Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-12-23 to 2009-02-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
, adopted 1996-03-22
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2007-04-20
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): SymHelios 1031
- Chemical name: (2E/Z)-2-benzylidene-5,6-dimethoxy-3,3-dimethyl-indan-1-one
- Physical state: yellow powder
- Storage condition of test material: ambient temperature, dark, dry and in original container
- Melting point: 105 - 107 °C
- Water solubility: 1.95 mg/L (20 °C)
-Relative density: 1.20 at 24.6 °C
No further information on the test material was stated.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: males: 8-9 weeks; females: 8-9 weeks
- Weight at study initiation: males: 261.7 - 316.5 g; females: 194.6 - 236.1 g
- Housing: except during the mating period (1 male / 1 female were placed in one cage), the parental males and females were kept individually in MAKROLON cages (type III) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 15 cm. Granulated textured wood (Brandenburg, 49424 Goldenstedt-Arkeburg, Germany) was used as bedding material.
- Diet (ad libitum): commercial ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous hydroxypropylmethylcellulose gel
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle (0.5% aqueous hydroxypropylmethylcellulose gel) to the appropriate concentrations and was administered orally at a constant application volume of 5 mL/kg b.w./day. The control animals received the vehicle (0.5% aqueous hydroxypropylmethylcellulose
gel) in the same way. The amount of the test item was adjusted to the animal's current body weight daily.
The test item suspensions were freshly prepared every day.

VEHICLE:
Batch no.: 07K01-N04 (Fagron GmbH, 22885 Barsbüttel, Germany
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each test item that was prepared with the vehicle, tests by appropriate analytical methods were conducted to determine the concentration, stability and homogeneity of the test item in the solution.
For the analysis of the test item-vehicle preparations, samples of approximately 10 mL were taken at the following times and were stored at -20°C or colder until shipment for analysis.

At study initiation:
- Analysis of stability and concentration: immediately after preparation of the suspensions as well as 8 and 24 hours after storage of the test item preparations at room temperature (3 samples/dose level group); total number of samples: 9.
- Analysis of homogeneity: at the start of administration, during (middle) administration and before administration to the last animal of each dose level group (3 samples/dose level group).

At study termination:
- Analysis of concentration: during treatment with the test item always before administration to the last animal/dose group (1 sample/dose group); total number of samples: 3.
- 4 further vials containing the test item or vehicle of test days 1 and 28 as well as 200 mL vehicle (0.5% Methocel) were prepared.

The results indicate that the content of SymHelios 1031 in the dosing suspensions corresponded reasonably well with the nominal concentrations in the concentration range of 20 to 200 mg/mL, so that maintenance of the dosing regimen can be concluded. Moreover, the dosing suspensions were shown to be stable and homogenous during the application period. In addition, the stability of frozen samples over a period of 73 days was confirmed, a period ranging from sampling at LPT until the time of further sample dilution at the analytical test facility.

For the results of the analysis please see also Section 8 Analytical methods (Entry: Analytical methods.001)
Duration of treatment / exposure:
Main study males: 2 weeks prior to mating, during the mating period and approximately 2 weeks post mating at least until the minimum total dosing period of 28 days had been completed (up to and including the day before sacrifice).
Main study females: throughout the study beginning 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.
Recovery animals (not mated animals): treated in the same manner as the main study animals and kept without treatment for at least 14 days after the first scheduled sacrifice of main study dams.
Frequency of treatment:
once daily, 7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, and 1000 mg/kgb.w./day
Basis:
other: actual administered
No. of animals per sex per dose:
Main study: 40 males / 40 females divided into groups as followed:
Control group: 10 males / 10 females
100 mg/kg b.w./day: 10 males / 10 females
300 mg/kg b.w./day: 10 males / 10 females
1000 mg/kg b.w./day: 10 males / 10 females
14-day recovery period: 10 males / 10 females divided into groups as followed:
Control group: 5 males / 5 females
1000 mg/kg b.w./day: 5 males / 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels for this study were selected based on the results of a dose-range-finding study. In this study, the test item was applied orally at concentrations of 100, 300 or 1000 mg SymHelios 1031/kg b.w., once daily for 7 consecutive days.
- No mortality was noted in this dose-range-finding study. No changes were noted on behaviour, external appearance, faeces, body weight and food consumption as well as at necropsy.
- Based on the results the following dose-levels were selected for the combined repated dose toxicity and reproduction/developmental toxicity screening study: 0, 100, 300 and 1000 mg SymHelios 1031/kg b.w./day.
- Recovery animals: recovery animals were treated with the test item in the same manner as the main study animals. However, they were not used for the assessment of reproduction / developmental data and they were not mated.
Positive control:
No data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS (Parental animals): Yes
- Time schedule: each animal was observed for clinical signs at least once daily throughout the test period. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays, animals were checked regularly from 7:00 a.m. to 11:00 noon with a final check performed at approximately 3:30 p.m.
- Further checks were made early in the morning and again in the afternoon of each working day to identify dead or moribund animals. This allowed post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m.
- Cage side observations: behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity, including mortality, were recorded. Any signs of illness or reaction to treatment were recorded for each individual animal. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS (Parental animals): Yes
- Time schedule: once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals.
- These observations were made outside the home cage in a standard arena at the same time, each time. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT (Parental animals): Yes
- Time schedule for examinations: males and females were weighed on the first day of dosing, weekly thereafter and at study termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum) and on day 4 post-partum during lactation. Body weights were recorded individually for each adult animal.

