Registration Dossier

Administrative data

Description of key information

Oral (OECD 408), rat: NOAEL (local, males/females): 300 mg/kg bw/day

Oral (OECD 408), rat: NOAEL (systemic, males/females) ≥ 1000 mg/kg bw/day

The systemic NOAEL was considered relevant for the risk assessment.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Jan - 6 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted in 1998
Deviations:
yes
Remarks:
some additional endpoints were considered, including e.g., estrous cyclicity and sperm analysis
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted in 2008
Deviations:
yes
Remarks:
some additional endpoints were considered, including e.g., estrous cyclicity and sperm analysis
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Name: RZETA (A mixture composed of (1,4-diazabicyclo[2.2.2]octane-2- yl)methanol (89.8%) and 1,5-diazabicyclo[3.2.2]nonan-3-ol (9.8%)
Supplier: Tosoh Corporation, Japan
Lot No.: 6Z02RZETACR
Analytical purity for 76950-43-1 (1,4-diazabicyclo[2.2.2]octane-2-yl)methanol): 89.8%
Appearance: pale yellow solid
Expiration date: December 2, 2018
Storage conditions: at room temperature in an airtight, light-protected, and N2 sealed container.
Certificate of Analysis of the test article provided: yes, attached to this report (Annex 1)
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
as recommended by the OECD Guideline 408
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Females nulliparous and non-pregnant: not stated
- Age at study initiation: 5 weeks (males) and 6 weeks (females)
- Weight at study initiation: 170 - 192 g (males) and 150 - 172 g (females)
- Fasting period before study: no
- Housing: animals were housed in metal bracket cages (260 W × 380 D × 180 H, mm) with wire mesh floors; three or less animals per cage during the quarantine and acclimation period, and one after group assignment
- Diet: Pellet diet, CRF-1, Oriental Yeast Co., Ltd., ad libitum
- Water: Sapporo-City tap water, ad libitum
- Acclimation period: at least 6 days

DETAILS OF FOOD AND WATER QUALITY:
According to the study report, each lot of the diet used was analyzed for contaminants; the contamination analyses were performed by Eurofins Food and Product Testing Japan, K.K. (Analysis report: Nos. AR-16-JP-110873-01-JA, AR-16-JP-113015-01-JA and AR-17-JP-001203-01-JA) and the diet manufacturer (Analytical test report Nos. 16G03-103, 16G03-117 and 17G03-013). The analysis data of each lot was obtained from the diet manufacturer. The analysis items and the acceptable values were those specified in the Standard Operating Procedures of Safety Research Institute for Chemical Compounds Co., Ltd., and all values were within the acceptable ranges. No further details are available from the study report.
According to the study report, a sample of drinking water was collected at the animal room No. 301 used in this study on January 5, 2017, and July 4, 2017, which was analyzed for contaminants. The analysis was performed by Nihon Eisei Co., Ltd. (Results of water quality test: No. A285508 and A291241) and the analysis data were obtained. The analysis items and the acceptable values were those specified in the Standard Operating Procedures of Safety Research Institute for Chemical Compounds Co., Ltd., and all values were within the acceptable ranges. No further details are available from the study report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Jan To: 10 May 2017
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Otsuka distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was accurately weighed out, to which the vehicle was added to achieve the prescribed concentration; the test article was dissolved using a stirrer. Dosing solution was used within the verified stability period (within 8 days). Dosing solutions were refrigerated (actual range 1.7 ºC to 7.1 ºC) in an air-tight and light-protected container.

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL, respectively
- Amount of vehicle: 10 mL/kg bw
- Lot/batch nos. : 6I83 and 6G74
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the dosing solutions:
According to the study report, the stability of dosing solutions was investigated prior to the conduct of the present study (Report NCAS 14-012). Within this study, the stability of 1 and 100 mg/mL preparations of the test item during 8-day storage under refrigeration followed by 5-hr storage at room temperature after preparation was confirmed. With respect to the dose levels considered within the present study, the concentrations of test item in the dosing solutions were analysed at the first preparation, at mid-term of administration period and at the final preparation; in fact, all prepared concentrations were subjected to analysis. The analytic study report (study No. 16-182) is provided within the 90-Day study report in Annex 2. In brief, the analytical results confirmed adequate concentrations of the test item in the dosing solutions: the nominal concentrations of 10, 30 and 100 mg/L which were analysed showed final dose concentrations of 9.98, 31.1 and 101 mg/mL, respectively, indicating recoveries >= 99.8%.
Duration of treatment / exposure:
90 or 91 days, followed by a 14 days post-exposure (recovery) observation period
Frequency of treatment:
once daily, 7 days/week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 (main study)
5 (recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosage levels were selected based on the results of a previous screening test according to OECD Guideline 422 conducted with the test item (Fuji, 2014). In this study, the test item was administered at 0, 100, 300, and 1000 mg/kg bw/day by gavage to male and female Crl:CD rats. Squamous cell hyperplasia at the limiting ridge of the stomach with high incidence in males at 1000 mg/kg bw/day and increased liver and kidney weight in females at 1000 mg/kg bw/day were reported. Therefore, 1000 mg/kg bw/day was selected as the highest test dose for the present study, and 300 and 100 mg/kg bw/day were selected by dividing the highest dose by a common ratio of 3.
- Post-exposure recovery: 14 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily during acclimation; at least twice daily, before and after dosing, during administration period; at least twice daily, in the morning and afternoon, during recovery period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the start of administration, on administration day 7, and once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: on administration days 1, 4, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 90, and necropsy day; before dosing on each measurement day; during recovery period: on recovery days 1, 7, and 14

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of administration (during the acclimation period) and in administration week 12 and recovery week 2
- Dose groups that were examined: all animals before the start of administration and during recovery period; all animals in the control- and high dose-toxicity study groups and recovery groups during administration period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: red blood cell count (RBC), hematocrit (HCT), hemoglobin concentration (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (Platelet), white blood cell count (WBC), reticulocyte count (Reticulocyte), differential count of WBC (neutrophils, eosinophils, basophils, monocytes, lymphocytes), prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters examined: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GTP), glucose, total cholesterol (T-Cho), triglyceride (TG), total bilirubin (T-Bil), urea nitrogen (UN), creatinine (Crea), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), inorganic phosphorus (IP), total protein (TP), albumin/globulin ratio (A/G ratio), protein fraction (albumin, α1-globulin, α2-globulin, β-globulin, γ-globulin)

URINALYSIS: Yes
- Time schedule for collection of urine: in administration week 13 and recovery week 2
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters examined: pH, protein, glucose, ketone body, urobilinogen, bilirubin, occult blood, urine volume, specific gravity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in administration week 13 and recovery week 2
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes
- the following organ weights were recorded for all animals (except the one that died): brain, heart, liver, kidneys, lung, testes, epididymides, seminal vesicles (with coagulating glands), spleen, thymus, adrenals, pituitary gland, and thyroids (with parathyroids), prostate, ovaries, uterus

HISTOPATHOLOGY: Yes
- The following organs and tissues were preserved and examined: brain, pituitary gland, spinal cord (cervical, mid thoracic, and lumber regions), thyroids, parathyroids, thymus, heart, lung, larynx, trachea, bronchus, aorta, liver, spleen, kidneys, adrenals, tongue, submandibular lymph nodes, salivary glands (submandibular glands, sublingual glands), esophagus, stomach, duodenum, jejunum, ileum (with Peyer’s patches), cecum, colon, rectum, mesenteric lymph nodes, pancreas, mammary gland (right abdomen), skin (abdomen), urinary bladder, testes (left side), epididymides, prostate, seminal vesicles (with coagulating glands), ovaries, uterus, vagina, eyeballs, harderian glands, sternum (with bone marrow), femur (with bone marrow, right), skeletal muscle (biceps femoris) with sciatic nerve, and gross lesions (with a border to the normal tissue)

- Microscopic specimens of all the organs and tissues fixed and preserved at necropsy were prepared in the control and high dose-toxicity study groups and microscopically examined. Histopathological examination of the high dose group revealed effects of the test article administration in the stomach of males and females. Consequently, microscopic specimens were prepared from all animals in the middle- and low-dose groups and recovery groups and microscopically examined.
- For the animal that died, microscopic specimens were prepared from all the organs and tissues fixed and preserved and microscopically examined.
Other examinations:
ESTROUS CYCLICITY:
- Time schedule for examination: main test, from administration day 77 to 90; recovery, from recovery day 1 to 14
- Dose groups that were examined: main test, all groups; recovery, control and 1000 mg/kg bw/day group

SPERM ANALYSIS:
- Time schedule for examination: main test, at necropsy; recovery, at necropsy
- Dose groups that were examined: main test, all groups; recovery, control and 1000 mg/kg bw/day group
Statistics:
please refer to section "Any other information on materials and methods"
Clinical signs:
no effects observed
Description (incidence and severity):
13-Week treatment:
No clinical symptoms of toxicity were noted during clinical and detailed observations of all treated male and female rats; the control animals were inconspicuous.

