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Toxicity to reproduction

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Endpoint:
reproductive toxicity, other
Remarks:
28-day oral toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06-06-2013 to 31-10-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
06-06-2013 to 31-10-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: “28-day Repeated Dose Toxicity Study in Mammalian Species” prescribed in “Concerning Testing Methods Relating to the New Chemical Substances” (March 31, 2011)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Hino Breeding Center
- Age at study initiation: 5 weeks
- Weight at study initiation: 142.4-177.2 g and 123.9-147.2 g, respectively for males and females
- Housing: Hanging stainless steel cages with wire-mesh floor (260 W×380 D×180 H mm) for quarantine and acclimation, and hanging stainless cages (165 W×300 D×150 H mm) after the grouping
- Diet: Pelleted diet (MF, lot no. 130403 and 130515, Oriental Yeast)
- Water: Chlorinated water maintained at 3-5 ppm of chloric level prepared by adding sodium hypochlorite (Purelox) to Hita City supply water was given by an automatic watering system with sipper tubes
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.3-23.9
- Humidity (%): 50.4-64.9
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was weighed and dissolved in corn oil to prepare 3.40 w/v% formulation. A part of 3.40 w/v% formulation was diluted with corn oil to prepare formulations of 0.850 and 0.210 w/v%. The formulations were subdivided into plastic containers, and stored at the cold place (actual temperature: 2-9ºC, tolerance temperature: 1-10ºC). These were dosed to the animals within 12 days after preparation. On each dosing day, formulations were taken out from the storage place and kept at room temperature until dosing.