FOOD CONSUMPTION AND COMPOUND INTAKE (Parental animals)(if feeding study):
- Food consumption for each animal determined: Yes; the quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period with the execution of the mating period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week.

FOOD EFFICIENCY (Parental animals): No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)(Parental animals): Yes
- Time schedule for examinations: drinking water consumption was monitored daily by visual appraisal throughout the study.

OPHTHALMOSCOPIC EXAMINATION (Parental animals): No

HAEMATOLOGY(Parental animals) : Yes
- Blood samples were taken under light ether anaesthesia from the retrobulbar venous plexus from animals fasted overnight at necropsy.
- At the end of the pre-mating period: 5 male and 5 female main study animals randomly selected of each group.
- At the end of the recovery period (on the first day of dissection): all recovery animals.
- Parameters: haemoglobin content, erythrocytes, leucocytes, reticulocytes, platelets, differential blood count (relative/absolute: neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes, monocytes and large unstained cells), haematocrit value, thromboplastin time, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular haemoglobin, and mean corpuscular haemoglobin concentration.

CLINICAL CHEMISTRY (Parental animals): Yes
- Blood samples were taken under light ether anaesthesia from the retrobulbar venous plexus from animals fasted overnight at necropsy.
- At the end of the pre-mating period: 5 male and 5 female main study animals randomly selected of each group.
- At the end of the recovery period (on the first day of dissection): all recovery animals.
- Parameters: albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea (blood urea), calcium, chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase, and aspartate aminotransferase.

URINALYSIS (Parental animals): No

NEUROBEHAVIOURAL EXAMINATION (parental animals): Yes
- Time schedule for examinations: screening of sensory reactivity to stimuli of different types as well as the assessment of grip strength and motor activity assessment were conducted towards the end of the main study, 1 - 2 hours after dosing and before any blood sampling for laboratory examinations. (Observation screening: males shortly before scheduled sacrifice; females during lactation, shortly before scheduled kill).
- Dose groups examined: five males and five females randomly selected from each main study group.
- Battery of functions tested:
1. Observation screening: righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, pilo-erection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function.
2. Functional tests: grip strength and locomotor activity (two types of movement: stereotype, static movement and active locomotion).
Sacrifice and pathology:
GROSS PATHOLOGY (Parental animals): Yes
- Male animals were sacrificed after a total dosing period of 30 days. Dams with offspring were sacrificed on day 4 post-partum, or shortly thereafter. Females that did not show any evidence of copulation were sacrificed 24 days after the last day of the mating period.
- Dissection of all animals allocated to the recovery period started on test day 56.
- At the time of sacrifice or death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system. Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI (1964).

The number of corpora lutea and implantation sites was recorded in the female adult animals:
Corpora lutea
- number per dam
- absolute number per group
- mean per group
Implantations
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group

The animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis, exsanguinated, weighed, dissected and inspected macroscopically under the direction of a pathologist. All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.