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group):
No clinical symptoms of toxicity were noted during clinical and detailed observations of the male and female rats of the treated group during the recovery period. The control animals were inconspicuous.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male in the mid-dose group died within the course of day 87 of treatment; this case of death was rather incidental and not related to the treatment; no clear cause of death could however be assessed.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
13-Week treatment:
Except for the female rats of the mid-dose group (i.e., 300 mg/kg bw/day), no effects on body weight and body weight gain were noticed. In fact, for the females of the mid-dose group, body weight on treatment days 28 to 56 and on day 70, was statistically significantly reduced, compared to untreated females of the control group. The following mean body weight values were reported:
For day 28, at 300 mg/kg bw/day, the mean body weight for female rats was 230 ± 14.2 g versus 250.2 ± 21 g for control (p<0.05); for purpose of comparison, the mean body weight was 240.4 ± 15.3 and 241.8 ± 22.3 g for the 100 and the 1000 mg/kg bw/day group, respectively.
For day 56, at 300 mg/kg bw/day, the mean body weight for female rats was 263.3 ± 17.3 g versus 290.5 ± 21.7 g for control (p<0.05); for purpose of comparison, the mean body weight was 275.1 ± 21.6 and 280.7 ± 29.1 g for the 100 and the 1000 mg/kg bw/day group, respectively.
For day 70, at 300 mg/kg bw/day, the mean body weight for female rats was 281.1 ± 19.0 g versus 305.7 ± 21.6 g for control (p<0.05); for purpose of comparison, the mean body weight was 294.8 ± 23.8 and 300.8 ± 28.1 g for the 100 and the 1000 mg/kg bw/day group, respectively.
With respect to the body weight gain, this also was statistically significantly reduced for the females of the same test group, i.e., 300 mg/kg bw/day, on treatment days 4 to 77 and on day 90, compared to untreated control females. The following mean body weight gain values were reported:
For day 4, at 300 mg/kg bw/day, the mean body weight gain for female rats was 11.6 ± 3.8 g versus 15.9 ± 4.2 g for control (p<0.05); for purpose of comparison, the mean body weight gain was 12.6 ± 3.2 and 15.7 ± 3.2 g for the 100 and the 1000 mg/kg bw/day group, respectively.
For day 14, at 300 mg/kg bw/day, the mean body weight gain for female rats was 40.7 ± 10.2 g versus 52.7 ± 9.2 g for control (p<0.01); for purpose of comparison, the mean body weight gain was 45.2 ± 10.2 and 46.5 ± 8.6 g for the 100 and the 1000 mg/kg bw/day group, respectively.
For day 35, at 300 mg/kg bw/day, the mean body weight gain for female rats was 80.9 ± 12.3 g versus 104.0 ± 15.1 g for control (p<0.01); for purpose of comparison, the mean body weight gain was 88.9 ± 17.3 and 92.4 ± 21.6 g for the 100 and the 1000 mg/kg bw/day group, respectively.
For day 77, at 300 mg/kg bw/day, the mean body weight gain for female rats was 125.1 ± 17.9 g versus 149.5 ± 16.4 g for control (p<0.05); for purpose of comparison, the mean body weight gain was 138.5 ± 26.0 and 143.9 ± 29.8 g for the 100 and the 1000 mg/kg bw/day group, respectively.
For day 90, at 300 mg/kg bw/day, the mean body weight gain for female rats was 128.7 ± 18.6 g versus 154.1 ± 15.1 g for control (p<0.05); for purpose of comparison, the mean body weight gain was 142.2 ± 25.2 and 146.0 ± 29.9 g for the 100 and the 1000 mg/kg bw/day group, respectively.
No such findings were reported for the male rats of all treated groups. Thus, these findings were not considered to be due to the treatment.

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group):
No statistically significant differences were noted with respect to body weight and body weight gain of the male and female rats of the high-dose group (i,e., 1000 mg/kg bw/d) compared with those in the control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
13-Week treatment:
Except for the female rats of the mid-dose group (i.e., 300 mg/kg bw/day), no effects on food consumption were noticed. The findings as reported for the mid-dose females were not considered to be treatment-related. In fact, food consumption for female rats in the mid-dose group was statistically significantly reduced on day 21 of treatment, when compared to the females of the control group (18.18 ± 1.37 g versus 20.54 ± 1.95 g). No such findings were reported for the females of the low- and high-dose groups (i.e., 100 and 1000 mg/kg bw/day); no such findings were reported for the male rats of all treated groups.

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group):
No statistically significant differences were noted with respect to food consumption for male or female rats in the 1000 mg/kg bw/day group compared with those in the control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmological examinations revealed no abnormalities in any of the test animals.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
13-Week treatment, male rats (Table 1A):
Hemoglobin concentration (HGB) was statistically significantly decreased in the male rats of the high-dose group (.i.e., 1000 mg/kg bw/day) compared to the control value (14.85 ± 0.72 g/dL versus 16.11 ± 0.28 g/dL for control; p<0.01).
Hematocrit (HCT) was statistically significantly decreased in the male rats of the high-dose group compared to the control value (41.36 ± 1.85% versus 45.10 ± 0.73% for control; p<0.01).
Mean corpuscular volume (MCV) was statistically significantly decreased in the male rats of the high-dose group compared to the control value (47.32 ± 1.29 fL versus 50.77 ± 2.00 fL for control; p<0.01).
Mean corpuscular hemoglobin (MCH) was statistically significantly decreased in the male rats of the high-dose group compared to the control value (16.99 ± 0.47 pg versus 18.13 ± 0.68 pg for control; p<0.01).
Platelet count was statistically significantly increased in the male rats of the high-dose group compared to the control value (125.31 ± 17.80 104/μL versus 102.62 ± 7.55 104/μL for control; p<0.01).
Prothrombin time (PT) was statistically significantly increased in the male rats of the high-dose group compared to the control value (23.86 ± 4.05 sec versus 18.49 ± 2.83 sec for control; p<0.05).
Activated partial thromboplastin time (APTT) was statistically significantly increased in the male rats of the high-dose group compared to the control value (21.27 ± 3.30 sec versus 17.73 ± 2.22 sec for control; p<0.01).

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), male rats (Table 1B):
Hematological examinations in those males of the high-dose group which were subjected to an additional treatment-free 14 day-recovery period, revealed no statistically significant differences between treated and control animals.

13-Week treatment, female rats (Table 1C):
Activated partial thromboplastin time (APTT) was statistically significantly increased in the female rats of the high-dose group compared to the control value (13.47 ± 1.45 sec versus 11.54 ± 1.30 sec for control; p<0.01).

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), female rats (Table 1D):
Hematological examinations in those female rats of the high-dose group which were subjected to an additional treatment-free 14 day-recovery period, revealed no statistically significant differences between treated and control animals.


