62

0.19

150

No.

Mean

S.D.

12

23.6

3.8

12

4.6

0.7

12

267

74

12

52*

13 D

12

1.46

0.10

12

13.97

1.29

12

2.72

0.15

750

No.

Mean

S.D.

6

30.5*

6.5 D

6

6.1*

1.3 D

6

296

53

6

60

10

6

1.38

0.04

6

18.53**

0.87 D

6

3.73**

0.26 D

 

 

Female (lactation day 5)

 

 

Thyroid (R+L)

Liver

Kidney (R+L)

 

 

Absolute

mg

Relative

mg/100 g BW

Absolute

mg

Relative

mg/100 g BW

Relative

mg/100 g BW

0

No.

Mean

S.D.

12

15.8

2.8

12

5.2

0.9

12

9.84

0.92

12

3.25

0.27

12

0.64

0.05

30

No.

Mean

S.D.

12

16.6

2.6

12

5.3

0.9

12

10.23

0.49

12

3.26

0.17

12

0.68

0.04

150

No.

Mean

S.D.

9

16.5

2.8

9

5.3

0.9

9

10.46

1.76

9

3.32

0.31

9

0.66

0.06

750

No.

Mean

S.D.

12

21.0**

3.1 D

12

6.9**

0.9 D

12

12.66**

1.09 ST

12

4.13**

0.24 D

12

0.70*

0.07 D

 

 

Female (Satellite group, end of administration)

 

 

Thyroid (R+L)

Heart

Liver

 

 

Absolute

mg

Relative

mg/100 g BW

Absolute

mg

Absolute

mg

Relative

mg/100 g BW

0

No.

Mean

S.D.

5

13.4

1.5

5

5.0

0.6

5

0.87

0.05

5

7.05

0.67

5

2.60

0.14

750

No.

Mean

S.D.

5

19.9**

1.9 T

5

7.3**

1.1 T

5

0.98**

0.03 T

5

11.36**

1.48 T

5

4.10**

0.27 T

 

 

Male (end of recovery)

 

 

 

Thyroid (R+L)

 

 

 

Absolute

mg

Relative

mg/100 g BW

 

0

No.

Mean

S.D.

5

21.0

2.7

5

4.0

0.5

 

750

No.

Mean

S.D.

5

28.3**

4.0 T

5

5.4*

0.8 T

 

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the NOAEL for systemic toxicity was determined to be 150 mg/kg bw/day based on transitional epithelial cell hyperplasia in the kidney and urinary bladder of male rats and transitional epithelial cell hyperplasia in the urinary bladder in female rats observed in the 750 mg/kg bw dose group.