Bis(2-ethylhexyl) 2-(4-hydroxy-3,5-dimethoxybenzylidene)malonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004-11-04 to 2005-04-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 27 July 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

30 September 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals: Rat, HanRcc: WIST(SPF)
Rationale: Recognized by international guidelines as a
recommended test system.

Total Number of Animals:
Group 1 and 4: 10 males and 10 females
Group 2 and 3: 5 males and 5 females
Age (at Delivery): 6 weeks
Body Weight Range (at Acclimatization): Males: 138.6 g to 160.5 g (mean: 149.7 g)
Females: 115.5 g to 130.2 g (mean: 122.8 g)

Identification:
Acclimatization: Cage card and tail mark (later ear tattoo)
Treatment: Cage card and individual ear tattoo
Randomization: Computer-generated random algorithm.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRY

CONDITIONS
Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environmental conditions (temperature range: 22 ± 3 °C; relative humidity range: 10-70 %). Humidity was occasionally lower than this range. This is considered not to have any influence on the study. There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.

ACCOMODATION
In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).

DIET
Pelleted standard Kliba Nafag 3433 (batch no. 63/04) rat / mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.

WATER
Community tap-water was available ad libitum in water bottles. None of the contaminants analyzed in the water and diet is considered to have been present at a concentration that would have affected the validity of the results.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on oral exposure:

DOSE FORMULATION
The dose formulations were prepared weekly.
The test substance was weighed into a tared glass container on a suitable precision balance and the vehicle, PEG 300, was added to give the appropriate final concentration of the test item. The mixtures were prepared using a magnetic stirrer and stored at room temperature (15 - 25 °C).
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSE FORMULATIONS
Concentration, homogeneity and stability of dose formulations were determined in samples taken after experimental start using a HPLC method with UV detection. Concentration and homogeneity of dose formulations were also determined in samples taken during week 3 of the treatment.

Duration of treatment / exposure:

Duration of Treatment: 28 days
Duration of Recovery: 15 days

Frequency of treatment:

Frequency of Administration: Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

800 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Group 1: 0 mg/kg body weight: 10
Group 2: 40 mg/kg body weight: 5
Group 3: 200 mg/kg body weight: 5
Group 4: 800 mg/kg body weight: 10

Control animals:

yes, concurrent vehicle

Details on study design:

see executive summary

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS (Viability/Mortality): Yes
- Time schedule: Twice daily

DETAILD CLINICAL OBSERVATIONS:
- Time schedule: Acclimatization Period Once before the first test item exposure
Treatment Period Once weekly (Allocation A and B)
Recovery Period Not performed during recovery

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly

FOOD CONSUMPTION: Yes
- Time Schedule: once weekly

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: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks (main treatment groups) and after 6 weeks (recovery groups)
- Anaesthetic used for blood collection: Yes (after light Isoflurane anesthesia)
- Animals fasted: Yes (for approx. 18 hours)
- How many animals: all animals
- Parameters examined: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Platelet (thrombocyte) count, Reticulocyte count, Reticulocyte maturity index, Methemoglobin, Total leukocyte count, Differential leucocyte count, Coagulation: Thromboplastin time, Activated partial thromboplastin time.
Clinical laboratory data are expressed, with a few exceptions, in general accordance with the International System of Units (SI).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks (main treatment groups) and after 6 weeks (recovery groups)
- Animals fasted: Yes (for approx. 18 hours)
- How many animals: all animals
- Parameters examined.: Glucose, Urea, Creatinine, total Bilirubin, total Cholesterol, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, total Protein, Albumin, Globulin, Albumin/Globulin ratio, Bile acids.
Clinical laboratory data are expressed, with a few exceptions, in general accordance with the International System of Units (SI).