In fact, regarding the treated males, the decreased platelets count and PT value as reported were statistically significantly different from the control values; however they remained with the respective historical control ranges of the testing facility (64 to 155.8 × 104/μL for PLT and 15 to 31.9 sec for PT; Historical control data on hematology (2012 to 2017). Data on file. Safety Research Institute for Chemical Compounds Co., Ltd.). With respect to the decreased MCV, MCH, HGB and HCT values, the latter was reported being slightly below the historical control range of the testing facility in only 2/10 males; no information regarding historical control range was reported with respect to MCV, MCH, HGB. Nevertheless, since no supporting changes in red blood cell count (RBC), mean corpuscular hemoglobin concentration (MCHC) and/or reticulocytes count were noticed and since at necropsy, histopathological examination provided no evidence for bleeding, the effects as described above are neither considered adverse nor to be of toxicological relevance. Regarding the APTT value, this also slightly exceeded the historical control range of the testing facility in only 2/10 males (i.e > 28.4 sec; according to historical control data on hematology (2012 to 2017). Data on file. Safety Research Institute for Chemical Compounds Co., Ltd.).
For the females, the APTT value remained within the historical control range of the testing facility for this gender (9.9 – 18.8 sec for females, according to historical control data on hematology (2012 to 2017). Data on file. Safety Research Institute for Chemical Compounds Co., Ltd.).
No such effects were reported for the low- and mid-dose levels (i.e., 100 and 300 mg/kg bw/day) for both, male and female rats.
Thus, with respect to hematology, effects almost were noticed at the highest dose level of 1000 mg/kg bw/day. Taking into account both the results after treatment and after recovery, the effects are neither considered to be adverse nor of particular toxicological relevance, and none of them is considered treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
13-Week treatment, male rats (Table 2A):
The alkaline phosphatase (ALP) level was statistically significantly increased in the male rats of the high-dose group (i.e., 1000 mg/kg bw/day) compared to the control value (477.7 ± 114.5 IU/L versus 304.0 ± 63.4 IU/L for control; p<0.01).
The Gamma- glutamyl transpeptidase (γ-GTP) level was statistically significantly increased in the male rats of the high-dose group (i.e., 1000 mg/kg bw/day) compared to the control value (0.53 ± 0.18 IU/L versus 0.24 ± 0.23 IU/L for control; p<0.05).
Calcium (Ca) also was statistically significantly increased in the male rats of the high-dose group (i.e., 1000 mg/kg bw/day) compared to the control value (10.01 ± 0.30 mg/dL versus 9.59 ± 0.26 mg/dL for control; p<0.05).
Total cholesterol was statistically significantly decreased in the male rats of the high-dose group (i.e., 1000 mg/kg bw/day) compared to the control value (38.6 ± 9.4 mg/dL versus 53.9 ± 11.1 mg/dL for control; p<0.01).

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), male rats (Table 2B):
No relevant effects were noticed.

13-Week treatment, female rats (Table 2C):
Alanine aminotransferase (ALT) was statistically significantly decreased in mid- and high-dose females (i.e., 300 and 1000 mg/kg bw/day) compared to the control values (27.2 ± 6.4 IU/L for the mid dose, p<0.05 and 23.4 ± 7.6 IU/L for the high dose, p<0.01, versus 49.3 ± 28.6 IU/L for control).

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), female rats (Table 2D):
The only remarkable effect noticed at the end of the recovery period, was a statistically significant increase in inorganic phosphorus reported for the females of the high-dose group; no such finding was observed at the end of administration period. Thus, the increased inorganic phosphorus level was not given more importance, since no further concomitant findings were noticed.

Regarding the males, for the increased γ-GTP and Ca levels, the values in 9/10 male rats were statistically significantly different from control but remained within the respective historical control ranges of the testing facility (0.0 – 0.8 IU/L for γ-GTP and 8.8 – 10.4 mg/dL for Ca; Historical control data on biochemistry (2012 to 2017). Data on file. Safety Research Institute for Chemical Compounds Co., Ltd.). Only in one male each, the γ-GTP and the Ca level was slightly above the upper historical control limit, respectively.
With respect to the total cholesterol, only in 3/10 animals the values were found below the lower limit of the historical control range of the testing facility (36 – 100 mg/dL; Historical control data on biochemistry (2012 to 2017). Data on file. Safety Research Institute for Chemical Compounds Co., Ltd.

In conclusion, since the reported changes were neither accompanied nor supported by any relevant histopathological findings in organs of concern, i.e., liver and/or thyroid, and in the absence of any relevant changes affecting other particular parameters such as e.g. the triglyceride (TG) level, the clinical-chemical findings in the male rats were considered to be neither adverse nor toxicologically relevant.

With respect to the decreased ALT levels reported for mid- and high-dose females, these are considered of no toxicological relevance, since, according to the study report, low values are clinically not important. Further, according to the study report, individual values at both dose levels were within the historical control range of the testing facility. Hereby it is to be noticed that the unit for ALT values as reported in the tables of the study report was given as IU/L whereas the historical control range refers to the values in mg/dL (18 – 119 mg/dL).

Additional findings such as a statistically significant decrease in total bilirubin seen in males of the low-dose group only, or a significant decrease in aspartate aminotransferase (AST) as reported for the females of the mid-dose group only, are considered incidental and not related to treatment.

Thus, with respect to clinical-chemistry, effects were noticed at the highest dose level of 1000 mg/kg bw/day. Taking into account both the results after treatment and after recovery, the effects are neither considered to be adverse nor of particular toxicological relevance, and none of them is considered treatment-related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
13-Week treatment, male rats:
Urinalysis revealed statistically significantly increased protein content and specific gravity for the males of the high-dose group (i.e., 1000 mg/kg bw/day) when compared to control.

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), male rats:
All parameters considered within urinalysis were inconspicuous for the males.

13-Week treatment, female rats:
Urinalysis revealed statistically significant increases in protein content and increased pH for the females of the high-dose group (i.e., 1000 mg/kg bw/day): a statistically significant increase in protein content was also reported for the females of the mid-dose group (i.e., 300 mg/kg bw/day).

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), female rats:
Except for a statistically significantly decreased specific gravity, all parameters considered within urinalysis were inconspicuous for the females. No particular toxicological meaning can be associated to this finding.

Thus, with respect to urinalysis, the main finding for both male and female rats refers to statistically significantly elevated urinary protein content noticed at the high-dose level of 1000 mg/kg bw/day. In this group, specific gravity was also elevated, however in males only, whereas high pH was reported for females only. In those animals of the high-dose group subjected to an additional 14-day recovery period (i.e., without treatment), all parameters considered within urinalysis were inconspicuous for males whereas for the females, a statistically significantly decreased specific gravity was the only effect noticed. Taking into account the fact that necropsy revealed no concomitant findings affecting the kidneys, and that no clinical-chemical changes were found that may reflect any impairment of the renal function, and in the view of the results following recovery, the effects described above are considered neither as adverse nor as toxicologically relevant. Thus no treatment-related effect could be evidenced within urinalysis.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Significant differences in functional observations in treated males or females (any dose level) compared with the control group were neither seen within the treatment nor during recovery.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
13-Week treatment, male rats (Table 3A):
At necropsy, weighing of organs revealed that the mean absolute liver weight was statistically significantly increased in male rats of the high-dose group (1000 mg/kg bw/day), as compared to control (16.29 ± 2.024 g versus 14.06 ± 1.412 g; p<0.05); the mean relative liver weight also was statistically significantly increased (2.902 ± 0.204 versus 2.545 ± 0.236 g/100 g bw; p<0.01). Also, for these males, the mean absolute kidney weight was statistically significantly increased as compared to control (3.742 ± 0.367 g versus 3.258 ± 0.330 g; p<0.05), as well as the mean relative kidney weight (0.669 ± 0.054 versus 0.590 ± 0.045 g/100 g bw; p<0.01). For the males of the low- and mid-dose group, values with respect to liver and kidneys were comparable to control and thus, inconspicuous.

Further statistically significant but incidental differences were noticed for the mean absolute and relative thymus weights which were significantly increased compared to control values in males of the low- and high-dose groups (100 and 1000 mg/kg bw/day). No such findings were noticed at 300 mg/kg bw/day. Further, histopathological examination for the high dose group revealed no concomitant abnormalities. A further incidental finding in males refers to the mean absolute testis weight which was found to be statistically significantly increased as compared to control value, but only in the low-dose group; testis weight in the other two test groups was inconspicuous.