VEHICLE
- Corn oil has been commonly used as a vehicle in general toxicity studies and we have historical control data.
- Concentration in vehicle: 0.21, 0.85, 3.40 w/v%
- Amount of vehicle: 10 mL/kg
- Lot/batch no. (if required): V2H8729
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability Analyses: Stability of the 10.0 and 0.100 w/v% formulations in the cold place were analyzed by high performance liquid chromatography (HPLC) in CERI Hita (Study No. X18-1035). The test substance in the 10.0 and 0.100 w/v% formulations were judged as stable for 12 days under the storage condition, because those concentrations after storage were within 100 ± 10% (101 and 101%, respectively) of those immediately after preparation for 13 days.
- Concentration Analysis: Concentration analysis for the 3.40, 0.850 and 0.210 w/v% formulations was performed by HPLC in CERI Hita immediately after the first preparation (Study No. X18-1035) The concentrations of the formulations were confirmed to be within 100±10% of each nominal concentration (3.40 w/v% formulation: 104%, 0.850 w/v% formulation: 103%, 0.210 w/v% formulation: 99.0%) and the preparation procedure was judged to be appropriate.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Dose / conc.:
21 mg/kg bw/day (actual dose received)
Dose / conc.:
85 mg/kg bw/day (actual dose received)
Dose / conc.:
340 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: As a range finding study, “a 7-day repeated-dose oral toxicity study of Delta Damascone in rats” was performed in CERI Hita (Study No. P11-1035, non-GLP). In the study, three male and three female Crl:CD(SD) rats in each group were treated orally with the substance dissolved in corn oil at 0, 30, 100, 300 and 1000 mg/kg/day for 7 days. General clinical observations and body weight measurements were performed in the dosing period. Necropsy and organ weights measurements were performed on the day after the last dosing. As a result, the changes of the general condition were observed, such as salivation immediately after the administration in males and females of the 100 mg/kg groups and higher, decreased spontaneous locomotion in males and females of the 300 mg/kg groups and higher, restlessness in males of the 300 mg/kg group and females of the 300 mg/kg group and higher, staining around nose and mouth in males and females of the 1000 mg/kg groups and incomplete eyelid opening and staining hair in females of the 1000 mg/kg group. On the day 3 of the dosing period, one female of the 1000 mg/kg group was dead. In body weight, decreases from the last value, a statistically significant decrease or a low tendency were observed in males and femalesof the 1000 mg/kg groups. In the pathological examinations, statistically significant increases of relative weights of liver and macroscopic enlargement of the liver were observed in males and females of the 300 mg/kg groups. Whitish region of mucosa in the forestomach was observed in females of the 300 mg/kg group and higher and males of the 1000 mg/kg group. Statistically significant decreases of absolute and relative weights of the spleen were observed in females of the 300 mg/kg group. Based on these results, it was considered that the test substance had an irritant property to gastric mucosa and the major target organs of the test substance were the stomach and liver. Although no abnormal changes were found in the body weight in the 300 mg/kg groups, the findings in general clinical condition such as decreased spontaneous locomotion and restlessness were observed and the effects on the liver were pathologically observed. Therefore, it was considered that the repeated dosing of Delta Damascone at much higher dose than 300 mg/kg for 28 days might cause severe toxicity such as death or moribundity. Accordingly, in the present study, the high dose was set at 340 mg/kg/day and lower doses of 85 and 21 mg/kg/day were set.
- Grouping: Three dose levels for the test substance groups and one vehicle control group were used. Recovery groups were set for the high dose and vehicle control groups without treatment for 14 days after the dosing period.
Observations and examinations performed and frequency:
GENERAL CLINICAL OBSERVATIONS: During the dosing period, animals were observed three times a day, i.e., before dosing, between just after dosing and 1 hour after dosing, and between 2 and 6 hours after dosing. During the recovery period, observation was performed once a day.
DETAILED CLINICAL OBSERVATIONS: Detailed clinical observations in all animals were performed once before dosing. Thereafter, animals were examined once a week during the dosing and recovery periods on a blind test basis. The blind test was performed using the random numbers and observation labels without identifying the dosing groups. Observation parameters and methods: (a) Observations at removal from cage (Animal reactions such as excitement from external stimuli (holding animals or bringing a hand close to animals to hold) were observed and assessed by scoring method. Ease of removal and vocalization. (b) Handling observations: Muscle tone, subnormal temperature, hair appearance (piloerection, staining hair and unkempt hair), skin and mucous color (paleness, reddening and cyanosis), eyes (lacrimation, exophthalmos and pupillary size), salivation and secretion. (c) Observation in arena: Animals were placed in a standard arena (on an observation platform of 90 cm×60 cm) and observed for one minute or more (within five minutes), and the frequencies of defecation (number of feces) and urination (number of pools) were recorded for one minute. Posture, motor activity level, respiration, gait characteristics, lid closure, tremor, twitch, convulsion, stereotypical behavior and abnormal behavior.
SENSORIMOTOR FUNCTION EXAMINATIONS: All animals were examined in week 4 (on days 27 and 28) of the dosing period. Reflex tests and measurements of grip strength were performed on a blind test basis. In one female of the high dose (340 mg/kg) group, a hyper reaction was observed in pain response in reflex test. Therefore, in the week 2 of the recovery period (on day 11 of the recovery period), the pain response test was performed for females. In males, the examinations were not performed in the recovery period, since no abnormal changes related to the test substance treatment were observed in the 340 mg/kg groups in the dosing period. Observation parameters and methods: (a) Reflex: Optic (approach contact/touch) response, Pinna response, Pain response, Pupillary reflex, Air righting reflex. (b) Grip strength (c) Locomotor activity counts.
BODY WEIGHT MEASUREMENTS: on the grouping day, on days 1, 3, 8, 12, 17, 21, 26 and 28 of the dosing period, on days 1, 5, 10 and 14 of the recovery period, on the necropsy days (before carrying animals from the animal room).
FOOD CONSUMPTION MEASUREMENTS: Feeding weights on the grouping day, Remainder weights on days 1, 3, 8, 15, 22 and 28 of the dosing period, Feeding weights on day 1 of the recovery period, Remainder weights on days 4, 8 and 14 of the recovery period. Feeding weights on days 8, 15 and 22 of the dosing period and on day 8 of the recovery period were measured after food was replenished. Reminder weight on days 1 and 3 of the dosing period and day 4 of the recovery period were also used as feeding weight and the replenishment was not conducted on those days. Mean food consumption per day was calculated from their feeding and remainder weights for each period.
URINALYSES: Collecting Urine Samples: Urine samples were collected for about 16 hours in individual metabolic cages (150 W×200 D×263 H mm) with drinking water and without food, from the afternoon of the last day of the dosing period for the main groups and that of the recovery period for the recovery groups to each next morning. Parameters and Methods: Urine samples were examined for the following parameters: Urine volume, Color, Turbidity, Urine osmotic pressure, pH, Protein, Glucose, Occult blood, Urinary sediment. Urinary sediments were stained and examined in males and females of the control and high dose (340 mg/kg) groups of the main groups. Examination of urinary sediments was not conducted for the intermediate dose (85 mg/kg) or low dose (21 mg/kg) groups or the recovery groups, since no abnormal changes related to the treatment were noted in either sex of the 340 mg/kg groups.
BLOOD EXAMINATIONS: (A) Blood Sampling and Preparation of Test Samples: Blood was collected from the abdominal aorta under isoflurane anesthesia after the urine collection on the next day of the last day of the dosing period for the main groups and that of the recovery period for the recovery groups. Blood sampling were performed after overnight fasting (16 to 20 hours) for each period. (B) Haematological Examinations: Whole blood or plasma samples were used to determine the following parameters: Red blood cell count, Haemoglobin conc., Haematocrit value, Mean corpuscular volume, Mean corpuscular haemaglobin, Mean corpuscular hemoglobin conc., Platelet count, Reticulocyte count ratio, White blood cell count, Differentiation of leukocytes (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Prothrombin time, Activated partial thromboplastin time. Blood smears were not prepared since reticulocyte count ratio and differentiation of leukocytes were able to measure by the instruments. (C) Blood Chemical Examinations: Serum samples were used to determine the following parameters: Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Choline esterase, gamma-Glutamyl transpeptidase, Total cholesterol, Triglyceride, Blood urea nitrogen, Creatinine, Total protein, Albumin, A/G ratio, Glucose, Total bilirubin, Total bile acids, Inorganic phosphorus, Calcium, Sodium, Potassium, Chloride.
Sacrifice and pathology:
(A) Gross Necropsy: All animals were subjected to a detailed gross necropsy after blood sampling and bleeding from the ventral aorta on the day after last administration for the main groups and on the day after last withdrawal day for the recovery groups. External surface of the body, all orifices, subcutis, cranial, thoracic, abdominal and pelvic cavities, and their contents were observed. For females, vaginal smears were collected before the gross necropsy, and the stages of estrous cycle were determined with light microscope after Giemsa staining.
(B) Tissue Collecting and Organ Weight Measurements: The following organs were removed from all animals: Respiratory system (Trachea, lungs), Digestive system (Submandibular glands, stomach, intestines (duodenum to rectum, including Peyer’s patches), pancreas, liver*), Cardiovascular system (Heart*), Urinary system (Kidneys*, urinary bladder) Reproductive system (Testes*, epididymides*, prostate*, seminal vesicles* (including coagulating gland), ovaries*, uterus*, vagina), Nervous system (Brain* (including cerebrum, cerebellum and pons), spinal cord, sciatic nerve), Hematopoietic system (Bone marrow (femur), axillar lymph nodes, mesenteric lymph nodes, spleen*, thymus*), Endocrine system (Pituitary gland, thyroid* (including parathyroid), adrenals*), Sense organ (Eye balls), Musculoskeletal system (Skeletal muscle (femoral region), bone (femur)). From one animal of the 340 mg/kg bw group the skin was collected. Trachea, lungs and urinary bladder were inflated with 10% neutralized buffered formalin before removal. Stomach and intestines were filled and fixed with 10% neutralized buffered formalin and the contents were washed away with water. The weights of organs with an asterisk were measured using an electric balance (SARTORIUS). Kidneys, testes, epididymides, ovaries and adrenals were weighed right and left together. Prostate was weighed including a part of urethra. Seminal vesicles including coagulating gland were removed after ligation of the root and weighed. Uterus was weighed with the contents. Thyroid adhered to trachea, including parathyroid, was fixed with 10% neutralized buffered formalin, and the right and left lobes were removed from trachea and weighed on the day after necropsy. Relative organ weight was calculated based on the body weight measured on the necropsy day.
(C) Histopathological examinations: The testes and epididymides were fixed in modified Davidson’s fixative. The other organs/tissues were preserved in 10% neutralized buffered formalin. Light microscopic examinations were performed for the following organs and the tissues of the control and 340 mg/kg bw group after embedding in paraffin, sectioning and hematoxylin and eosin (HE) staining. Decalcification was done for bone and bone marrow (femur) with 10% formic acid. Examined organs: Trachea, Lungs, Submandibular gland, Duodenum-ileum, Cecum- rectum, Pancreas, Heart, Kidneys, Urinary bladder, Prostate, Coagulating gland, Seminal vesicle, Ovaries, Uterus, Vagina, Cerebrum, Cerebellum, Pons, Spinal cord, Sciatic nerve, Bone marrow, Axillar lymph nodes, Mesenteric lymph nodes, Spleen, Thymus, Pituitary gland, Thyroid, Parathyroid, Adrenals, Eye ball, Skeletal muscle, and Bone. For the forestomach, glandular stomach, and liver, histopathological examinations were performed for males and females of all treatment groups and the recovery groups since treatment-related changes were suspected in males and females of the 340 mg/kg groups. For the testes and epididymides, histopathological examinations were performed for males of the control and 340 mg/kg bw group and of the recovery groups since a statistically significant change was found in the absolute organ weight of the epididymides in males of the 340 mg/kg recovery group. The testes were also examined as the related organs to the epididymides. Of one animal in the 340 mg/kg bw the skin was examined and of one female of the 85 mg/kg bw group and one female of the 340 mg/kg bw group, the vagina was examined (on account of gross lesions observed at necropsy).
Statistics:
Data regarding body weights, food consumption, grip strength and locomotor activity counts during the dosing period, and parameters of hematological examinations, parameters of blood chemical examinations, urine volume, urine osmotic pressure, organ weights and body weights on the necropsy day of the main groups were analyzed by the Bartlett’s test for homogeneity of variances. When the variances were homogeneous at a significance level of 5%, the Dunnett’s test was used. When the variances were not homogeneous, the nonparametric Dunnett’s test was used. The frequencies of defecation (number of feces) and urination (number of pools) during the dosing period were analyzed by the nonparametric Dunnett’s test.
Data regarding body weights, food consumption during the recovery period, and parameters of haematological examinations, parameters of blood chemical examinations, urine volume, urine osmotic pressure, organ weights and body weights on the necropsy day of the recovery groups were analyzed by the F-test for variance ratio. When there were no significant differences at a significance level of 5% in this analysis, the Student’s t-test was used. When there was a significant difference, the Aspin-Welch t-test was used. The frequencies of defecation (number of feces) and urination (number of pools) during the recovery period were analyzed by the Mann-Whitney U-test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
DURING DOSING PERIOD:
In males, salivation and decreased spontaneous locomotion in 10 animals out of 10, staining around nose and mouth in nine animals, much food on the tray (increase of the amount of food dropped on tray by biting) in eight animals, restlessness which meant increased chin rubbing or cage scratching in six animals, decreased respiratory rate in four animals, incomplete eyelid opening, lacrimation and reddish tear in three animals, staining hair in two animals and diarrhoea, loss of hair and moist hair in one animal were observed in the 340 mg/kg group. In the 85 mg/kg group, salivation in five animals out of five, decreased spontaneous locomotion in four animals and incomplete eyelid opening in one animal were observed. In the 21 mg/kg group, salivation was observed in one animal out of five. No abnormal changes were observed in the control group. The decreased spontaneous locomotion was observed sporadically or continuously throughout the dosing period after day 4 of the dosing period in the 340 and 85 mg/kg groups. The salivation was observed continuously after day 2 of the dosing period in the 340 mg/kg group. In the 85 mg/kg group, the salivation was observed sporadically or continuously after day 4 of the dosing period. The persistence time of the salivation tended to prolong with the dosing progresses. The salivation was also observed immediately before the administration in the 340 mg/kg group. In the 21 mg/kg group, the salivation was observed only on day 14 of the dosing period and disappeared within 30 minutes after administration. The restlessness which meant increased chin rubbing or cage scratching was sporadically observed after week 2 of the dosing period in the 340 mg/kg group. The much food on the tray was observed sporadically in week 3 and 4 of dosing period in the 340 mg/kg group. The diarrhoea was observed only in one animal of the 340 mg/kg group on day 5 of the dosing period. In females, salivation, decreased spontaneous locomotion and staining around nose and mouth in 10 animals out of 10, staining around external genitalia in four animals, much food on the tray (increase of the amount of food dropped on tray by biting) in three animals, restlessness which meant increased chin rubbing or cage scratching in two animals and incomplete eyelid opening, reddish tear, lacrimation, staining hair, staining lower abdomen and staining around anus in one animal were observed in the 340 mg/kg group. In the 85 mg/kg group, salivation in four animals out of five and decreased spontaneous locomotion in one animal were observed. No abnormal changes were observed in the 21 mg/kg group or control group. The decreased spontaneous locomotion was observed sporadically or continuously after day 5 of the dosing period in the 340 mg/kg groups while the number of the animals which showed decreased spontaneous locomotion was decreased after week 3 of the dosing period. In the 85 mg/kg group, the decreased spontaneous locomotion was observed in week 1 and 2 (from day 6 to day 8 of the dosing period) in one animal. The salivation was observed continuously after day 2 of the dosing period in the 340 mg/kg group. The persistence time of the salivation tended to prolong with the dosing progresses. In the 85 mg/kg group, the salivation was observed from day 5 to day 10 of the dosing period and disappeared within 1 hour after administration. The restlessness was sporadically observed in week 2 and 3 of the dosing period in the 340 mg/kg group. The much food on the tray was observed sporadically or continuously in week 3 and 4 of dosing period in the 340 mg/kg group.
In males, staining around nose and mouth was observed in four animals and three animals out of 10 of the 340 mg/kg group in week 1 and 2, respectively. Staining around nose and mouth or mandible to abdomen in four animals, lacrimation in three animals and salivation in four animals were observed in the 340 mg/kg group in week 4. Continuous vocalization (+2) was observed in one animal out of five in week 3 in the 21 mg/kg group. A statistically significant increase of the frequency of urination was observed in the 85 mg/kg group in week 1, although no dose-dependency was observed. No statistically significant changes in the frequency of defecation were observed in any treatment groups. No abnormal changes were observed in the control group. In females, staining around nose and mouth or abdomen was observed in two animals out of 10 of the 340 mg/kg group in week 1. Staining lower abdomen was observed in one animal of the 340 mg/kg group in week 2. Continuous vocalization (+2) was observed in one animal of the 340 mg/kg group. Staining around nose and mouth or external genitalia in five animals and salivation in one animal were observed in the 340 mg/kg group in week 4. Statistically significant increases of the frequencies of urination in the 85 mg/kg group in week 1 and in the 21 mg/kg group in week 2 were observed, although no dose-dependency was observed. No statistically significant changes in the frequency of defecation were observed in any treatment groups. No abnormal changes were observed in the control group.