The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

HISTOPATHOLOGY (Parental animals): Yes
- the following organs or parts of organs of all adult animals were fixed in 7% Formalin (testes and epididymides were fixed in Bouin's fixative): epididymis (2), ovary (2), testicle (2), vagina, mammary gland, prostate, and uterus (incl. cervix and oviducts), adrenal gland (2), bone marrow (os femoris), brain (cerebrum, cerebellum, brain stem), gross lesions observed, heart (right and left ventricle, septum), inlarge testine (colon, rectum), small intestine (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method), kidney and ureter (2), liver, lungs (with mainstem bronchi and bronchioles [preserved by inflation with fixative and then immersion]), lymph node (cervical) (1), lymph node (mesenteric) (1), nerve (sciatic), oesophagus, seminal vesicle, spinal cord (3 sections), spleen, stomach, thymus, thyroid (incl. parathyroids), tissue masses or tumours (including regional lymph nodes), tongue (incl. base), trachea (incl. larynx), and urinary bladder.
- Any other organs displaying macroscopic changes were also preserved.
- The afore-listed organs of the selected parental animals of groups 1 and 4 (5 male and 5 female animals per group) and all recovery animals (in total 20 recovery animals) were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining.
- Sections of the testes from the 5 male animals of groups 1 and 4 (control and high dose group) were also stained with PAS and a rough qualitative sperm staging was performed.
- In addition, frozen sections of the heart, liver and one kidney were made and stained with scarlet R. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plan of section and in all cases where they were noted as grossly enlarged.
- Due to findings in the liver of the groups 4 male animals, the livers of 5 male animals of the low and intermediate dose level groups (groups 2 and 3) were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining.
Other examinations:
Organ weights (parental animals):
- the weights of the following organs of all adult male animals were determined before fixation: epididymis (2) and testicle (2).
- the weights of the following organs of 5 adult male and 5 adult female animals (randomly selected of each main study group) and all recovery animals were determined before fixation: adrenal gland (2), kidney (2), spleen, brain, liver, thymus, heart and ovary; adrenal glands, gonads and kidneys were weighed individually and identified as left or right.
Statistics:
The test item-treated groups 2 to 4 were compared with the control group 1.
The following statistical methods were used:
- STUDENT's t-test (All numerical functional tests(p ≤ 0.01).

- Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control. Biometrics 482-491 (Sep 1964) (Body weight / food consumption / haematology / clinical biochemistry / organ weights (absolute and relative) (p ≤ 0.01).

- For all numerical values homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.

- In case of heterogeneity of variances, the STUDENT's t-test was carried out, limit of significance was p ≤ 0.01.

- Exact test of R.A. FISHER (histopathology (p ≤ 0.05).

- For the comparison of classification measurements (for example the fertility index) the FISHER's exact test, n < 100 or the chi2-test with Yates' correction for continuity, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01) were employed.

- These statistical procedures were used for all data. The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY (Parental animals)
Main study / recovery:
- No mortality occurred during the treatment period at any of the tested dose levels or in the animals scheduled for the 14-day recovery period.
- None of the animals treated with either 100, 300 or 1000 mg SymHelios 1031/kg b.w./day revealed any signs of systemic toxicity.
- Results of the detailed clinical observations performed weekly during the treatment period until the end of the 14-day recovery period did not reveal any behavioural changes.
- Faeces of control and test item-treated animals showed a normal consistency during the entire experimental period.

BODY WEIGHT AND WEIGHT GAIN (Parental animals)
Main study:
- Males (pre-mating and mating period): no test item-related influence was noted on the body weight of the male rats treated with either 100, 300 or 1000 mg SymHelios 1031/kg b.w./day during the pre-mating and mating period.
- Body weight at autopsy on test day 31 was not influenced for the male animals compared to the control group.
- Females (pre-mating, mating, gestation and lactation period): no test item-related influence was noted on the body weight of the female rats
treated with either 100, 300 or 1000 mg SymHelios 1031/kg b.w./day during the pre-mating, mating, gestation or lactation period.
- Body weight at autopsy on test days 42 to 53 was not influenced for the female animals compared to the control group.
Recovery period:
- No test item-related changes were noted for the animals of the 14-day recovery period previously treated with 1000 mg SymHelios 1031/kg b.w./day for 6 weeks.
- Body weight at autopsy on test day 56 (recovery sacrifice) was not influenced for the animals of both sexes compared to the control group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study) (Parental animals)
Main study:
- Males (pre-mating period): no test item-related influence was observed on the food consumption of the male animals treated with either 100, 300 or 1000 mg SymHelios 1031/kg b.w./day during the pre-mating period.
- Females (pre-mating, gestation and lactation period): no test item-related influence was observed on the food consumption of the female animals treated with either 100, 300 or 1000 mg SymHelios 1031/day during the pre-mating, gestation or lactation period.
Recovery period:
- No test item-related changes were noted for the animals of the 14-day recovery period previously treated with 1000 mg SymHelios 1031/kg b.w./day for 6 weeks.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study) (Parental animals)
Main Study/recovery period:
- Visual appraisal of the drinking water consumption revealed no differences between the control and the test item-treated animals of both sexes.