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), male rats (Table 3B):
At the end of the recovery period, the mean relative liver weight of the high-dose male rats was reported to be statistically significantly increased as compared to control (2.620 ± 0.131 versus 2.362 ± 0.158 g/100 g; p<0.05); however, the extent of the increase was lowered as compared to the value after as reported for the 13 –Week treatment (2.902 ± 0.204 g/100 g bw), indicating reversibility. The mean absolute and relative kidney weights of the high-dose male rats both were comparable to those of the control animals.

13-Week treatment, female rats (Table 3C):
At necropsy, weighing of organs revealed that the mean relative kidney weight was statistically significantly increased in female rats of the high-dose group (1000 mg/kg bw/day), as compared to control (0.728 ± 0.060 versus 0.661 ± 0.060 g/100 g bw; p<0.05).The mean absolute kidney weight was comparable to control. The mean absolute and relative kidney weights of the low- and mid-dose female rats were comparable to those of the control animals. Since no concomitant histopathological findings could be evidenced, the increase in relative kidney weight as reported above is not considered as toxicologically relevant.
As statistically significant but incidental differences, the mean absolute heart weight and the mean absolute and relative pituitary gland weight were reported to be statistically significantly decreased for the females of the mid-dose group (i.e., 300 mg/kg bw/day); for the low- and high-dose group, the values were similar to control and thus inconspicuous.

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), female rats (Table 3D):
At the end of the recovery period, the mean relative spleen weight was reported to be statistically significantly increased in the high-dose females as compared to control (180.608 ± 17.691 versus 154.032 ± 13.916 mg/100 g bw; p<0.05). The mean absolute spleen weight was slightly but not statistically significantly increased as compare to control. Since no such finding was evidenced at the end of the treatment period, and since the finding was only seen in females, this was not considered a relevant toxicological finding.

Thus, the only changes in organ weight for which a relationship to treatment cannot be excluded refer to the statistically significant increases in mean absolute and relative liver weight and in mean absolute and relative kidney weight as reported for the male rats of the high-dose group (1000 mg/kg bw/day) at the end of the 13-Week treatment period. After recovery, the mean absolute liver weight still was slightly but no more statistically significantly increased as compared to control, whereas the mean relative liver weight of the high-dose male rats still was statistically significantly increased. However, with respect to the relative liver weight, the extent of the increase was lowered as compared to the value as reported for the 13–Week treatment. Thus the findings at the end of the recovery period are clearly indicative of reversibility. With respect to the kidney weights, the mean absolute and relative kidney weights of the high dose male rats after recovery both were comparable to those of the control animals, thus also indicating reversibility.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
13-Week treatment, male rats:
At necropsy gross pathology revealed no abnormalities which could be associated to the treatment. In fact, only spontaneous findings including e.g., small sized testes and epididymis in one control animal and an unilateral yellowish white focus in the cauda of the epididymis in one high-dose animal, were noticed.
With respect to the one animal of the mid-dose group which died on day 87 of treatment, gross pathology was inconspicuous.

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), male rats:
Gross pathology was inconspicuous.

13-Week treatment, female rats:
At necropsy gross pathology revealed no abnormalities which could be associated to the treatment. In fact, only spontaneous findings including e.g., adhesion of the spleen to fat tissue and to the pancreas in one control animal, were noticed.

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), female rats:
Gross pathology was inconspicuous.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
13-Week treatment, male rats (Table 4):
The main findings as revealed by histopathology for male rats were focused on the stomach of the animals of the high-dose group (1000 mg/kg bw/day). In fact, infiltration of inflammatory cells in the lamina propria at the limiting ridge of the stomach was noticed in 4 animals, hyperkeratosis at the limiting ridge of the stomach was noticed in 4 animals, and infiltration of inflammatory cells in the submucosa of the glandular stomach was noticed in 5 animals of this test group. All findings were graded +1 on a severity scale of 4. No such findings were detected in males of the control, the low- and the mid-dose groups. Thus a relationship to the treatment and the dose level cannot be excluded.
Further slight changes were occasionally noted in the lung, liver, kidneys, and other organs in males in the high-dose group. These were also noted in the control group, and were known to occur spontaneously, and thus were considered unrelated to the treatment.
With respect to the one male that died in the mid dose group, histopathology revealed slight aggregation of alveolar macrophages in the lung and slight focal inflammation in the heart.

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), male rats (Table 4):
At the end of the recovery period, histopathology revealed no abnormal findings; especially regarding the stomach no such findings as described above could be evidenced.

13-Week treatment, female rats (Table 4):
The main finding as revealed by histopathology for female rats was focused on the stomach of the animals of the high-dose group (1000 mg/kg bw/day). In fact, hyperkeratosis at the limiting ridge of the stomach was reported for 2 animals in this group; the finding was graded +1 on a severity scale of 4. No such finding was detected in females of the control, the low- and the mid-dose groups. Thus a relationship to the treatment and the dose level cannot be excluded, especially in the view of the findings reported above for the male rats of the high-dose group.
Further slight changes were occasionally noted in the lung, liver, kidneys, and other organs in females in the high dose group. These were also noted in the control group, and were known to occur spontaneously, and thus were considered unrelated to the treatment.

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), female rats (Table 4):
At the end of the recovery period, histopathology revealed no abnormal findings; especially regarding the stomach no such findings as described above could be evidenced.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
ESTROUS CYCLICITY
13-Week treatment, female rats:
Estrous cyclicity was not affected by the treatment; in fact no statistically significant differences were noticed when comparing all test groups including the control to each other.

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), female rats:
No significant differences in estrous cyclicity were noticed when comparing the high-dose group with the control.

SPERM ANALYSIS

13-Week treatment, male rats:
The only reported finding refers to the straight line velocity and straightness of epididymal sperm which were reported as statistically significantly high for the males of the high-dose group; however no differences in motility between this group and the control group were noticed. Further, there were no significant differences between the treated and control groups regarding all other parameters examined. Thus, the finding is considered incidental and not related to the treatment.

13-Week + 14-Day recovery (control and 1000 mg/kg bw/day test group), male rats:
No significant differences were noticed between the high-ose and the control group with respect to all sperm parameters considered.
Key result
Dose descriptor:
NOAEL
Remarks:
local
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Critical effects observed:
no

Table 1A_ 13-Week treatment_male rats_Hematological parameters (mean value ± SD of n animals):

Parameters

Dose level (mg/kg bw/day)

0 (n=10)

100 (n=10)

300 (n=9)

1000 (n=10)

RBC (104/μL)

889.3 ± 29.4

907.5 ± 48.4

891.6 ± 30.1

874.3 ± 44.7

HGB (g/dL)

16.11 ± 0.28

16.21 ± 0.43

15.69 ± 0.50

14.85 ± 0.72**

HCT (%)

45.10 ± 0.73

44.76 ± 1.24

43.78 ± 1.55

41.36 ± 1.85**

MCV(fL)

50.77 ± 2.00

49.42 ± 2.14

49.14 ± 2.31

47.32 ± 1.29**

MCH (pg)

18.13 ± 0.68

17.88 ± 0.72

17.61 ± 0.73

16.99 ± 0.47**

MCHC (g/dL)

35.74 ± 0.36

36.21 ± 0.39

35.84 ± 0.61

35.90 ± 0.35

Reticulocyte (%)

2.877 ± 0.586

2.878 ± 0.180

2.863 ± 0.318

2.939 ± 0.345

Platelet (104/μL)

102.62 ± 7.55

113.00 ± 13.23

112.84 ± 13.56

125.31 ± 17.80**

WBC (102/μL)

84.92 ± 19.27

79.36 ± 16.59

82.58 ± 16.31

101.72 ± 33.69

Neutrophil (102/μL)

11.60 ± 2.86

10.64 ± 3.31

10.86 ± 3.00

14.34 ± 4.37

Lymphocyte(102/μL)

68.76 ± 16.16

64.18 ± 14.11

67.27 ± 16.26

81.95 ± 29.59

Monocyte(102/μL)

3.07 ± 0.94

3.03 ± 1.10

3.08 ± 1.10

3.30 ± 1.00

Eosinophil(102/μL)