DURING RECOVERY PERIOD:
In males, much food on the tray was observed in one animal out of five of the 340 mg/kg group on day 1 of the recovery period. No abnormal changes were observed in the control group. In females, staining around external genitalia was observed in one animal out of five of the 340 mg/kg group on day 1 of the recovery period. No abnormal changes were observed in the control group.
No statistically significant changes in frequencies of defecation or urination were noted in either sex of the 340 mg/kg group. No abnormal changes were observed in the control or the 340 mg/kg groups in the other parameters.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
DURING DOSING PERIOD:
In males, statistically significant decreases were found from day 8 to 28 of dosing period in the 340 mg/kg group. The body weight of the 340 mg/kg group was 77.1% compared to that of the control group on day 28 of the dosing period. No statistically significant changes were noted in the 21 or 85 mg/kg groups. In females, no statistically significant changes were noted in any treatment groups.

DURING RECOVERY PERIOD:
In males, statistically significant decreases were found from day 1 to 14 of the recovery period. Although the bodyweight of the 340 mg/kg group on day 1 of the recovery period was 76.4% compared to that of the control group, that on day 14 recovered to 85.4% compared to that of the control group. In females, no statistically significant changes were noted in the 340 mg/kg groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
DURING DOSING PERIOD:
In males, a lower tendency (89.2% compared to the control group) was found in the 340 mg/kg group on day 3 of the dosing period. A higher tendency (111.0% compared to the control group) was found in the 340 mg/kg group on day 28 of the dosing period. No statistically significant changes were noted in the 21 or 85 mg/kg groups. In females, a lower tendency (84.6% compared to the control group) on day 3 of the dosing period and statistically significant increases or a higher tendency on day 15, 22 and 28 (115.3%, 113.7% and 126.6% compared to the control group, respectively) were found in the 340 mg/kg group. No statistically significant changes were noted in the 21 or 85 mg/kg groups.

DURING RECOVERY PERIOD:
No statistically significant changes were noted in either sex of the 340 mg/kg groups.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
MAIN GROUP:
In males, statistically significant decreases of red blood cell count, haemoglobin concentration and haematocrit value were found in the 85 mg/kg group, although no dose-dependency was noted. No statistically significant changes were noted in the 21 or 340 mg/kg groups. In females, statistically significant decreases of mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration (MCHC) were found in the 340 mg/kg group, although the change of MCHC was within the range of the historical control data of CERI Hita. No statistically significant changes were noted in the 21 or 85 mg/kg groups.

RECOVERY GROUP:
In males, a statistically significant decrease of MCHC and a statistically significant increase of reticulocyte count ratio were found in the 340 mg/kg group, although the change of MCHC was within the range of the historical control data of CERI Hita. In females, a statistically significant decrease of MCHC was found in the 340 mg/kg group, although the change of MCHC was within the range of the historical control data of CERI Hita.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
MAIN GROUP:
In males, statistically significant increases of aspartate aminotransferase, gamma-glutamyl transpeptidase (gamma-GTP) and total bilirubin, a higher tendency of alanine aminotransferase (ALT), statistically significant decreases of total protein, albumin and calcium and a lower tendency of triglyceride were found in the 340 mg/kg group. Statistically significant decreases of total protein and albumin were found in the 85 mg/kg group. A statistically significant decrease of choline esterase (ChE) was found in the 21 mg/kg group, although no dose-dependency was noted. In females, a statistically significant increase of gamma-GTP and statistically significant decreases of ChE and glucose were found in the 340 mg/kg group. A statistically significant increase of total cholesterol was found in the 85 mg/kg group, although no dose-dependency was noted. No statistically significant changes were noted in the 21 mg/kg group.

RECOVERY GROUP:
In males, statistically significant increases of ALT and total cholesterol and statistically significant decreases of creatinine and glucose were found in the 340 mg/kg group, although those changes were within the range of the historical control data in CERI Hita. In females, a statistically significant decrease of ChE and a statistically significant increase of triglyceride were found in the 340 mg/kg group, although those changes were within the range of the historical control data in CERI Hita.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
DURING DOSING PERIOD:
In males, a statistical significant increase of the urine volume and a lower tendency of the urine osmotic pressure were found in the 340 mg/kg group, although they were within the range of the historical control data of CERI Hita. No abnormal changes were noted in the other examined parameters in the control or any treatment groups. In females, no statistically significant changes in urine volume or urine osmotic pressure were noted in any treatment groups. No abnormal changes were noted in the other examined parameters in the control or any treatment groups.

DURING RECOVERY PERIOD:
No statistically significant changes in urine volume or urine osmotic pressure were noted in either sex of the 340 mg/kg groups. No abnormal changes were noted in the other examined parameters in either sex of the control or 340 mg/kg groups.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
DURING DOSING PERIOD:
In males, a statistically significant decrease of locomotor activity counts was observed in interval from 50 to 60 minutes, although no dose-dependency was observed. No statistically significant changes in grip strength were noted in any treatment groups. No abnormal changes were observed in the control or any treatment groups in the reflex test. In females, a hyper reaction (+1) to pain response was observed in one animal out of 10 of the 340 mg/kg group. No statistically significant changes in grip strength or locomotor activity counts were noted in any treatment groups. No abnormal changes were observed in the control, 21 or 85 mg/kg groups in the reflex test.

DURING RECOVERY PERIOD:
In males, the examinations were not performed since no abnormal changes suspected of the effect of the treatment were noted in week 4 of the dosing period. In females, no abnormal changes were observed in pain response of reflex test in the control or 340 mg/kg groups.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
MAIN GROUP:
In males, statistically significant increases in relative weight of the liver (131.4% compared to that of the control group) and kidneys (113.6% compared to that of the control group) were found in the 340 mg/kg group. A statistically significant decrease in the body weight of the necropsy day (76.0% compared to that of the control group) was found in the 340 mg/kg group. Statistically significant changes or tendencies were also found in the 340 mg/kg group, such as statistically significant increases of relative weights of the heart, testes and brain, statistically significant decreases of absolute weights of epididymides and adrenals, statistically significant decreases of absolute and relative weights of the prostate and seminal vesicles and lower tendencies of the absolute and relative weights of the spleen. No statistically significant changes were noted in the 21 or 85 mg/kg groups. In females, statistically significant increases in relative weights of the liver (132.8% compared to that of the control group) and kidneys (115.8% compared to that of the control group) were found in the 340 mg/kg group. A lower tendency in the body weight of the necropsy day (89.9% compared to that of the control group) was found. A statistically significant increase in relative weight of the heart and statistically significant decreases of absolute and relative weights of the spleen were also found in the 340 mg/kg group. No statistically significant changes were noted in the 21 or 85 mg/kg groups.