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 weeks (main treatment groups) and after 6 weeks (recovery groups)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume (18 hours), Specific gravity, color, appearance, pH, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes.
Clinical laboratory data are expressed, with a few exceptions, in general accordance with the International System of Units (SI).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4
- Dose groups that were examined: all dose groups examined
- Battery of functions tested: grip strength / locomotor activity

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Sacrifice and pathology:

Pathology

Necropsy
Sacrifice: after 4 Weeks (main Groups)
after 6 Weeks (Recovery Groups)
All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded.
All animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution except for eyes with optic nerve and harderian gland which were fixed in Davidson's solution or epididymides and testes which were fixed in Bouin’s solution:
Adrenal glands
Aorta
Bone (sternum, femur including joint)
Bone marrow (femur)
Brain - including section of medulla/pons,
cerebral and cerebellar cortex
Cecum
Colon
Duodenum
Epididymides (fixed in Bouin's solution)
Esophagus
Eyes w/optic nerve (fixed in Davidson's
solution)
Harderian gland (fixed in Davidson's solution)
Heart
Ileum, with Peyer's patches
Jejunum with Peyer's patches
Kidneys
Larynx
Lacrimal gland, exorbital
Liver
Lungs, filled w/formalin at necropsy
Lymph nodes - mesenteric, mandibular
Mammary gland area
Nasal cavity
Ovaries
Pancreas
Pharynx
Pituitary gland
Prostate gland incl. coagulating glands
Rectum
Salivary glands - mandibular, sublingual
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord - cervical, midthoracic, lumbar
Spleen
Stomach
Testes (fixed in Bouin's solution)
Thymus
Thyroid (incl. parathyroid gland, if possible)
Tongue
Trachea
Urinary bladder, filled w/formalin at
necropsy
Uterus
Vagina
Gross lesions


Organ Weights
The following organs were weighed before fixation and the weight recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight and brain weight.
Adrenal glands
Brain
Heart
Kidneys
Liver
Ovaries
Spleen
Testes
Epididymides
Thymus

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity,
body weight, clinical laboratory data, organ weights, and ratios:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate were applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
• Fisher's exact-test

The following statistical methods were used for statistical analysis of clinical laboratory data:
• Quantitative data were analyzed by a one-way analysis of variance (ANOVA) when the variances are considered homogenous according to Bartlett. Alternatively, if the variances are considered to be heterogenous (p<=0.05) a non-parametric Kruskal-Wallis test was used. Treated groups were compared to the control groups using Dunnett's test if the ANOVA was significant at the 5% leveland by Dunn's test in the case of a significant Kruskal-Wallis test (p<=0.05).
• Ordinal data such as urine sediment were analyzed using the Kruskal-Wallis test. If the test was significant, comparisons were made between the control group and each of the treatment groups using Dunn's test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

GENERAL CAGESIDE OBSERVATIONS (DAILY):
There were no test item-related clinical signs noted during daily observations at any dose level tested.

DETAILED CLINICAL OBSERVATIONS (WEEKLY):
There were no test item-related clinical signs noted during weekly observations at any dose level

Mortality:

mortality observed, non-treatment-related

Description (incidence):

