Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-05-05 to 2000-06-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
1995
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
1996
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines for Screening Toxicity Testing of Chemicals: Testing Methods for New Substances
Version / remarks:
1986
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
HanIbm:WIST (SPF)
Details on species / strain selection:
Recognized by the international guidelines as the recommended test system.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division CH-4414 Fullinsdorf / Switzerland
- Age at study initiation: 7 weeks
- Weight at study initiation: mean 170 g (males); mean 140 g (females)
- Housing: 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 Provimi Kliba 3433 rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) was available ad libitum
- Water: Community tap-water from Itingen was available ad libitum in water bottles
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Miglyol - 812-neutral oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article formulations were prepared weekly. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17-23°C).
Homogeneity of the test article in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: test item is only soluble in Miglyol - 812-neutral oil.
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed by a HPLC method supplied by the Sponsor.
The mean concentrations of the homogeneity samples were found to be 101.8%, 99.9%, and 94.1% of the nominal concentrations of dose group 2 (10 mg/mL), dose group 3 (30 mg/mL), and dose group 4 (100 mg/mL), respectively. The individual concentrations varied in the range from -3% to +1% of the mean concentrations. Therefore, the test item was found to be homogeneously distributed in the vehicle. The substance is stable in the vehicle at room temperature (17-23 °C) for at least seven days.
Duration of treatment / exposure:
28 days (plus 14 days recovery)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Groups 1 and 4: 10 males; 10 females
Groups 2 and 3: 5 males; 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Whereas three of five male rats died after a single oral administration of 2000 mg/kg body weight, all female rats survived the 15-day observation period until their scheduled death. Symptoms started one to 15 minutes after administration and lasted up to day 3. Single oral administration of 1000 mg/kg was tolerated by male rats without any symptoms (Heusener, 19961). Based on these findings, 100, 300 and 1000 mg/kg were selected as the doses in the present 28 day study. It is expected that at least 100 mg/kg will be tolerated by the rats without any adverse findings and that 1000 mg/kg/day will be in the toxic range (information provided by the sponsor).
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29-42 (recovery).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded weekly during pretest, treatment and recovery and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

FOOD CONSUMPTION: Yes
The food consumption was recorded once during the pretest period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the RCC computer.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of exposure period (after 4 weeks and after 6 weeks)
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: all
- Parameters checked: White blood cells, Red blood cells, Hemoglobin, Hematocrit, Mean cell volume, Mean corpuscular volume, Mean corpuscular hemoglobin, Platelets, Reticulocytes, Differential blood count, Prothrombin time, Partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of exposure period (after 4 weeks and after 6 weeks)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Sodium, Potassium, Calcium, Chloride, Phosphate, Glucose, Urea, Creatinine, Bilirubin, total, Cholesterol, Triglyceride, Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Protein, total, Albumin, Bile acid

URINALYSIS: Yes
- Time schedule for collection of urine: end of exposure period (after 4 weeks and after 6 weeks)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Osmolality, pH, protein, glucose, ketone, bilirubin, nitrite, blood and urobilinogen, appearance, color, ketone, crystals

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (cerebrum, cerebellum, pons), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Esophagus, Eyes with 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, Ovaries, Pancreas, Pituitary gland, Prostate gland, 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), Tongue, Lacrimal gland (exorbital), Liver, Lungs (infused with formalin at necropsy) Lymph nodes (mesenteric, mandibular), Mammary gland area, Nasal cavity, Trachea, Urinary bladder (infused with formalin at necropsy), Uterus, Vagina, Gross lesions