RECOVERY GROUP:
In males, a statistically significant decrease in the body weight of the necropsy day (85.5% compared to that of the control group), statistically significant increases in relative weights of the testes and brain and statistically significant decreases in absolute weights of the epididymides and prostate were found in the 340 mg/kg group. In females, a statistically significant increase in relative weights of the liver (113.8% compared to that of the control group) was found in the 340 mg/kg group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
MAIN GROUP:
In males, enlargement of the liver in four animals out of five, blackish region of mucosa in the glandular stomach in two animals and loss of hair in one animal were observed in the 340 mg/kg group. No abnormal changes were noted in the control or the 21 or 85 mg/kg groups. In females, enlargement of the liver in five animals out of five, blackish region of mucosa in the glandular stomach in three animals and recessed region of mucosa in the forestomach in one animal were observed in the 340 mg/kg group. In the 85 mg/kg group, blackish region of mucosa in the glandular stomach in one animal and nodule in the vagina in one animal were observed. No abnormal changes were observed in the control or 21 mg/kg groups.

RECOVERY GROUP:
In males, no abnormal changes were noted in the control or 340 mg/kg groups. In females, nodule in the vagina in one animal was observed in the 340 mg/kg group. In the control group, no abnormal changes were noted.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
MAIN GROUP:
In males, in the liver, hypertrophy of periportal hepatocytes in three animals out of five of the 340 mg/kg group and one animal of the 85 mg/kg group and prominent nucleoli of periportal hepatocytes in five animals of the 340 mg/kg group and one animal of the 85 mg/kg group were observed, which were corresponding to the enlargement of the liver observed macroscopically. In the glandular stomach, focal necrosis of fundic mucosa in two animals, which were corresponding to the blackish region of mucosa of the glandular stomach observed macroscopically and edema in submucosal layer in one animal were observed in the 340 mg/kg group. In the two animals which showed the glaudular stomach lesions, spongiosis of squamous epithelium in limiting ridge, oedema in lamina propria of limiting ridge and oedema in submucosal layer were also observed in the forestomach of one animal and oedema in lamina propria of limiting ridge was also observed in the forestomach of the other animal. No abnormal changes were noted in the kidney in which a statistically significant increase in relative weight was found in the 340 mg/kg group. In one animal of the 340 mg/kg group, decreased hair follicles was observed in the skin, which was corresponding to the gross lesion. Microgranuloma in the liver in one animal of the 85 mg/kg group and solitary cyst in medulla in one animal of the control group were also observed. In the 21 mg/kg group, no abnormal changes were observed in the forestomach, glandular stomach or liver which were examined. In females, in the liver, hypertrophy of periportal hepatocytes in one animal out of five and prominent nucleoli of periportal hepatocytes in four animals were observed in the 340 mg/kg group, which were corresponding to the enlargement of the liver observed macroscopically. In the glandular stomach, focal necrosis of fundic mucosa in three animals of the 340 mg/kg group and one animal of the 85 mg/kg group was observed, which were corresponding to the macroscopic lesion. In the forestomach, focal spongiosis of squamous epithelium was observed in one animal of the 340 mg/kg group, which were corresponding to the recessed region of mucosa in the forestomach. No abnormal changes were noted in the kidney in which a statistically significant increase in relative weight was found in the 340 mg/kg group. In one animal of the 85 mg/kg group, squamous epithelial cyst was observed in the vagina, which was corresponding to the gross lesion. Periportal fatty change of hepatocytes and granuloma in the liver in one animal of the control group, mineralization in cortico-medullary junction in the kidney in one animal of the control group and ectopic thymic tissue in the thyroid in one animal of the 340 mg/kg group were also observed. Accumulation of lipid in hepatocyte was revealed by the specimen stained with oil red O of the liver for one female of the control group, since many positive components were observed in the hepatocytes.

RECOVERY GROUP:
In males, hyperplasia of squamous epithelium in limiting ridge in the forestomach was observed in one animal out of five of the 340 mg/kg group. Microgranuloma in the liver was observed in one animal each of the control and 340 mg/kg groups. No abnormal changes were noted in the epididymides in which a statistically significant decrease in absolute weight was found in the 340 mg/kg group. In females, squamous epithelial cyst was observed in the vagina in one animal of the 340 mg/kg group, which was corresponding to the gross lesion. No abnormal changes were observed in the forestomach, glandular stomach or liver in the control or 340 mg/kg groups.
Other effects:
no effects observed
Description (incidence and severity):
Estrous Cycle Stage
MAIN GROUP:
In the control group, one female out of five was in estrus, two were in metestrus and two were in diestrus. In the 21 mg/kg group, three females were in estrus and two were in metestrus. In the 85 mg/kg group, one female was in proestrus, one was in metestrus and three were in diestrus. In the 340 mg/kg group, one female was in estrus, one was in metestrus and three were in diestrus.

RECOVERY GROUP:
In the control group, one female out of five was in proestrus, one was in metestrus and three were in diestrus. In the 340 mg/kg group, two females were in estrus, one was in metestrus, two were in diestrus.
Dose descriptor:
NOAEL
Effect level:
85 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical biochemistry
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: See 'Any other information on results incl. tables'
Dose descriptor:
NOAEL
Effect level:
85 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: See 'Any other information on results incl. tables'
Critical effects observed:
not specified

The authors of the study concluded that the No-Observed-Adverse-Effect Level (NOAEL) of the test substance under the conditions tested was 21 mg/kg/day since the following adverse effects were observed in the 85 mg/kg groups; decreased spontaneous locomotion in males and females, incomplete eyelid opening and statistically significant decreases of total protein and albumin in males and focal necrosis of fundic mucosa in the glandular stomach in females.

However, the registrant considers the effects observed at 85 mg/kg not to be adverse. The decreased spontaneous locomotion seen at the clinical observations in 4/5 male rats and, on a few occasions only, in 1/5 female rats of the 85 mg/kg group is likely to be secondary to the irritant effect of the substance on the gastrointestinal tract following the oral administration rather than a reflection of systemic toxicity. This is supported by the absence of an effect on spontaneous motor activity during the weekly detailed clinical observations (arena observations) and during the quantitative motor activity assessment conducted at the end of the 28-day treatment period. Heamatological parameters are not affected at 85 mg/kg bw. Biochemical parameters: Protein is slightly decreased in males only at 85 mg/kg bw but < 10%. In females there is a slight increase in gamma-GT and in total cholesterol of which the latter decreases again at 340 mg/kg bw. The liver parameters are only slightly affected and can still be considered an adaptive response. An enlarged liver is seen in one male at this dose, which is considered an adaptive change. In females a microscopic effect is seen in the glandular stomach (focal minimal necrosis of the fundic mucosa) in one animal. This effect is considered to be due to the irritant properties of the substance and therefore does not need to be considered for the systemic NOAEL. In summary, the registrant estimates the systemic NOAEL to be 85 mg/kg bw/day.

Conclusions:
The NOAEL in the oral 28-day repeated dose toxicity study in rats is considered to be 85 mg/kg bw/day.
Executive summary:

In a study performed in compliance with the Japanese guideline '28 -day Repeated Dose Toxicity Study in Mammalian Species' (Concerning Testing Methods Relating to the New Chemical Substances) and GLP, the test substance was administered to five male and female Crl:CD(SD) rats per group by oral gavage as a dilution in corn oil at dose levels of 0, 21, 85 and 340 mg/kg/day. Additionally, the study included two recovery groups of 5 rats/sex each which were dosed for 28 days with the vehicle (control) or 340 mg/kg of test substance and then left untreated for 14 days. During the dosing period, general clinical observations, detailed clinical observations, function examinations, body weight measurements and food consumption measurements were performed. On the day after the last dosing, urinalyses, blood examinations and pathological examinations were performed after collecting urine and blood samples.