1.46 ± 0.77

1.49 ± 0.46

1.34 ± 0.40

2.10 ± 0.63

Basophil(102/μL)

0.03 ± 0.05

0.02 ± 0.04

0.03 ± 0.05

0.03 ± 0.05

PT (sec)

18.49 ± 2.83

20.30 ± 1.25

20.24 ± 2.24

23.86 ± 4.05*

APTT (sec)

17.73 ± 2.22

19.36 ± 1.46

19.64 ± 1.69

21.27 ± 3.30**

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

Table 1B_ 13-Week + 14-Day recovery_male rats_Hematological parameters (mean value ± SD of n animals):

Parameters

Dose level (mg/kg bw/day)

0 (n=5)

1000 (n=5)

RBC (104/μL)

916.2 ± 57.2

894.6 ± 30.0

HGB (g/dL)

15.80 ± 0.28

15.64 ± 0.34

HCT (%)

44.54 ± 1.62

44.06 ± 1.33

MCV(fL)

48.76 ± 3.70

49.28 ± 1.91

MCH (pg)

17.30 ± 0.93

17.50 ± 0.55

MCHC (g/dL)

35.48 ± 0.79

35.48 ± 0.41

Reticulocyte (%)

3.792 ± 1.775

3.448 ± 0.737

Platelet (104/μL)

105.32 ± 7.94

104.56 ± 7.34

WBC (102/μL)

88.56 ± 22.32

79.94 ± 18.70

Neutrophil (102/μL)

12.74 ± 5.31

17.70 ± 6.46

Lymphocyte(102/μL)

70.68 ± 16.60

57.60 ± 14.33

Monocyte(102/μL)

4.04 ± 1.37

3.36 ± 1.00

Eosinophil(102/μL)

1.06 ± 0.32

1.26 ± 0.55

Basophil(102/μL)

0.04 ± 0.05

0.02 ± 0.04

PT (sec)

19.82 ± 4.12

21.64 ± 3.66

APTT (sec)

18.20 ± 3.43

19.14 ± 2.23

Table 1C_ 13-Week treatment_female rats_Hematological parameters (mean value ± SD of n animals):

Parameters

Dose level (mg/kg bw/day)

0 (n=10)

100 (n=10)

300 (n=10)

1000 (n=10)

RBC (104/μL)

817.5 ± 44.6

823.0 ± 46.4

853.2 ± 38.1

801.6 ± 44.6

HGB (g/dL)

15.14 ± 1.16

15.28 ± 0.58

15.82 ± 0.32

14.77 ± 0.64

HCT (%)

42.34 ± 2.64

42.62 ± 1.54

43.74 ± 1.02

40.87 ± 1.77

MCV (fL)

51.78 ± 1.41

51.87 ± 2.28

51.33 ± 1.96

51.04 ± 1.82

MCH (pg)

18.51 ± 0.65

18.60 ± 0.80

18.58 ± 0.60

18.46 ± 0.57

MCHC (g/dL)

35.73 ± 0.82

35.84 ± 0.22

36.18 ± 0.44

36.14 ± 0.34

Reticulocyte (%)

3.113 ± 1.530

2.595 ± 0.558

2.650 ± 0.500

2.515 ± 0.785

Platelet (104/μL)

105.60 ± 20.45

91.49 ± 8.88

92.25 ± 14.38

98.38 ± 14.24

WBC (102/μL)

61.45 ± 32.01

47.40 ± 16.76

59.40 ± 16.69

53.26 ± 20.82

Neutrophil (102/μL)

14.55 ± 22.20

6.52 ± 1.85

8.06 ± 3.28

6.81 ± 3.58

Lymphocyte(102/μL)

43.67 ± 13.33

38.41 ± 15.38

48.41 ± 15.12

43.88 ± 17.95

Monocyte(102/μL)

2.24 ± 1.34

1.65 ± 0.72

1.68 ± 0.63

1.77 ± 0.87

Eosinophil(102/μL)

0.98 ± 0.37

0.81 ± 0.49

1.23 ± 0.64

0.79 ± 0.40

Basophil(102/μL)

0.01 ± 0.03

0.01 ± 0.03

0.02 ± 0.04

0.01 ± 0.03

PT (sec)

15.56 ± 0.79

15.69 ± 0.98

15.82 ± 0.70

15.61 ± 0.76

APTT (sec)

11.54 ± 1.30

12.14 ± 1.23

12.41 ± 0.82

13.47 ± 1.45**

* : p<0.0; **: p< 0.01

Table 1D_ 13-Week + 14-Day recovery_female rats_Hematological parameters (mean value ± SD of n animals):

Parameters

Dose level (mg/kg bw/day)

0 (n=5)

1000 (n=5)

RBC (104/μL)

796.6 ± 38.9

823.8 ± 20.4

HGB (g/dL)

14.86 ± 0.38

15.14 ± 0.78

HCT (%)

41.82 ± 0.89

42.06 ± 1.69

MCV(fL)

52.58 ± 1.88

51.06 ± 1.65

MCH (pg)

18.68 ± 0.67

18.38 ± 0.61

MCHC (g/dL)

35.52 ± 0.22

35.98 ± 0.70

Reticulocyte (%)

3.280 ± 0,647

3.346 ± 0.436

Platelet (104/μL)

94.02 ± 11.54

105.18 ± 20.06

WBC (102/μL)

46.16 ± 16.09

54.16 ± 22.91

Neutrophil (102/μL)

8.16 ± 2.32

7.94 ± 3.00

Lymphocyte(102/μL)

35.40 ± 17.28

43.18 ± 19.98

Monocyte(102/μL)

1.64 ± 0.21

1.98 ± 0.75

Eosinophil(102/μL)

0.94 ± 0.25

1.06 ± 0.26

Basophil(102/μL)

0.02 ± 0.04

0.00 ± 0.00

PT (sec)

16.00 ± 0.51

16.92 ± 1.37

APTT (sec)

12.38 ± 0.80

11.90 ± 1.39

Table 2A_ 13-Week treatment_male rats_Clinical chemical parameters (mean value ± SD of n animals):

Parameters

Dose level (mg/kg bw/day)

0 (n=10)

100 (n=10)

300 (n=9)

1000 (n=10)

AST (IU/L)

61 ± 8.0

57.8 ± 7.5

56.0 ± 6.9

66.1 ± 8.7

ALT (IU/L)

29.5 ± 4.5

30.7 ± 4.1

27.6 ± 2.8

32.7 ± 7.5

ALP (IU/L)

304.0 ± 63.4

313.1 ± 43.6

339.4 ± 79.1

477.7 ± 114.5**

γ-GTP (IU/L)

0.24 ± 0.23

0.45 ± 0.29

0.42 ± 0.20

0.53 ± 0.18*

T-Bil (mg/dL)

0.060 ± 0.013

0.047 ± 0.009

0.048 ± 0.011

0.049 ± 0.010

Glucose (mg/dL)

162.4 ± 11.7

163.3 ± 16.2

174.9 ± 18.1

179.6 ± 16.9

T-Cho (mg/dL)

53.9 ± 11.1

52.3 ± 5.2

48.0 ± 8.3

38.6 ± 9.4**

TG (mg/dL)

60.1 ± 30.2

72.7 ± 45.7

60.9 ± 20.3

61.6 ± 24.4

TP (g/dL)

5.60 ± 0.16

5.63 ± 0.14

5.59 ± 0.15

5.57 ± 0.24

UN (mg/dL)

13.72 ± 0.82

14.17 ± 1.39

14.16 ± 1.92

14.41± 2.59

Crea (mg/dL)

0.307± 0.048

0.312± 0.019

0.290± 0.027

0.283± 0.054

Na (mEq/L)

141.2± 1.5

142.5± 0.7

142.3± 1.3

142.5± 1.4

K (mEq/L)

4.737± 0.207

4.672± 0.228

4.781± 0.178

4.835± 0.283

Cl (mEq/L)

104.3± 1.6

104.5± 1.1

104.9± 1.1

104.7± 1.3

Ca (mg/dL)

9.59± 0.26

9.88± 0.37

9.73± 0.25

10.01± 0.30*

IP (mg/dL)