HISTOPATHOLOGY: Yes
Slides of all organs and tissues which were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist.
Organ and tissue samples taken from animals which died spontaneously were evaluated similarly to those organs taken from animals of the high-dose group.
As treatment-related morphologic changes were detected in the livers of high-dose animals, these same organs from animals of the mid- and low-dose groups were examined.
Statistics:
The following statistical methods were used to analyze grip strength, locomotor activity, body weights, organ weights and all ratios, as well as clinical laboratory data :
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was 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 could not be assumed to follow a normal distribution.
• Student's T-Test was applied to locomotor activity and grip strength.
• Fisher's exact-test was applied to macroscopic findings.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, piloerection and increased salivation was noted during daily and weekly observations in animals of both sexes, whereas hunched posture was seen only in females during daily observation. These findings were considered to be test article-related effects. At 1000 mg/kg/day, increased coprophagia and bedding consumption was noted during daily observation following administration of the test article. These findings were considered to be due to the residual taste of the test article, rather than indications of toxicity. All other findings noted during daily and weekly observations were considered to be incidental.
Description (incidence):
One female treated with 1000 mg/kg/day was found dead on treatment day 16. All remaining animals survived until scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights and body weight gain of the males treated with 1000 mg/kg/day were slightly lower than those of the control males during the treatment period. Although the differences from the control values never attained statistical significance, a relationship with the test article treatment could not be eliminated.
During the recovery period, the mean body weight gain was depressed in males previously treated with 1000 mg/kg/day when compared with the controls. The differences from the control values attained statistical significance on days 1 and 8 of the recovery period, and remained lower than that of the control males on day 14 of recovery. This finding is considered to be a late effect of the test article treatment.
The mean body weights of the test article-treated females were generally similar to that of the controls during the treatment and recovery periods.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males and females treated with 1000 mg/kg/day consumed less feed (-1.1% and -8%, respectively) during the initial week of treatment when compared with the control values. The mean daily food consumption of these animals improved to levels comparable to that of the controls during subsequent weeks of treatment and during the recovery period. This transient finding was considered to be test article-related.
The mean daily food consumption of the remaining animals compared favorably with that of the controls during the treatment and recovery periods.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test article-related differences to the control values were evident in the hematology parameters at any dose level. The few changes in hematology parameters, although statistically significant, were considered to be spontaneous and were with the ranges of the historical control data.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males and females treated with 1000 mg/kg/day for four weeks had increased levels of total cholesterol, triglycerides and phospholipids, when compared with the control values. The differences attained statistical significance and were considered to indicate test article-related effects upon lipid metabolism.
Increased activity of alanine aminotransferase was noted in males and females treated with 1000 mg/kg/day. This statistically significant finding was considered to be indicative of increased metabolic activity caused by the test article.
Slightly higher globulin levels were noted in females treated with 1000 mg/kg/day, and hence the albumin/globulin ratio was slightly lower. Although statistically significant, the toxicological significance of this finding remains unclear.
All other differences from the control values were considered to be incidental changes unrelated to the treatment with the test article.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The specific gravity of the males and females treated with the test article at 1000 mg/kg/day was increased with statistical significance when compared with the control females. As the remaining parameters were largely unaffected, this finding was considered to be incidental, rather than an indication of renal insufficiency in urine concentration.
The urinary pH was lower in males only, and considered to be an incidental finding.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Salivation was noted in four males and three females treated with 1000 mg/kg/day. This finding was considered to be test article-related. All other findings were considered to be incidental.

The mean forelimb grip strength was slightly higher in males treated with 1000 mg/kg/day, when compared with the controls. The difference from the control value attained statistical significance (p<0.05). This finding was considered to be unrelated to treatment, as the hind-limb grip strength compared favorably with those of the controls. The fore- and hind-limb grip strength of the remaining males compared favorably with those of the control males.
The mean forelimb grip strength of the test article-treated females was lower than that of the controls and attained statistical significance (p<0.01) at all dose levels. The hindlimb grip strength of the females treated with 1000 mg/kg/day was also lower with statistical significance (p<0.05).
However, in the absence of a dose-response relationship or concomitant changes in the males, the findings noted in the females were considered to be fortuitous.
The mean locomotor activity of the animals treated with 1000 mg/kg/day was less than that of the controls. The differences attained statistical significance (p<0.01) and were considered to be test article-related.
The mean locomotor activity of the animals treated with 100 mg/kg/day or 300 mg/kg/day compared favorably with the control values.