In the general clinical observations, decreased spontaneous locomotion in males and females of the 85 mg/kg groups and higher, incomplete eyelid opening in males of the 85 mg/kg group and higher and females of the 340 mg/kg group, lacrimation, reddish tear and staining hair in males and females of the 340 mg/kg groups, decreased respiratory rate, loss of hair and moist hair in males of the 340 mg/kg group and staining around external genitalia, staining lower abdomen and staining around anus in females of the 340 mg/kg group were observed. Salivation in males and females of the 85 mg/kg groups and higher and staining around nose and mouth, much food on the tray (increase of the amount of food dropped on tray by biting) and restlessness which meant increased chin rubbing or cage scratching in males and females of the 340 mg/kg groups were also observed. In the detailed clinical observations, staining hair in males and females in week 1, 2 and 4, salivation in males and females in week 4 and lacrimation in males in week 4 were observed in the 340 mg/kg groups. In the body weight, lower tendencies were found from day 8 to day 28 of the dosing period in males of the 340 mg/kg group. In the food consumption, lower tendencies on day 3 of the dosing period in males and females, statistically significant increases or a higher tendency from day 15 to day 28 of the dosing period in females and a higher tendency on day 28 of the dosing period in males were found in the 340 mg/kg groups. In the blood chemical examinations, statistically significant decreases of total protein and albumin in males of the 85 mg/kg group and higher, statistically significant increases of gamma-glutamyl transpeptidase in males and females of the 340 mg/kg groups, statistically significant increases of aspartate aminotransferase and total bilirubin, a higher tendency of alanine aminotransferase (ALT) and a lower tendency of triglyceride in males of the 340 mg/kg group and a statistically significant decrease of choline esterase (ChE) in females of the 340 mg/kg group were found. In the organ weight, statistically significant increases of relative weights of the liver were found in males and females of the 340 mg/kg groups. Statistically significant increases of relative weight of the kidneys were also found in males and females of the 340 mg/kg groups. In the gross necropsy, blackish region of mucosa in the glandular stomach in females of the 85 mg/kg group and higher and males of the 340 mg/kg group, enlargement of the liver in males and females of the 340 mg/kg groups and recessed region of mucosa in the forestomach in female of the 340 mg/kg group were observed. In the histopathological examinations, hypertrophy and prominent nucleoli of periportal hepatocytes in the liver in males of the 85 mg/kg group and higher and females of the 340 mg/kg group, focal necrosis of fundic mucosa in the glandular stomach in females of the 85 mg/kg group and higher and males of the 340 mg/kg group, spongiosis of squamous epithelium and oedema in lamina propria in limiting ridge and oedema in submucosal layer in the forestomach, and oedema in submucosal layer in the glandular stomach in males of the 340 mg/kg group and focal spongiosis of squamous epithelium in the forestomach in female of the 340 mg/kg group were observed. No abnormal changes were observed in the sensorimotor function examinations, urinalyses, haematological examinations and oestrous cycle stage. In the recovery group, most of changes observed in dosing period and at the end of dosing period disappeared, although much food on the tray on day 1 of the recovery period, statistically significant decreases of the body weight from day 1 to day 14 of the recovery period and a statistically significant increase of ALT at the end of recovery period in males and staining around external genitalia on day 1 of the recovery period, a statistically significant decrease of ChE and a statistically significant increase of relative weight of the liver in females were observed. From these results, the main adverse effects of the test substance were estimated to be a hepatocellular injury and an irritant property to gastric mucosa.

The authors of the study concluded that the No-Observed-Adverse-Effect Level (NOAEL) of the test substance under the conditions tested was 21 mg/kg/day since the following adverse effects were observed in the 85 mg/kg groups; decreased spontaneous locomotion in males and females, incomplete eyelid opening and statistically significant decreases of total protein and albumin in males and focal necrosis of fundic mucosa in the glandular stomach in females.

However, the registrant considers the effects observed at 85 mg/kg not to be adverse. The decreased spontaneous locomotion seen at the clinical observations in 4/5 male rats and, on a few occasions only, in 1/5 female rats of the 85 mg/kg group is likely to be secondary to the irritant effect of the substance on the gastrointestinal tract following the oral administration rather than a reflection of systemic toxicity. This is supported by the absence of an effect on spontaneous motor activity during the weekly detailed clinical observations (arena observations) and during the quantitative motor activity assessment conducted at the end of the 28-day treatment period. Heamatological parameters are not affected at 85 mg/kg bw. Biochemical parameters: Protein is slightly decreased in males only at 85 mg/kg bw but < 10%. In females there is a slight increase in gamma-GT and in total cholesterol of which the latter decreases again at 340 mg/kg bw. The liver parameters are only slightly affected and can still be considered an adaptive response. An enlarged liver is seen in one male at this dose, which is considered an adaptive change. In females a microscopic effect is seen in the glandular stomach (focal minimal necrosis of the fundic mucosa) in one animal. This effect is considered to be due to the irritant properties of the substance and therefore does not need to be considered for the systemic NOAEL. In summary, the registrant estimates the systemic NOAEL to be 85 mg/kg bw/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: "28-day Repeated Dose Toxicity Study in Mammalian Species” prescribed in “Concerning Testing Methods Relating to the New Chemical Substances” (March 31, 2011)
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one
EC Number:
275-156-8
EC Name:
[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one
Cas Number:
71048-82-3
Molecular formula:
C13H20O
IUPAC Name:
[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Hino Breeding Center
- Age at study initiation: 5 weeks
- Weight at study initiation: 142.4-177.2 g and 123.9-147.2 g, respectively for males and females
- Housing: Hanging stainless steel cages with wire-mesh floor (260 W×380 D×180 H mm) for quarantine and acclimation, and hanging stainless cages (165 W×300 D×150 H mm) after the grouping
- Diet: Pelleted diet (MF, lot no. 130403 and 130515, Oriental Yeast)
- Water: Chlorinated water maintained at 3-5 ppm of chloric level prepared by adding sodium hypochlorite (Purelox) to Hita City supply water was given by an automatic watering system with sipper tubes
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.3-23.9
- Humidity (%): 50.4-64.9
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance was weighed and dissolved in corn oil to prepare 3.40 w/v% formulation. A part of 3.40 w/v% formulation was diluted with corn oil to prepare formulations of 0.850 and 0.210 w/v%. The formulations were subdivided into plastic containers, and stored at the cold place (actual temperature: 2-9ºC, tolerance temperature: 1-10ºC). These were dosed to the animals within 12 days after preparation. On each dosing day, formulations were taken out from the storage place and kept at room temperature until dosing.

VEHICLE
- Corn oil has been commonly used as a vehicle in general toxicity studies and we have historical control data.
- Concentration in vehicle: 0.21, 0.85, 3.40 w/v%
- Amount of vehicle: 10 mL/kg
- Lot/batch no.: V2H8729
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability Analyses: Stability of the 10.0 and 0.100 w/v% formulations in the cold place were analyzed by high performance liquid chromatography (HPLC) in CERI Hita (Study No. X18-1035). The test substance in the 10.0 and 0.100 w/v% formulations were judged as stable for 12 days under the storage condition, because those concentrations after storage were within 100 ± 10% (101 and 101%, respectively) of those immediately after preparation for 13 days.
- Concentration Analysis: Concentration analysis for the 3.40, 0.850 and 0.210 w/v% formulations was performed by HPLC in CERI Hita immediately after the first preparation (Study No. X18-1035) The concentrations of the formulations were confirmed to be within 100±10% of each nominal concentration (3.40 w/v% formulation: 104%, 0.850 w/v% formulation: 103%, 0.210 w/v% formulation: 99.0%) and the preparation procedure was judged to be appropriate.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
21 mg/kg bw/day (actual dose received)
Dose / conc.:
85 mg/kg bw/day (actual dose received)
Dose / conc.:
340 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: As a range finding study, “a 7-day repeated-dose oral toxicity study of Delta Damascone in rats” was performed in CERI Hita (Study No. P11-1035, non-GLP). In the study, three male and three female Crl:CD(SD) rats in each group were treated orally with the test substance dissolved in corn oil at 0, 30, 100, 300 and 1000 mg/kg/day for 7 days. General clinical observations and body weight measurements were performed in the dosing period. Necropsy and organ weights measurements were performed on the day after the last dosing. As a result, the changes of the general condition were observed, such as salivation immediately after the administration in males and females of the 100 mg/kg groups and higher, decreased spontaneous locomotion in males and females of the 300 mg/kg groups and higher, restlessness in males of the 300 mg/kg group and females of the 300 mg/kg group and higher, staining around nose and mouth in males and females of the 1000 mg/kg groups and incomplete eyelid opening and staining hair in females of the 1000 mg/kg group. On the day 3 of the dosing period, one female of the 1000 mg/kg group was dead. In body weight, decreases from the last value, a statistically significant decrease or a low tendency were observed in males and femalesof the 1000 mg/kg groups. In the pathological examinations, statistically significant increases of relative weights of liver and macroscopic enlargement of the liver were observed in males and females of the 300 mg/kg groups. Whitish region of mucosa in the forestomach was observed in females of the 300 mg/kg group and higher and males of the 1000 mg/kg group. Statistically significant decreases of absolute and relative weights of the spleen were observed in females of the 300 mg/kg group. Based on these results, it was considered that the test substance had an irritant property to gastric mucosa and the major target organs of the test substance were the stomach and liver. Although no abnormal changes were found in the body weight in the 300 mg/kg groups, the findings in general clinical condition such as decreased spontaneous locomotion and restlessness were observed and the effects on the liver were pathologically observed. Therefore, it was considered that the repeated dosing of Delta Damascone at much higher dose than 300 mg/kg for 28 days might cause severe toxicity such as death or moribundity. Accordingly, in the present study, the high dose was set at 340 mg/kg/day and lower doses of 85 and 21 mg/kg/day were set.
- Grouping: Three dose levels for the test substance groups and one vehicle control group were used. Recovery groups were set for the high dose and vehicle control groups without treatment for 14 days after the dosing period.