6.64± 0.75

6.60± 0.67

6.58± 0.76

7.12± 0.71

A/G

0.877± 0.065

0.859± 0.057

0.878± 0.060

0.894± 0.076

Albumin (%)

46.72± 1.80

46.16± 1.72

46.70± 1.75

47.13± 2.04

α1-G (%)

22.32± 2.60

24.03± 2.29

23.53± 1.73

22.02± 1.74

α2-G (%)

9.52± 1.36

9.24± 0.92

8.83± 1.15

9.27± 1.11

β-G (%)

16.21± 0.94

15.75± 0.69

15.48± 0.83

16.57± 1.31

γ-G (%)

5.23± 0.70

4.82± 0.96

5.46± 0.95

5.01± 1.63

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

Table 2B_ 13-Week + 14-Day recovery_male rats_Clinical chemical parameters (mean value ± SD of n animals):

Parameters

Dose level (mg/kg bw/day)

0 (n=5)

1000 (n=5)

AST (IU/L)

62.6 ± 6.3

80.8 ± 31.2

ALT (IU/L)

28.4 ± 3.2

39.6 ± 21.0

ALP (IU/L)

257.6 ± 53.7

300.6 ± 113.4

γ-GTP (IU/L)

0.30 ± 0.14

0.46 ± 0.23

T-Bil (mg/dL)

0.072 ± 0.028

0.060 ± 0.012

Glucose (mg/dL)

160.8 ± 11.0

164.2 ± 36.0

T-Cho (mg/dL)

64.2 ± 9.8

55.4 ± 9.0

TG (mg/dL)

63.4 ± 29.2

55.2 ± 12.7

TP (g/dL)

5.58 ± 0.22

5.50 ± 0.07

UN (mg/dL)

14.50 ± 1.43

14.48± 1.51

Crea (mg/dL)

0.334± 0.051

0.320± 0.029

Na (mEq/L)

143.2± 0.8

143.4± 2.1

K (mEq/L)

4.700± 0.274

4.462± 0.315

Cl (mEq/L)

107.4± 1.7

107.2 ± 2.6

Ca (mg/dL)

9.70 ± 0.45

9.60 ± 0.25

IP (mg/dL)

5.78 ± 0.63

6.10 ± 0.62

A/G

0.876 ± 0.065

0.864 ± 0.059

Albumin (%)

46.68 ± 1.83

46.30 ± 1.62

α1-G (%)

22.32 ±1.68

24.10± 2.62

α2-G (%)

9.24± 0.50

8.60± 0.57

β-G (%)

16.36± 0.71

15.70± 0.73

γ-G (%)

5.40± 1.15

5.30± 1.10

Table 2C_ 13-Week treatment_female rats_Clinical chemical parameters (mean value ± SD of n animals):

Parameters

Dose level (mg/kg bw/day)

0 (n=10)

100 (n=10)

300 (n=10)

1000 (n=10)

AST (IU/L)

112.3 ± 53.9

84.2 ± 25.3

64.2 ± 13.8*

72.2 ± 25.8

ALT (IU/L)

49.3 ± 28.6

32.1 ± 14.3

27.2 ± 6.4*

23.4 ± 7.6**

ALP (IU/L)

150.0 ± 82.1

162.6 ± 70.3

178.2 ± 72.2

162.2 ± 57.0

γ-GTP (IU/L)

2.01 ± 4.64

0.66 ± 0.17

1.05 ± 0.37

0.70 ± 0.37

T-Bil (mg/dL)

0.082 ± 0.037

0.074 ± 0.017

0.065± 0.014

0.063 ± 0.014

Glucose (mg/dL)

159.2 ± 18.7

155.1 ± 19.8

149.4 ± 20.8

154.6 ± 15.2

T-Cho (mg/dL)

62.3 ± 8.6

65.5 ± 13.1

60.1 ± 21.9

59.4 ± 7.2

TG (mg/dL)

15.4 ± 8.4

12.2 ± 4.3

10.6 ± 4.7

13.3 ± 6.2

TP (g/dL)

6.17 ± 0.47

6.15 ± 0.61

6.17 ± 0.22

6.06 ± 0.25

UN (mg/dL)

15.71 ± 1.58

16.18 ± 2.27

15.50 ± 2.40

16.64 ± 2.41

Crea (mg/dL)

0.368 ± 0.041

0.374± 0.033

0.353± 0.037

0.368± 0.047

Na (mEq/L)

142.4± 1.0

142.3± 1.5

143.0 ± 0.8

141.5 ± 1.8

K (mEq/L)

4.227 ± 0.380

4.184 ± 0.297

4.248 ± 0.271

4.372 ± 0.305

Cl (mEq/L)

105.7 ± 1.2

105.7 ± 1.8

106.1 ± 1.4

105.3 ± 1.8

Ca (mg/dL)

9.81 ± 0.27

9.94 ± 0.44

9.97 ± 0.20

10.09 ± 0.35

IP (mg/dL)

5.42 ± 1.30

5.65 ± 1.15

5.55± 0.88

6.12± 0.74

A/G

1.083± 0.224

1.142± 0.095

1.101± 0.106

1.092± 0.128

Albumin (%)

51.34± 6.88

53.24± 2.07 

52.31± 2.30

52.04± 2.75

α1-G (%)

17.94± 2.99

17.09± 1.90

16.91± 0.91

16.79± 1.55

α2-G (%)

9.14± 2.19

8.40± 1.63

8.29± 1.27

8.87± 1.11

β-G (%)

15.05± 2.04

14.23± 1.27

14.88± 0.85

15.21± 1.73

γ-G (%)

6.53 ± 1.07

7.04 ± 0.88

7.61 ± 1.37

7.09 ± 1.45

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

Table 2D_ 13-Week + 14-Day recovery_female rats_Clinical chemical parameters (mean value ± SD of n animals):

Parameters

Dose level (mg/kg bw/day)

0 (n=5)

1000 (n=5)

AST (IU/L)

121.0 ± 71.5

56.6 ± 12.6

ALT (IU/L)

52.8 ± 30.6

25.2 ± 6.2

ALP (IU/L)

108.0 ± 23.9

112.0 ± 23.5

γ-GTP (IU/L)

0.78 ± 0.51

0.64 ± 0.22

T-Bil (mg/dL)

0.086 ± 0.011

0.070± 0.020

Glucose (mg/dL)

155.2 ± 10.7

154.8 ± 26.4

T-Cho (mg/dL)

79.6 ± 13.2

69.0 ± 8.3

TG (mg/dL)

18.8 ± 11.5

21.4 ± 13.6

TP (g/dL)

6.50 ± 0.35

6.08 ± 0.22

UN (mg/dL)

15.82 ± 2.18

18.54 ± 1.77

Crea (mg/dL)

0.416 ± 0.054

0.422 ± 0.030

Na (mEq/L)

141.6 ± 1.1

142.4 ± 1.3

K (mEq/L)

4.178 ± 0.283

4.120 ± 0.298

Cl (mEq/L)

107.0± 1.4

107.2 ± 1.3

Ca (mg/dL)

10.12 ± 0.29

9.90 ± 0.07

IP (mg/dL)

3.78 ± 0.19

4.38 ± 0.37

A/G

1.204 ± 0.141

1.098 ± 0.051

Albumin (%)

54.46 ± 2.94

52.36 ± 1.17

α1-G (%)

17.84 ± 0.77

18.44± 1.34

α2-G (%)

7.18± 0.58

7.34± 0.29

β-G (%)

14.12± 1.22

14.96± 0.51

γ-G (%)

6.40± 1.76

6.90± 1.54

Table 3A_ 13-Week treatment_male rats_Organ weights (values as mean ± SD)