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
After 4 Weeks:
Test article-related organ weight changes were restricted to increased liver weights in males and females at 1000 mg/kg/day (statistically significant in females, p<0.01).
Although spleen weights were slightly reduced in the males and females treated with 1000 mg/kg/day, the differences noted in the animals treated with 100 mg/g/day or 300 mg/kg/day were not dose-related, and there were no morphologic changes of toxicological relevance. Therefore, this finding was considered to be incidental.
All other organ weights and ratios were similar to those of the control animals!
After 6 Weeks:
The absolute and relative organ weights of males and females previously treated with 1000 mg/kg/day compared favorably with those of the controls.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Autolysis and cannibalism with missing organs were observed in the single female which died spontaneously.
The remaining findings were considered to be within the range of spontaneous background alterations in rats of this strain and age. They consisted of renal pelvic dilation, uterine horn dilation, and discoloration and/or discolored foci in several organs.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic changes which were deemed to be test article-related were recorded in the livers of two males treated with 100 mg/kg/day, three males treated with 300 mg/kg/day and nine from ten animals treated with 1000 mg/kg/day for four weeks. These lesions consisted of minimal to slight hepatocellular hypertrophy in the central and intermediate zones of the lobules and correlated with the increased liver weight. At 1000 mg/kg/day, this finding was slightly more prominent in the females than in the males, and decreased in severity and incidences in the recovery groups. The remaining findings were within the range of spontaneous background lesions which may be recorded in rats of this strain and age.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, a no-observed-effect-level (NOEL) could not be established. However, in the absence of commensurate changes in the clinical biochemistry parameters, liver hypertrophy noted in a few males treated with 100 mg/kg bw/day or 300 mg/kg bw/day was considered to be indicative of metabolic adaptation. The no-observed-adverse-effect-level (NOAEL) was considered to be 300 mg/kg bw/day following treatment with the test item.
Executive summary:

In a subacute toxicity study, the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, Miglyol-812-neutral oil.

The groups- comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4.

At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

From the animals of the low and middle dose groups, livers were examined to establish a no-effect level.

Results:

One Female (1000 mg/kg/day) was found dead on treatment day 16. The cause of death could not be established. All remaining animals survived until scheduled necropsy.

At 1000 mg/kg/day, piloerection and increased salivation was noted during daily and weekly observations in animals of both sexes, whereas hunched posture was seen only in females during daily observation. These findings were considered to be test article-related effects.

At 1000 mg/kg/day, increased coprophagia and bedding consumption was noted during daily observation following administration of the test article. These findings were considered to be due to the residual taste of the test article, rather than indications of toxicity.

All other findings noted during daily and weekly observations were considered to be incidental.

At 1000 mg/kg/day, salivation was noted in four males and three females during functional observational battery. This finding was considered to be test article-related.

The fore- and hindlimb grip strength of test article-treated animals were unaffected when compared with the values of the controls.

The males and females treated with the test article at 1000 mg/kg/day were less active than the controls. This finding was considered to be related to the treatment with the test article. The locomotor activity of the animals treated with 100 mg/kg/day or 300 mg/kg/day were similar to that of the controls.

The mean daily food consumption of the males and females treated with 1000 mg/kg/day was less than that of the controls during the first week of treatment, but improved during subsequent weeks. This finding was considered to be test article-related.

The food consumption of all other animals was unaffected.

The body weight development of the males treated with 1000 mg/kg/day was less than that of the control males during the treatment and recovery periods. This finding was considered to be test article-related. The females at this dose level were unaffected, as were both sexes treated with 100 mg/kg/day or 300 mg/kg/day.

The hematology parameters of the test article-treated animals were unaffected when compared with those of the controls.

At 1000 mg/kg/day, males and females had increased levels of total cholesterol, triglycerides and phospholipids, when compared with the control values. These differences were considered to indicate test article-related effects upon lipid metabolism. Increased activity of alanine aminotransferase noted in males and females treated with 1000 mg/kg/day was considered to be indicative of increased metabolic activity caused by the test article.

Slightly higher globulin levels were noted in females treated with 1000 mg/kg/day, and hence the albumin/globulin ratio was slightly lower. Although statistically significant, the toxicological significance of this finding remains unclear.

The urinalysis parameters of the test article-treated animals were unaffected when compared with those of the controls.

Increased liver weights were noted in males and females at 1000 mg/kg/day after four weeks' treatment. This finding correlated with morphologic changes seen in these animals and was considered to be test article-related.

All other organ weights and ratios were considered to be unaffected by the test article administration.

No test article-related macroscopic findings were noted. Minimal to slight hepatocellular hypertrophy was noted in the livers of two males treated with 100 mg/kg/day, three males treated with 300 mg/kg/day and nine from ten animals treated with 1000 mg/kg/day for four weeks, and corresponded with increased liver weights. These changes decreased in severity and incidences in the recovery groups.

Based on the results of this study, a no-observed-effect-level (NOEL) could not be established. However, in the absence of commensurate changes in the clinical biochemistry parameters, liver hypertrophy noted in a few males treated with 100 mg/kg bw/day or 300 mg/kg bw/day was considered to be indicative of metabolic adaptation. The no-observed-adverse-effect-level (NOAEL) was considered to be 300 mg/kg bw/day following treatment with the test item.