Examinations

Parental animals: Observations and examinations:
GENERAL CLINICAL OBSERVATIONS: During the dosing period, animals were observed three times a day, i.e., before dosing, between just after dosing and 1 hour after dosing, and between 2 and 6 hours after dosing. During the recovery period, observation was performed once a day.
DETAILED CLINICAL OBSERVATIONS: Detailed clinical observations in all animals were performed once before dosing. Thereafter, animals were examined once a week during the dosing and recovery periods on a blind test basis. The blind test was performed using the random numbers and observation labels without identifying the dosing groups. Observation parameters and methods: (a) Observations at removal from cage (Animal reactions such as excitement from external stimuli (holding animals or bringing a hand close to animals to hold) were observed and assessed by scoring method. Ease of removal and vocalization. (b) Handling observations: Muscle tone, subnormal temperature, hair appearance (piloerection, staining hair and unkempt hair), skin and mucous color (paleness, reddening and cyanosis), eyes (lacrimation, exophthalmos and pupillary size), salivation and secretion. (c) Observation in arena: Animals were placed in a standard arena (on an observation platform of 90 cm×60 cm) and observed for one minute or more (within five minutes), and the frequencies of defecation (number of feces) and urination (number of pools) were recorded for one minute. Posture, motor activity level, respiration, gait characteristics, lid closure, tremor, twitch, convulsion, stereotypical behavior and abnormal behavior.
SENSORIMOTOR FUNCTION EXAMINATIONS: All animals were examined in week 4 (on days 27 and 28) of the dosing period. Reflex tests and measurements of grip strength were performed on a blind test basis. In one female of the high dose (340 mg/kg) group, a hyper reaction was observed in pain response in reflex test. Therefore, in the week 2 of the recovery period (on day 11 of the recovery period), the pain response test was performed for females. In males, the examinations were not performed in the recovery period, since no abnormal changes related to the test substance treatment were observed in the 340 mg/kg groups in the dosing period. Observation parameters and methods: (a) Reflex: Optic (approach contact/touch) response, Pinna response, Pain response, Pupillary reflex, Air righting reflex. (b) Grip strength (c) Locomotor activity counts.
BODY WEIGHT MEASUREMENTS: On the grouping day, on days 1, 3, 8, 12, 17, 21, 26 and 28 of the dosing period, on days 1, 5, 10 and 14 of the recovery period, on the necropsy days (before carrying animals from the animal room).
FOOD CONSUMPTION MEASUREMENTS: Feeding weights on the grouping day, Remainder weights on days 1, 3, 8, 15, 22 and 28 of the dosing period, Feeding weights on day 1 of the recovery period, Remainder weights on days 4, 8 and 14 of the recovery period. Feeding weights on days 8, 15 and 22 of the dosing period and on day 8 of the recovery period were measured after food was replenished. Reminder weight on days 1 and 3 of the dosing period and day 4 of the recovery period were also used as feeding weight and the replenishment was not conducted on those days. Mean food consumption per day was calculated from their feeding and remainder weights for each period.
URINALYSES: Collecting Urine Samples: Urine samples were collected for about 16 hours in individual metabolic cages (150 W×200 D×263 H mm) with drinking water and without food, from the afternoon of the last day of the dosing period for the main groups and that of the recovery period for the recovery groups to each next morning. Parameters and Methods: Urine samples were examined for the following parameters: Urine volume, Color, Turbidity, Urine osmotic pressure, pH, Protein, Glucose, Occult blood, Urinary sediment. Urinary sediments were stained and examined in males and females of the control and high dose (340 mg/kg) groups of the main groups. Examination of urinary sediments was not conducted for the intermediate dose (85 mg/kg) or low dose (21 mg/kg) groups or the recovery groups, since no abnormal changes related to the treatment were noted in either sex of the 340 mg/kg groups.
BLOOD EXAMINATIONS: (A) Blood Sampling and Preparation of Test Samples: Blood was collected from the abdominal aorta under isoflurane anesthesia after the urine collection on the next day of the last day of the dosing period for the main groups and that of the recovery period for the recovery groups. Blood sampling were performed after overnight fasting (16 to 20 hours) for each period. (B) Hematological Examinations: Whole blood or plasma samples were used to determine the following parameters: Red blood cell count, Haemoglobin conc., Haematocrit value, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular hemoglobin conc., Platelet count, Reticulocyte count ratio, White blood cell count, Differentiation of leukocytes (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Prothrombin time, Activated partial thromboplastin time. Blood smears were not prepared since reticulocyte count ratio and differentiation of leukocytes were able to measure by the instruments. (C) Blood Chemical Examinations: Serum samples were used to determine the following parameters: Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Choline esterase, gamma-Glutamyl transpeptidase, Total cholesterol, Triglyceride, Blood urea nitrogen, Creatinine, Total protein, Albumin, A/G ratio, Glucose, Total bilirubin, Total bile acids, Inorganic phosphorus, Calcium, Sodium, Potassium, Chloride.
Oestrous cyclicity (parental animals):
For females, vaginal smears were collected before the gross necropsy, and the stages of estrous cycle were determined with light microscope after Giemsa staining.
Sperm parameters (parental animals):
Parameters examined in all males:
testis weight and epididymis weight
Postmortem examinations (parental animals):
(A) Gross Necropsy: All animals were subjected to a detailed gross necropsy after blood sampling and bleeding from the ventral aorta on the day after last administration for the main groups and on the day after last withdrawal day for the recovery groups. External surface of the body, all orifices, subcutis, cranial, thoracic, abdominal and pelvic cavities, and their contents were observed.
(B) Tissue Collecting and Organ Weight Measurements: The following organs were removed from all animals: Respiratory system (Trachea, lungs), Digestive system (Submandibular glands, stomach, intestines (duodenum to rectum, including Peyer’s patches), pancreas, liver*), Cardiovascular system (Heart*), Urinary system (Kidneys*, urinary bladder) Reproductive system (Testes*, epididymides*, prostate*, seminal vesicles* (including coagulating gland), ovaries*, uterus*, vagina), Nervous system (Brain* (including cerebrum, cerebellum and pons), spinal cord, sciatic nerve), Hematopoietic system (Bone marrow (femur), axillar lymph nodes, mesenteric lymph nodes, spleen*, thymus*), Endocrine system (Pituitary gland, thyroid* (including parathyroid), adrenals*), Sense organ (Eye balls), Musculoskeletal system (Skeletal muscle (femoral region), bone (femur)). From one animal of the 340 mg/kg bw group the skin was collected. Trachea, lungs and urinary bladder were inflated with 10% neutralized buffered formalin before removal. Stomach and intestines were filled and fixed with 10% neutralized buffered formalin and the contents were washed away with water. The weights of organs with an asterisk were measured using an electric balance (SARTORIUS). Kidneys, testes, epididymides, ovaries and adrenals were weighed right and left together. Prostate was weighed including a part of urethra. Seminal vesicles including coagulating gland were removed after ligation of the root and weighed. Uterus was weighed with the contents. Thyroid adhered to trachea, including parathyroid, was fixed with 10% neutralized buffered formalin, and the right and left lobes were removed from trachea and weighed on the day after necropsy. Relative organ weight was calculated based on the body weight measured on the necropsy day.
(C) Histopathological examinations: The testes and epididymides were fixed in modified Davidson’s fixative. The other organs/tissues were preserved in 10% neutralized buffered formalin. Light microscopic examinations were performed for the following organs and the tissues of the control and 340 mg/kg bw group after embedding in paraffin, sectioning and hematoxylin and eosin (HE) staining. Decalcification was done for bone and bone marrow (femur) with 10% formic acid. Examined organs: Trachea, Lungs, Submandibular gland, Duodenum-ileum, Cecum- rectum, Pancreas, Heart, Kidneys, Urinary bladder, Prostate, Coagulating gland, Seminal vesicle, Ovaries, Uterus, Vagina, Cerebrum, Cerebellum, Pons, Spinal cord, Sciatic nerve, Bone marrow, Axillar lymph nodes, Mesenteric lymph nodes, Spleen, Thymus, Pituitary gland, Thyroid, Parathyroid, Adrenals, Eye ball, Skeletal muscle, and Bone. For the forestomach, glandular stomach, and liver, histopathological examinations were performed for males and females of all treatment groups and the recovery groups since treatment-related changes were suspected in males and females of the 340 mg/kg groups. For the testes and epididymides, histopathological examinations were performed for males of the control and 340 mg/kg bw group and of the recovery groups since a statistically significant change was found in the absolute organ weight of the epididymides in males of the 340 mg/kg recovery group. The testes were also examined as the related organs to the epididymides. Of one animal in the 340 mg/kg bw the skin was examined and of one female of the 85 mg/kg bw group and one female of the 340 mg/kg bw group, the vagina was examined (on account of gross lesions observed at necropsy).
Statistics:
Data regarding body weights, food consumption, grip strength and locomotor activity counts during the dosing period, and parameters of hematological examinations, parameters of blood chemical examinations, urine volume, urine osmotic pressure, organ weights and body weights on the necropsy day of the main groups were analyzed by the Bartlett’s test for homogeneity of variances. When the variances were homogeneous at a significance level of 5%, the Dunnett’s test was used. When the variances were not homogeneous, the nonparametric Dunnett’s test was used. The frequencies of defecation (number of feces) and urination (number of pools) during the dosing period were analyzed by the nonparametric Dunnett’s test.
Data regarding body weights, food consumption during the recovery period, and parameters of haematological examinations, parameters of blood chemical examinations, urine volume, urine osmotic pressure, organ weights and body weights on the necropsy day of the recovery groups were analyzed by the F-test for variance ratio. When there were no significant differences at a significance level of 5% in this analysis, the Student’s t-test was used. When there was a significant difference, the Aspin-Welch t-test was used. The frequencies of defecation (number of feces) and urination (number of pools) during the recovery period were analyzed by the Mann-Whitney U-test.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
DURING DOSING PERIOD:
In males, salivation and decreased spontaneous locomotion in 10 animals out of 10, staining around nose and mouth in nine animals, much food on the tray (increase of the amount of food dropped on tray by biting) in eight animals, restlessness which meant increased chin rubbing or cage scratching in six animals, decreased respiratory rate in four animals, incomplete eyelid opening, lacrimation and reddish tear in three animals, staining hair in two animals and diarrhoea, loss of hair and moist hair in one animal were observed in the 340 mg/kg group. In the 85 mg/kg group, salivation in five animals out of five, decreased spontaneous locomotion in four animals and incomplete eyelid opening in one animal were observed. In the 21 mg/kg group, salivation was observed in one animal out of five. No abnormal changes were observed in the control group. The decreased spontaneous locomotion was observed sporadically or continuously throughout the dosing period after day 4 of the dosing period in the 340 and 85 mg/kg groups. The salivation was observed continuously after day 2 of the dosing period in the 340 mg/kg group. In the 85 mg/kg group, the salivation was observed sporadically or continuously after day 4 of the dosing period. The persistence time of the salivation tended to prolong with the dosing progresses. The salivation was also observed immediately before the administration in the 340 mg/kg group. In the 21 mg/kg group, the salivation was observed only on day 14 of the dosing period and disappeared within 30 minutes after administration. The restlessness which meant increased chin rubbing or cage scratching was sporadically observed after week 2 of the dosing period in the 340 mg/kg group. The much food on the tray was observed sporadically in week 3 and 4 of dosing period in the 340 mg/kg group. The diarrhoea was observed only in one animal of the 340 mg/kg group on day 5 of the dosing period. In females, salivation, decreased spontaneous locomotion and staining around nose and mouth in 10 animals out of 10, staining around external genitalia in four animals, much food on the tray (increase of the amount of food dropped on tray by biting) in three animals, restlessness which meant increased chin rubbing or cage scratching in two animals and incomplete eyelid opening, reddish tear, lacrimation, staining hair, staining lower abdomen and staining around anus in one animal were observed in the 340 mg/kg group. In the 85 mg/kg group, salivation in four animals out of five and decreased spontaneous locomotion in one animal were observed. No abnormal changes were observed in the 21 mg/kg group or control group. The decreased spontaneous locomotion was observed sporadically or continuously after day 5 of the dosing period in the 340 mg/kg groups while the number of the animals which showed decreased spontaneous locomotion was decreased after week 3 of the dosing period. In the 85 mg/kg group, the decreased spontaneous locomotion was observed in week 1 and 2 (from day 6 to day 8 of the dosing period) in one animal. The salivation was observed continuously after day 2 of the dosing period in the 340 mg/kg group. The persistence time of the salivation tended to prolong with the dosing progresses. In the 85 mg/kg group, the salivation was observed from day 5 to day 10 of the dosing period and disappeared within 1 hour after administration. The restlessness was sporadically observed in week 2 and 3 of the dosing period in the 340 mg/kg group. The much food on the tray was observed sporadically or continuously in week 3 and 4 of dosing period in the 340 mg/kg group.
In males, staining around nose and mouth was observed in four animals and three animals out of 10 of the 340 mg/kg group in week 1 and 2, respectively. Staining around nose and mouth or mandible to abdomen in four animals, lacrimation in three animals and salivation in four animals were observed in the 340 mg/kg group in week 4. Continuous vocalization (+2) was observed in one animal out of five in week 3 in the 21 mg/kg group. A statistically significant increase of the frequency of urination was observed in the 85 mg/kg group in week 1, although no dose-dependency was observed. No statistically significant changes in the frequency of defecation were observed in any treatment groups. No abnormal changes were observed in the control group. In females, staining around nose and mouth or abdomen was observed in two animals out of 10 of the 340 mg/kg group in week 1. Staining lower abdomen was observed in one animal of the 340 mg/kg group in week 2. Continuous vocalization (+2) was observed in one animal of the 340 mg/kg group. Staining around nose and mouth or external genitalia in five animals and salivation in one animal were observed in the 340 mg/kg group in week 4. Statistically significant increases of the frequencies of urination in the 85 mg/kg group in week 1 and in the 21 mg/kg group in week 2 were observed, although no dose-dependency was observed. No statistically significant changes in the frequency of defecation were observed in any treatment groups. No abnormal changes were observed in the control group.