Dose level

N

Mean body weight (g) at treatment end

Liver

Kidneys

Heart

Lung

Spleen

Thymus

Salivary gland

Adrenals

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

0 mg/kg bw/d

10

552.9± 42.0

14.060±1.412

2.545± 0.236

3.258± 0.330

0.590±0.045

1.565±0.136

0.282±0.02

1.731±0.173

0.314±0.026

861.1±135.2

155.928±22.348

314.4±78.9

56.754±12.917

794±69.7

143.836±10.755

70.6±13.7

12.798±2.379

100 mg/kg bw/d

10

577.1± 54.9

14.849 ±2.068

2.567± 0.175

3.513± 0.405

0.608±0.04

1.613±0.152

0.281±0.032

1.697±0.135

0.293±0.019

802.9±134.2

139.319±20.149

506±142.5

87.01±20.94

888.3±88.2

154.135±10.254

68±11.6

11.743±1.244

300 mg/kg bw/d

9

570.0± 44.0

14.499 ±1.521

2.541± 0.116

3.599± 0.337

0.631±0.049

1.510±0.084

0.267±0.018

1.719±0.144

0.303±0.037

827.6±94.4

145.274±13.512

359.7±62.3

63.608±12.72

812.2±78.3

142.936±14.906

65.1±5.5

11.492±1.393

1000 mg/kg bw/d

10

561.1± 53.9

16.290 ±2.024*

2.902± 0.204**

3.742± 0.367*

0.669±0.054**

1.472±0.128

0.263±0.027

1.666±0.131

0.299±0.027

840.4±142.3

149.741±18.569

464±170.3*

82.403±28.22*

843±126.2

150.419±19.27

61.7±7.8

11.111±1.895

Dose level

N

Mean body weight (g) at treatment end

Pituitary gland

Thyroid

Testis

Epididymis

Prostate

Seminal vesicles

Brain

N; number of animals

AW; absolute weight (g or mg)

RW; relative weight (g/100g body weight or mg/100g)

* ; p<0.05

**;p< 0.01

 

AW (mg)

RW (mg/100g)

AW (mg) 

RW (mg/100g)

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

0 mg/kg bw/d

10

552.9± 42.0

11.47± 1.76

2.076± 0.281

22.49±2.62

4.103±0.678

3.083±0.800

0.565±0.158

1.359±0.270

0.248±0.057

837.9±177.1

151.300±27.309

2.092±0.178

0.381±0.048

2.237±0.093

0.405±0.025

100 mg/kg bw/d

10

577.1± 54.9

11.49± 2.09

1.986 ± 0.296

23.45±4.09

4.083±0.750

3.699±0.270

0.646±0.069

1.430±0.165

0.249±0.026

926.8±154.3

161.338±25.925

2.174±0.253

0.378±0.035

2.286±0.155

0.398±0.027

300 mg/kg bw/d

9

570.0± 44.0

11.36± 1.68

1.991 ± 0.241

21.97±3.19

3.863±0.554

3.501±0.327

0.618±0.069

1.411±0.091

0.250±0.023

824.6±158.8

145.242±29.781

2.311±0.325

0.408±0.069

2,224±0.085

0.393±0.035

1000 mg/kg bw/d

10

561.1± 53.9

11.68± 1.54

2.094 ± 0.301

23.91±4.34

4.252±0.612

3.588±0.263

0.643±0.058

1.407±0.101

0.252±0.015

925.5±167.8

165.480±29.833

2.221±0.256

0.399±0.064

2.205±0.102

0.396±0.035

Table 3B_13-Week + 14-Day recovery_male rats_ Organ weights (values as mean ± SD):

Dose level

N

Mean body weight (g) at the end of recovery

Liver

Kidneys

Heart

Lung

Spleen

Thymus

Adrenals

Pituitary gland

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

0 mg/kg bw/d

5

615.6±130.9

14.626±3.832

2.362±0.158

3.506±0.531

0.578±0.055

1.582±0.246

0.260±0.025

1.722±0.198

0.284±0.035

872.8±223.4

141.558±19.368

338.6±84.6

54.852±5.162

65.2±223.5

10.454±2.039

11.24±1.40

1.862±0.283

1000 mg/kg bw/d

5

576.0±39.0

15.060±0.823

2.620±0.131

3.698±0.260

0.642±0.046

1.578±0.098

0.274±0.021

1.748±0.139

0.304±0.030

872.0±67.4

151.360±4.828

353.6±100.9

61.216±16.798

70.8±14.4

12.428±3.223

11.50±1.06

2.000±0.180

Dose level

N

Mean body weight (g) at the end of recovery

Thyroid

Testis

Epididymis

Prostate

Seminal vesicles

Brain

N; number of animals

AW; absolute weight (g or mg)

RW; relative weight (g/100g body weight or mg/100g)

* ; p<0.05

**;p< 0.01

 

AW (mg)

RW (mg/100g)

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (mg)

RW (mg/100g)

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

0 mg/kg bw/d

5

615.6±130.9

21.18±5.25

3.522±0.985

3.478±0.329

0.582±0.110

1.430±0.239

0.238±0.047

852.4±372.8

140.624±60.790

2.336±0.364

0.390±0.082

2.306±0.074

0.388±0.079

1000 mg/kg bw/d

5

576.0±39.0

20.28±3.49

3.528±0.613

3.616±0.254

0.628±0.045

1.542±0.080

0.268±0.022

1018.8±75.4

177.570±18.205

2.636±0.2326

0.462±0.044

2.300±0.070

0.402±0.024

Table 3C_ 13-Week treatment_female rats_Organ weights (values as mean ± SD)

Dose level

N

Mean body weight (g) at treatment end

Liver

Kidneys

Heart

Lung

Spleen

Thymus

Salivary gland

Adrenals

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

0 mg/kg bw/d

10

290.8±14.3

7.272±0.852

2.498±0.244

1.920±0.183

0.661±0.060

0.9580±0.054

0.329±0.019

1.280±0.104

0.440±0.036

523.2±70.7

180.134±20.961

309.1±78.9

106.137±25.854

434.4±53.8

149.803±20.229

73.7±9.7

25.428±3.794

100 mg/kg bw/d

10

284.6±27.4

6.804±0.526

2.395±0.108

1.818±0.11

0.634±0.063

0.918±0.089

0.324±0.022

1.253±0.105

0.443±0.041

546.5±78

192.104±20.889

251.1±70.1

87.972±21.934

469.9±49.0

165.845±18.389

71.9±11.4

25.255±3.324

300 mg/kg bw/d

10

273.9±22.3

6.687±0.623

2.441±0.097

1.842±0.237

0.673±0.055

0.865±0.094*

0.315±0.026

1.197±0.074

0.438±0.045

496.5±40.2

181.910±15.920

309.5±103.5

114.098±40.765

456.2±52.6

167.681±24.523

65.1±7.1

23.927±3.430

1000 mg/kg bw/d

10

281.4±21.4

7.527±0.620

2.674±0.098

2.046±0.151

0.728±0.060*

0.876±0.057

0.312±0.015

1.194±0.088

0.425±0.026

521.0±62.1

184.955±14.893

324.8±77.8

115.662±28.205

461.0±36.8

164.419±15.097

77.0±10.0

27.541±4.482

Dose level

N

Mean body weight (g) at treatment end

Pituitary gland

Thyroid

Ovary

Uterus

Brain

N; number of animals

AW; absolute weight (g or mg)

RW; relative weight (g/100g body weight or mg/100g)

* ; p<0.05

**;p<0.01

 

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

AW (g)

RW (g/100g)

0 mg/kg bw/d

10

290.8±14.3

15.17±2.05

5.227±0.737

16.11±3.46

5.569±1.323

96.3±10.7

33.212±4.190

686.8±219.3

236.275±74.543

2.021±0.065

0.696±0.033

100 mg/kg bw/d

10

284.6±27.4

13.39±2.03

4.714±0.653

13.08±2.30

4.587±0.605

112.0±26.9

39.644±10.332

897.1±354

319.380±137.277

2.049±0.092

0.724±0.067

300 mg/kg bw/d

10

273.9±22.3

11.86±1.99**

4.349±0.768*

15.34±3.64

5.696±1.670

98.9±19.2

36.588±8.876

743.5±241.9

273.838±95.196

2.028±0.065

0.746±0.076

1000 mg/kg bw/d

10

281.4±21.4

13.81±2.15

4.922±0.765

15.43±3.60

5.491±1.235

101.4±22.6

36.112±7.567

676.4±295.3

240.593±99.50

2.052±0.084

0.733±0.053

Table 3D_ 13-Week + 14-Day recovery_female rats_ Organ weights (values as mean ± SD):