At 40 mg/kg/day, a male rat died during the first week of the treatment period. The cause of death was due to tubular necrosis of the kidneys secondary to kidney failure. This death was considered to be unrelated to the test item and all other rats survived until scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and the mean body weight gain of the test item-treated rats compared favorably with those of the control rats during the treatment and recovery periods.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item-related changes during treatment or recovery in the mean daily food consumption at any dose level tested.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related differences in the hematology parameters were seen after the end of the treatment period, and none of the changes observed after the recovery were considered to be late effects.
In females, a marginal but statistically significant reduction in hemoglobin was noted at 800 mg/kg/day (p<0.01), as weil as a significant reduction in the hematocrit (p<0.05), when compared with the controls.
At 800 mg/kg/day, females had significantly higher mean absolute (p<0.05) and relative reticulocyte counts (p<0.01) when compared with the controls. The reticulocyte maturity indices were shifted towards high fluorescence reticulocytes (p<0.01) from low fluorescence reticulocytes (p<0.01). These changes were reversible after the treatment phase and males were unaffected.
Dose-unrelated increases in the mean relative eosinophil count were seen at 40 mg/kg/day (p<0.05) and 200 mg/kg/day (p<0.05) of females, and considered to be incidental. The mean absolute eosinophil count was significantly elevated (p<0.05), but this was considered to be due to an unusually low control value and of no toxicological relevance.
The mean relative lymphocyte count, significantly reduced in males at 800 mg/kg/day, remained within the range of the historical control data and considered to be incidental.
A number of lest item-unrelated differences were noted in males and females after 4 weeks of treatment. These differences were either independent of dose, without concomitant changes in related parameters or within the ranges of the historical control data, and therefore considered to be incidental.
After the recovery period, the red cell distribution width of the males and females previously treated with 800 mg/kg/day was significantly elevated (both p<0.05). These differences remained within the range of the historical control values and were considered to be incidental.
In males previously treated with 800 mg/kg/day, the mean hemoglobin distribution width was elevated (p<0.01), the mean absolute neutrophil count was significantly elevated (p<0.05), and the mean platelet count was increased (p<0.05) but these were not seen after the end of the treatment period, remained within the range of the historical control values and were considered to be incidental.
The mean relative thromboplastin time was significantly increased (i.e. the time was foreshortened) in females previously treated with 800 mg/kg/day when compared with controls. This was not seen after the end of the treatment period and was considered to be incidental.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Aspartate aminotransferase activity was significantly elevated in males (p<0.01) and females (p<0.01) treated with 800 mg/kg/day, whereas alanine aminotransferase activity was elevated only in males (p<0.01) at 800 mg/kg/day. These changes were considered to be test item-related metabolic adaptive responses, rather than systemic changes of toxicological relevance, and were reversible after the recovery period.
Glutamate dehydrogenase activity was elevated in males (p<0.01) and females (p<0.05) treated with 800 mg/kg/day after 4 weeks' treatment. Although the females remained within the range of the historical control values, this finding was considered to be a further indication of liver activation and metabolic adaptation. Similarly, alkaline phosphatase was elevated after 4 weeks' treatment at 800 mg/kg/day in males (p<0.01) and females (p<0.01), and reduced after 2 weeks' recovery in males (p<0.05) and females (p<0.01).
At 800 mg/kg/day, significantly elevated total bilirubin and elevated cholesterol levels were noted in males (both p<0.01) and females (both p<0.01) when compared with controls. Elevated phospholipids were also seen in males (p<0.01) and females (p<0.01) treated with 800 mg/kg/day. All were reversible after recovery. Albumin levels were decreased in both sexes (p<0.01) treated with 800 mg/kg/day, but only the females exceeded the historical control range. After recovery, the females had elevated albumin levels (p<0.01). Globulin levels were elevated in males (p<0.01) and females (p<0.01) treated with 800 mg/kg/day, but only females exceeded the range of the historical controls. Consequently, the albumin/globulin ratios were significantly lower in males (p<0.01) and females (p<0.01), whereby the latter exceeded the range of the historical control data.
Sodium and chloride levels were reduced in females treated with 800 mg/kg/day (p<0.01) for 4 weeks. Sodium levels reverted to normal after recovery, whereas lower chloride levels persisted (p<0.05). Elevated calcium levels were noted in males (p<0.05) and females (p<0.05) treated with 800 mg/kg/day after 4 weeks of treatment. This difference persisted in the females after the recovery period (p<0.01). All remained within the ranges of the historical control data.
In females treated with 800 mg/kg/day, bile acids were significantly elevated (p<0.01), but as this parameter varies widely and dose-independent variations were seen in females treated with 40 mg/kg/day and 200 mg/kg/day, the difference was considered to be of questionable toxicological relevance.
Lactate dehydrogenase activity was significantly elevated (p<0.01) in females, but remained within the range of the historical control values and was considered to be incidental.
After the recovery period, the mean protein level of females treated previously with 800 mg/kg/day was significantly elevated (p<0.01). As no changes were seen after the end of the treatment period, this finding was considered to be incidental.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test item-related changes were noted in the urinalysis parameters after treatment or recovery.