DURING RECOVERY PERIOD:
In males, much food on the tray was observed in one animal out of five of the 340 mg/kg group on day 1 of the recovery period. No abnormal changes were observed in the control group. In females, staining around external genitalia was observed in one animal out of five of the 340 mg/kg group on day 1 of the recovery period. No abnormal changes were observed in the control group.
No statistically significant changes in frequencies of defecation or urination were noted in either sex of the 340 mg/kg group. No abnormal changes were observed in the control or the 340 mg/kg groups in the other parameters.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
DURING DOSING PERIOD:
In males, statistically significant decreases were found from day 8 to 28 of dosing period in the 340 mg/kg group. The body weight of the 340 mg/kg group was 77.1% compared to that of the control group on day 28 of the dosing period. No statistically significant changes were noted in the 21 or 85 mg/kg groups. In females, no statistically significant changes were noted in any treatment groups.

DURING RECOVERY PERIOD:
In males, statistically significant decreases were found from day 1 to 14 of the recovery period. Although the body weight of the 340 mg/kg group on day 1 of the recovery period was 76.4% compared to that of the control group, that on day 14 recovered to 85.4% compared to that of the control group. In females, no statistically significant changes were noted in the 340 mg/kg groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
DURING DOSING PERIOD:
In males, a lower tendency (89.2% compared to the control group) was found in the 340 mg/kg group on day 3 of the dosing period. A higher tendency (111.0% compared to the control group) was found in the 340 mg/kg group on day 28 of the dosing period. No statistically significant changes were noted in the 21 or 85 mg/kg groups. In females, a lower tendency (84.6% compared to the control group) on day 3 of the dosing period and statistically significant increases or a higher tendency on day 15, 22 and 28 (115.3%, 113.7% and 126.6% compared to the control group, respectively) were found in the 340 mg/kg group. No statistically significant changes were noted in the 21 or 85 mg/kg groups.

DURING RECOVERY PERIOD:
No statistically significant changes were noted in either sex of the 340 mg/kg groups.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
MAIN GROUP:
In males, statistically significant decreases of red blood cell count, haemoglobin concentration and haematocrit value were found in the 85 mg/kg group, although no dose-dependency was noted. No statistically significant changes were noted in the 21 or 340 mg/kg groups. In females, statistically significant decreases of mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration (MCHC) were found in the 340 mg/kg group, although the change of MCHC was within the range of the historical control data of CERI Hita. No statistically significant changes were noted in the 21 or 85 mg/kg groups.

RECOVERY GROUP:
In males, a statistically significant decrease of MCHC and a statistically significant increase of reticulocyte count ratio were found in the 340 mg/kg group, although the change of MCHC was within the range of the historical control data of CERI Hita. In females, a statistically significant decrease of MCHC was found in the 340 mg/kg group, although the change of MCHC was within the range of the historical control data of CERI Hita.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
MAIN GROUP:
In males, statistically significant increases of aspartate aminotransferase, gamma-glutamyl transpeptidase (gamma-GTP) and total bilirubin, a higher tendency of alanine aminotransferase (ALT), statistically significant decreases of total protein, albumin and calcium and a lower tendency of triglyceride were found in the 340 mg/kg group. Statistically significant decreases of total protein and albumin were found in the 85 mg/kg group. A statistically significant decrease of choline esterase (ChE) was found in the 21 mg/kg group, although no dose-dependency was noted. In females, a statistically significant increase of gamma-GTP and statistically significant decreases of ChE and glucose were found in the 340 mg/kg group. A statistically significant increase of total cholesterol was found in the 85 mg/kg group, although no dose-dependency was noted. No statistically significant changes were noted in the 21 mg/kg group.