HAEMATOLOGY (Parental animals)
Main study/Recocery period:
- No test item-related changes were noted for the haematological parameters in male and female rats treated with 100, 300 or 1000 mg SymHelios 1031/kg b.w./day compared to the control.
- No test item-related influence was noted for the haemoglobin content, the number of erythrocytes, leucocytes, reticulocytes and platelets, the haematocrit value, the thromboplastin time and the activated partial thromboplastin time, the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC) compared to the control. Evaluation of the differential blood count revealed no test item-related changes.

CLINICAL CHEMISTRY (Parental animals)
Main study/recovery period:
- No test item-related changes were noted on biochemical parameters in male and female rats treated with 100, 300 or 1000 mg SymHelios 1031/kg b.w./day compared to the control.
- No test item-related influence was noted for the serum levels of albumin, creatinine, globulin, the albumin/globulin ratio, bile acids, cholesterol (total), glucose, protein (total), urea in blood, calcium, chloride, potassium, sodium and the serum activities of ALAT, aP and ASAT.

NEUROBEHAVIOUR (Parental animals).
Main study:
- The parameters of functional observations examined in test weeks 5 (males) or 6 (females) did not reveal any test item-related influence for the rats treated with 100, 300 or 1000 mg SymHelios 1031/kg b.w./day.
- Fore- and hindlimb strength or spontaneous motility were not influenced by the test item in any of the main study rats at any dose level.

ORGAN WEIGHTS (Parental animals)
Main study:
- No test item-related influence was noted for the relative and absolute organ weights of the male and female rats treated with 100 or 300 mg SymHelios 1031/kg b.w./day and of the high dosed female animals at 1000 mg SymHelios 1031/kg b.w./day.
- The following organ changes were noted in the male rats treated with 1000 mg SymHelios 1031/kg b.w./day on test day 31 which are regarded to be test itemrelated: liver (relative and absolute)-
Recovery period:
- No changes in organ weights were noted in the male and female rats of the recovery group previously treated with 1000 mg SymHelios 1031/kg b.w./day for 6 weeks compared to the control.
- Increases in relative and absolute liver weights noted in the high dosed male main study animals were not recorded in the animals at the end of the 14-day recovery period.

GROSS PATHOLOGY (Parental animals)
Main study:
- Macroscopic inspection at necropsy on test day 31 (males) or on test days 42 to 53 (females) revealed no test item-related changes in the organs or tissues after treatment with either 100, 300 or 1000 mg SymHelios 1031/kg b.w./day.
- The gastric mucosa of one female rat treated with the intermediate dose of 300 mg/kg revealed a yellowish discolouration. This finding is regarded to be spontaneous due to the low incidence. None of the high dosed rats revealed any pathological changes at macroscopic inspection.
Recovery period:
- Recovery dissection afterer a 14-day treatment-free period did not reveal any test item-related changes in the organs or tissues of the animals previously treated with 1000 mg SymHelios 1031/kg b.w./day.

HISTOPATHOLOGY: NON-NEOPLASTIC (Parental animals)
- The examination revealed a mild diffuse hepatocellular hypertrophy in the liver of the high-dosed male rats, treated with 1000 mg SymHelios 1031/kg b.w./day for 6 weeks which is considered to be test item-related. This finding correlated with the increase of the liver weight. The male animals of groups 2 and 3 treated with 100 or 300 mg SymHelios 1031/kg b.w./day for 6 weeks and the high-dosed female animals (1000 mg SymHelios 1031/kg b.w./day) showed no morphological difference in comparison to the control rats.
- A complete reversibility was noted for the liver of the high-dosed male rats at the end of the recovery period of 14 days.
- The histomorphological examination of other organs did not reveal any microscopical morphological changes which are considered to be related to the treatment with the test item. Type, incidence and severity of the lesions recorded were not increased in the test item-treated animals as compared to the control animals.

Effect levels

Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the present test conditions, the following effect levels were noted for SymHelios 1031 administered during the pre-mating and mating periods to parental males, and during the premating, mating, gestation and lactation periods until day 3 post-partum to parental female animals, and to the animals scheduled for a 14-day recovery period:
Increased relative and absolute liver weights were noted for the high dosed male rats treated with 1000 mg/kg b.w./day. Histopathology revealed mild diffuse hepatocellular hypertrophy in the liver of the high dosed male rats. At the end of the recovery period, a complete reversibility was noted for liver weights and histomorphological changes of the high dosed male rats.
Hence, the no-observed-effect level (NOEL) was 300 mg/kg b.w./day, p.o. via gavage.