A significantly elevated number of leukocytes was noted in the urine of males treated with 800 mg/kg/day, but these remained within the range of the historical control values and all other parameters compared favorably with the controls. This minor difference was considered to be incidental.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no test item-related clinical signs noted during the functional observational battery (week 4).
Grip Strength
No test item-related changes were noted in the mean fore- or hindlimb grip strength values.
Significantly elevated mean forelimb grip strength values were noted in females treated with 40 mg/kg/day, 200 mg/kg/day and 800 mg/kg/day (all p<0.01). The mean forelimb grip strength values of the males were unaffected, and the mean hindlimb grip strength values of the test item-treated females compared favorably with those of the controls
Locomotor Activity
No test item-related differences were noted during the determination of locomotor activity.
In males treated with 800 mg/kg/day, a slight (if primarily statistically insignificant) reduction of the mean locomotor activity was noted when compared with the controls. At 800 mg/kg/day, the mean locomotor activity was significantly reduced only during 40-50 minutes (p<0.05).
In females, significant reductions in mean locomotor activity were noted at 40 mg/kg/day during 10-20 minutes (p<0.05) and 20-30 minutes (p<0.05) when compared with the controls. The total locomotor activity (0-60 minutes) was significantly reduced (p<0.05) as well. At 200 mg/kg/day, significantly increased mean locomotor activity was noted during 20-30 minutes (p<0.01). These changes were not considered to be test item related as the mean locomotor activity of the females treated with 800 mg/kg/day was unaffected.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 4 Weeks
At 800 mg/kg/day, elevated mean absolute liver weights were noted in males (not significant) and females (p<0.05) after 4 weeks of treatment. The liver-to-body weight ratios were significantly elevated in males (p<0.01) and females (p<0.01) when compared with the controls. These differences were considered to be test item related, although likely to be the result of metabolic adaptation.
Females treated with 200 mg/kg/day had significantly reduced kidney-to-body weight ratios, when compared with controls. In males treated with 40 mg/kg/day, the mean absolute testes weights were significantly elevated (p<0.05) when compared with controls. In the absence of dose-response relationships or any correlating microscopical changes, these findings were considered to be incidental.
After 6 Weeks
In rats previously treated with 800 mg/kg/day, the mean adrenal-to-body weight ratio of the males was increased (p<0.05) and the mean liver-to-body weight ratio of females was increased (p<0.05) when compared with the respective controls. The forrner finding was not seen after the treatment period and considered to be incidental; whereas the latter was considered to be a non-adverse test item related metabolic adaptive consequence.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The male rat treated with 40 mg/kg/day, which died during the first week of treatment, presented dark red foci in the lung that correlated with congestion, edema and hemorrhages.
The few other findings recorded, such as dark red discoloration of the seminal vesicles, pelvic dilation of the kidneys and nodule in the epididymides of one control, were considered to be within the range of normal background changes, which may be seen in rats of this strain and age in 4-week studies and were considered to be incidental, reflecting the usual individual variability.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment and recovery periods test item-related microscopic lesions were observed in the liver:
Centrilobular hypertrophy of the hepatocytes was observed in all animals treated with 800 mg/kg/day and in 2/5 males treated with 200 mg/kg/day. This change was moderate in males and slight or moderate in females treated with 800 mg/kg/day, while it was minimal in males treated with 200 mg/kg/day. Minimal or slight single cell necrosis of the hepatocytes was present in all animals treated with 800 mg/kg/day. In addition, a higher incidence and severity of cholangitis and bile duct hyperplasia was present in animals at 800 mg/kg/day (incidence of cholangitis: 2/5 males and 4/5 females at 800 mg/kg/day versus 0/5 in control males and 1/5 in control females; bile duct hyperplasia: 2/5 in males and females at 800 mg/kg/day, versus 0/5 in control males and 1/5 in control females). The severity of both findings varied from minimal to moderate in males and minimal or slight in females.
After 2 weeks of recovery period, minimal to moderate cholangitis was observed in 4/5 males and in 2/5 females previously treated with 800 mg/kg/day versus none in control males and 2/5 in control females (however with a minimal or slight degree of severity). Minimal to moderate bile duct hyperplasia was seen in 4/5 males and in all females at 800 mg/kg/day; 1/5 was seen in control males and 2/5 in control females with a minimal or slight degree of severity. Minimal deposit of pigments (most likely hemosiderin) was present in the portal tract of 1/5 female at 800 mg/kg/day affected by cholangitis.
One male rat treated with 40 mg/kg/day died during the first week of treatment. A marked tubular necrosis of the kidneys was the final cause of death. This change was secondary to the kidneys failure most likely caused by heart insufficiency (reported microscopically as ventricular and auricular dilation). Congestion, edema and hemorrhage in the lungs were also associated to the heart failure. However, these kidneys, heart and lungs changes were of spontaneous occurrence and not related to the treatment with the lest item.
The remaining microscopic findings, regularly distributed among the groups, were within the range and severity of spontaneous background lesions which may be observed in rats of this strain and age in this laboratory and considered to be of no toxicological significance.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