Dose level

N

Mean body weight (g) at the end of recovery

Liver

Kidneys

Heart

Lung

Spleen

Thymus

Adrenals

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (g)

RW (g/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

0 mg/kg bw/d

5

310.8±29.9

7.538±0.845

2.424±0.121

1.896±0.199

0.612±0.052

1.022±0.121

0.330±0.023

1.238±0.026

0.400±0.042

480.0±75.1

154.032±13.916

240.2±381

78.018±16.311

70.8±8.7

22.746±1.082

1000 mg/kg bw/d

5

310.2±41.2

7.198±0.671

2.330±0.114

1.936±0.091

0.632±0.056

0.916±0.083

0.298±0.038

1.186±0.097

0.386±0.040

554.6±26.3

180.608±17.691

304.6±104.1

97.966±30.921

68.0±12.3

22.166±4.416

Dose level

N

Mean body weight (g) at treatment end

Pituitary gland

Thyroid

Ovary

Uterus

Brain

N; number of animals

AW; absolute weight (g or mg)

RW; relative weight (g/100g body weight or mg/100g)

* ; p<0.05

**; p<0.01

 

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

AW (mg)

RW (mg/100g)

AW (g)

RW (g/100g)

0 mg/kg bw/d

5

310.8±29.9

17.44±2.91

5.678±1.232

13.84±3.18

4.418±0.666

93.6±21.9

29.982±5.751

744.2±205.4

238.626±60.913

2.112±0.023

0.684±0.065

1000 mg/kg bw/d

5

310.2±41.2

14.10±3.17

4.550±0.860

16.06±3.33

5.258±1.441

96.4±11.5

31.254±3.108

721.8±193.0

227.396±39.058

2.068±0.110

0.674±0.063

Table 4: Incidence and severity of treatment-related stomach changes at end of treatment period

Sex

Male

Female

Dose (mg/kg bw/day)

0

100

300

1000

0

100

300

1000

Number of animals

10

10

9

10

10

10

10

10

Infiltration, inflammatory cell, lamina propria, limiting ridge

slight

0

0

0

4

0

0

0

0

mild

0

0

0

0

0

0

0

0

moderate

0

0

0

0

0

0

0

0

severe

0

0

0

0

0

0

0

0

Total

0

0

0

4

0

0

0

0

Hyperkeratosis, limiting ridge

slight

0

0

0

4

0

0

0

2

mild

0

0

0

0

0

0

0

0

moderate

0

0

0

0

0

0

0

0

severe

0

0

0

0

0

0

0

0

Total

0

0

0

4

0

0

0

2

Infiltration, inflammatory cell, submucosa, glandular stomach

slight

0

0

0

5

0

0

0

0

mild

0

0

0

0

0

0

0

0

moderate

0

0

0

0

0

0

0

0

severe

0

0

0

0

0

0

0

0

Total

0

0

0

5

0

0

0

0

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 1) and consistent studies and is thus sufficient to fulfil the standard information requirements set out in Annex IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Two reliable studies are available on the repeated dose toxicity of RZETA. The data comprise an oral subchronic repeated dose toxicity study and an oral combined repeated dose and reproduction/ developmental screening study.

The test substance was tested in a reliable (Klimisch score 1) 90-day oral repeated dose toxicity study in the rat according to OECD guideline 408 and in compliance with GLP (Tosoh corporation, 2017). Ten Sprague Dawley rats per sex and dose were treated via gavage with 100, 300, and 1000 mg/kg bw/day, respectively for 90 consecutive days. The control group received the vehicle, distilled water. Additional recovery groups consisted of five rats per sex and dose treated either with the vehicle or with the test item at 1000 mg/kg bw/day and were kept for a 14-day observation period after the end of administration. In addition to the toxicological endpoints required by the OECD guideline, some reproduction endpoints were included into the study (i.e. estrous cyclicity and sperm analysis).

No treatment-related effects were noted in clinical observation, detailed clinical observation, functional observations, body weight, food consumption, urinalysis, hematology, biochemistry, estrous cyclicity, sperm analysis or necropsy in any test substance group. In organ weight, the absolute and relative liver and kidney weight was significantly increased in females at 1000 mg/kg bw/day at the end of administration period. This was considered not toxicologically significant because histopathological examination revealed no effects of the test substance administration in the respective organs or any clinical chemistry findings indicative of impaired organ function. Histopathological examination revealed local signs of irritation, i.e. slight infiltration of inflammatory cells in the lamina propria at the limiting ridge of the stomach, hyperkeratosis at the limiting ridge of the stomach, and slight infiltration of inflammatory cells in the lamina propria at the limiting ridge of the stomach in males and females in the 1000 mg/kg group. This change was not observed at the end of recovery period, which showed the potential for recovery.

In conclusion, no adverse effects on systemic toxicity after repeated exposure were observed. Therefore a NOAEL of ≥ 1000 mg/kg bw/day was deduced for systemic toxicity in male and female rats. A NOAEL of 300 mg/kg bw/day was derived for local effects in males and females due to the histopathological findings in the stomach.

Supporting study:

A combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (Tosoh corporation, 2014) was performed. Twelve Sprague Dawley rats per sex and dose were treated via gavage with 100, 300, and 1000 mg/kg bw/day, respectively. The control group received the vehicle distilled water. Male animals were treated totally for 42 days, starting 14 days before the mating period. Females were treated for 14 days before mating and during the mating period until successful copulation, and during gestation through day 4 after parturition. For the control and 1000 mg/kg bw/day groups, 5 out of 12 males were selected to investigate the recovery for 14 days after administration. 10 non-mated females per control and 1000 mg/kg bw/day dose group were separately set, and 5 of them were necropsied at the end of the 42-day administration, and the other 5 females were investigated for recovery for 14 days.

The doses were selected on the basis of data from a preliminary study, in which 4 animals per sex and dose (100, 300, and 1000 mg/kg bw/day) were treated orally for 14 days. No mortality, clinical findings, body weight changes or changes in food consumption were observed. In urinalysis, the number of females with protein-positive urine increased in the 1000 mg/kg bw/day group. Hematological examination revealed low hematocrit and hemoglobin concentration in males in the 1000 mg/kg bw/day group. Biochemical examinations revealed high triglyceride concentrations in females in the 1000 mg/kg bw/day group. During necropsy no abnormal findings in males or females in any treated group were observed. In organ weight determinations, relative kidney weight was found increased in females in the 1000 mg/kg bw/day group.

In the main study no treatment-related effects were noted in clinical observation, detailed clinical observation, functional observations, body weight, food consumption, urinalysis, hematology, biochemistry, or necropsy in males or females in any test substance group. In organ weight, the absolute and relative liver and kidney weight was significantly increased in females in the 1000 mg/kg bw/day at the end of administration period. This was considered not toxicologically significant because histopathological examination revealed no effects of the test substance administration in the respective organs. Histopathological examination revealed local signs of irritation, i.e. hyperplasia of squamous cells at limiting ridge of the stomach with significantly high incidence in males in the 1000 mg/kg bw/day group at the end of administration period. This change was not observed at the end of recovery period, which showed the potential for recovery.

In conclusion, no adverse effects on systemic toxicity after repeated exposure were observed. Therefore a NOAEL of ≥ 1000 mg/kg bw/day was deduced for systemic toxicity in the OECD 422 study in male and female rats. A NOAEL of 300 mg/kg bw/day was derived for local effects for males due to the histopathological findings in the stomach.

 

Overall conclusion for repeated dose toxicity

Taken together, the available data on subchronic repeated dose toxicity for RZETA do not indicate systemic adverse effects up to and including the recommended limit value. A NOAEL of 300 mg/kg bw/day was derived for local effects in males and females due to the histopathological findings in the stomach. This is supported by the data obtained in the combined repeated dose and reproduction/ developmental screening study. Therefore, as the available data did not identify any hazard for repeated dose toxicity, RZETA is not considered to be hazardous following repeated exposure.

Justification for classification or non-classification

The available data on repeated oral dose toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.