RECOVERY GROUP:
In males, statistically significant increases of ALT and total cholesterol and statistically significant decreases of creatinine and glucose were found in the 340 mg/kg group, although those changes were within the range of the historical control data in CERI Hita. In females, a statistically significant decrease of ChE and a statistically significant increase of triglyceride were found in the 340 mg/kg group, although those changes were within the range of the historical control data in CERI Hita.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
DURING DOSING PERIOD:
In males, a statistical significant increase of the urine volume and a lower tendency of the urine osmotic pressure were found in the 340 mg/kg group, although they were within the range of the historical control data of CERI Hita. No abnormal changes were noted in the other examined parameters in the control or any treatment groups. In females, no statistically significant changes in urine volume or urine osmotic pressure were noted in any treatment groups. No abnormal changes were noted in the other examined parameters in the control or any treatment groups.

DURING RECOVERY PERIOD:
No statistically significant changes in urine volume or urine osmotic pressure were noted in either sex of the 340 mg/kg groups. No abnormal changes were noted in the other examined parameters in either sex of the control or 340 mg/kg groups.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
DURING DOSING PERIOD:
In males, a statistically significant decrease of locomotor activity counts was observed in interval from 50 to 60 minutes, although no dose-dependency was observed. No statistically significant changes in grip strength were noted in any treatment groups. No abnormal changes were observed in the control or any treatment groups in the reflex test. In females, a hyper reaction (+1) to pain response was observed in one animal out of 10 of the 340 mg/kg group. No statistically significant changes in grip strength or locomotor activity counts were noted in any treatment groups. No abnormal changes were observed in the control, 21 or 85 mg/kg groups in the reflex test.

DURING RECOVERY PERIOD:
In males, the examinations were not performed since no abnormal changes suspected of the effect of the treatment were noted in week 4 of the dosing period. In females, no abnormal changes were observed in pain response of reflex test in the control or 340 mg/kg groups.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
MAIN GROUP:
In males, in the liver, hypertrophy of periportal hepatocytes in three animals out of five of the 340 mg/kg group and one animal of the 85 mg/kg group and prominent nucleoli of periportal hepatocytes in five animals of the 340 mg/kg group and one animal of the 85 mg/kg group were observed, which were corresponding to the enlargement of the liver observed macroscopically. In the glandular stomach, focal necrosis of fundic mucosa in two animals, which were corresponding to the blackish region of mucosa of the glandular stomach observed macroscopically and oedema in submucosal layer in one animal were observed in the 340 mg/kg group. In the two animals which showed the glaudular stomach lesions, spongiosis of squamous epithelium in limiting ridge, oedema in lamina propria of limiting ridge and oedema in submucosal layer were also observed in the forestomach of one animal and oedema in lamina propria of limiting ridge was also observed in the forestomach of the other animal. No abnormal changes were noted in the kidney in which a statistically significant increase in relative weight was found in the 340 mg/kg group. In one animal of the 340 mg/kg group, decreased hair follicles was observed in the skin, which was corresponding to the gross lesion. Microgranuloma in the liver in one animal of the 85 mg/kg group and solitary cyst in medulla in one animal of the control group were also observed. In the 21 mg/kg group, no abnormal changes were observed in the forestomach, glandular stomach or liver which were examined. In females, in the liver, hypertrophy of periportal hepatocytes in one animal out of five and prominent nucleoli of periportal hepatocytes in four animals were observed in the 340 mg/kg group, which were corresponding to the enlargement of the liver observed macroscopically. In the glandular stomach, focal necrosis of fundic mucosa in three animals of the 340 mg/kg group and one animal of the 85 mg/kg group was observed, which were corresponding to the macroscopic lesion. In the forestomach, focal spongiosis of squamous epithelium was observed in one animal of the 340 mg/kg group, which were corresponding to the recessed region of mucosa in the forestomach. No abnormal changes were noted in the kidney in which a statistically significant increase in relative weight was found in the 340 mg/kg group. In one animal of the 85 mg/kg group, squamous epithelial cyst was observed in the vagina, which was corresponding to the gross lesion. Periportal fatty change of hepatocytes and granuloma in the liver in one animal of the control group, mineralization in cortico-medullary junction in the kidney in one animal of the control group and ectopic thymic tissue in the thyroid in one animal of the 340 mg/kg group were also observed. Accumulation of lipid in hepatocyte was revealed by the specimen stained with oil red O of the liver for one female of the control group, since many positive components were observed in the hepatocytes.

RECOVERY GROUP:
In males, hyperplasia of squamous epithelium in limiting ridge in the forestomach was observed in one animal out of five of the 340 mg/kg group. Microgranuloma in the liver was observed in one animal each of the control and 340 mg/kg groups. No abnormal changes were noted in the epididymides in which a statistically significant decrease in absolute weight was found in the 340 mg/kg group. In females, squamous epithelial cyst was observed in the vagina in one animal of the 340 mg/kg group, which was corresponding to the gross lesion. No abnormal changes were observed in the forestomach, glandular stomach or liver in the control or 340 mg/kg groups.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous Cycle Stage
MAIN GROUP:
In the control group, one female out of five was in estrus, two were in metestrus and two were in diestrus. In the 21 mg/kg group, three females were in estrus and two were in metestrus. In the 85 mg/kg group, one female was in proestrus, one was in metestrus and three were in diestrus. In the 340 mg/kg group, one female was in estrus, one was in metestrus and three were in diestrus.

RECOVERY GROUP:
In the control group, one female out of five was in proestrus, one was in metestrus and three were in diestrus. In the 340 mg/kg group, two females were in estrus, one was in metestrus, two were in diestrus.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
MAIN GROUP:
Statistically significant increases of relative weights of the testes, statistically significant decreases of absolute weights of epididymides, statistically significant decreases of absolute and relative weights of the prostate and seminal vesicles were observed. No statistically significant changes were noted in the 21 or 85 mg/kg groups.

RECOVERY GROUP:
In males, statistically significant increases in relative weights of the testes and statistically significant decreases in absolute weights of the epididymides and prostate were found in the 340 mg/kg group.
The magnitude of the organ weight changes at the end of the recovery period was smaller than that at the end of the treatment period. The changes in male reproductive organ weights were considered to be secondary to the lower body weight in the 340 mg/kg group. Based on these results, it can be concluded that the test substance did not exert adverse effects on male or female reproductive organs up to the highest dose tested (340 m/kg bw).

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
85 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: See 'Any other information on results incl. tables'
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
85 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: See 'Any other information on results incl. tables'
Key result
Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
>= 340 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks on result:
not measured/tested

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL for systemic toxicity in the oral 28-day repeated dose toxicity study in rats was considered to be 85 mg/kg bw /day. The next higher dose level of 340 mg/kg bw/day was a NOAEL for fertility based on the absence of direct adverse effects on male and female reproductive organs.
Executive summary:

In a study performed in compliance with the Japanese guideline '28 -day Repeated Dose Toxicity Study in Mammalian Species' (Concerning Testing Methods Relating to the New Chemical Substances) and GLP, the test substance was administered to five male and female Crl:CD(SD) rats per group by oral gavage as a dilution in corn oil at dose levels of 0, 21, 85 and 340 mg/kg/day. Additionally, the study included two recovery groups of 5 rats/sex each which were dosed for 28 days with the vehicle (control) or 340 mg/kg of test substance and then left untreated for 14 days. During the dosing period, general clinical observations, detailed clinical observations, function examinations, body weight measurements and food consumption measurements were performed. On the day after the last dosing, urinalyses, blood examinations and pathological examinations were performed after collecting urine and blood samples. The NOAEL for systemic general toxicity was determined to be 85 mg/kg bw (see IUCLID Chapter 7.5.1).

Regarding fertility, macroscopic examination at necropsy and histopathological examination of reproductive organs did not reveal treatment-related changes. The weights of the female reproductive organs (ovaries and uterus) showed no treatment-related changes either. Male reproductive organ weights showed statistically significant changes at the highest dose level (340 mg/kg), namely an increase in relative testes weight, a decrease in the absolute weight of the epididymides and decreases in the absolute and relative weights of the prostate and seminal vesicles. At the end of the recovery period, statistically significant changes in the 340 mg/kg group were limited to an increase in relative testes weight and decreases in the absolute weights of the epididymides and prostate. The magnitude of the organ weight changes at the end of the recovery period was smaller than that at the end of the treatment period. The changes in male reproductive organ weights were considered to be secondary to the lower body weight in the 340 mg/kg group. Based on these results, it can be concluded that the test substance did not exert adverse effects on male or female reproductive organs up to the highest dose tested (340 m/kg bw).