800 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, 200 mg/kg body weight/day was established as the no-observed-adverse-effect-level (NOAEL).

Executive summary:

Purpose

The purpose of this oral toxicity study was to assess the cumulative toxicity of the test item when administered daily to rats by gavage for a period of 28 days. The reversibility of possible treatment-related changes was assessed after a treatment-free 14-day recovery period. This study should provide a rational basis for a risk assessment in man. The results of this study should indicate potential target organs and should identify chemicals with neurotoxic potential.

Study Design

The study was performed according to OECD TG 407.

Results

Oral administration of the test item to Wistar rats at doses of 40, 200 and 800 mg/kg/day, for 28 days resulted in no test item-related deaths (although one rat died prematurely), no clinical signs of toxicological relevance (daily observations, weekly observations at weeks 1-3, or functional observational battery at week 4), no changes in fore-or hindlimb grip strength and no effects on locomotor activity, no differences in food consumption or body weight development of test item-treated or control rats, no effects on hematology or urinalysis parameters, and no macroscopical changes of toxicological relevance.

Test item-related findings were primarily seen in rats treated with 800 mg/kg/day and (with the exception of some microscopical changes seen in the liver at 800 mg/kg/day) were considered to be largely adaptive in nature.

Activation of liver enzymes (aspartate and alanine aminotransferases, glutamate dehydrogenase and alkaline phosphatase) seen at 800 mg/kg/day was considered to represent metabolic adaptation, and correlated with elevated mean liver weights in these animals (the liver-to-body weight ratio remained slightly elevated in females after the recovery period, but this was considered to represent partial reversal).

Test item-related microscopic changes were observed in the liver of males and females treated with 800 mg/kg/day. They consisted of centrilobular hypertrophy and single cell necrosis of the hepatocytes, cholangitis and bile duct hyperplasia. A few males treated with 200 mg/kg/day had a minimal centrilobular hypertrophy of hepatocytes that was considered to be an adaptive response. After 2 weeks of recovery period, cholangitis and bile duct hyperplasia were still observed in animals treated previously with 800 mg/kg/day.

Conclusion

Based on the results of this study with the test item, 40 mg/kg body weight/day was established as the no-observed-effect-level (NOEL) and 200 mg/kg body weight/day as the no-observed-adverse-effect-level (NOAEL).