Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct 22, 2004 - March 31, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: HanRcc:WIST (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 132 - 155 grams (mean 142 grams) Females: 116 - 127 grams (mean 121 grams)
- Fasting period before study: no (animals were fasted for approximately 18 hours before blood sampling)
- Housing: groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days under test conditions after health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 10-15 / hour
- Photoperiod (hrs dark / hrs light): 12 /12 hours

IN-LIFE DATES: From: 29 Oct 2004 To: 26 nov 2004 (Test Group) 10 Dec 2004 (Recovery Group)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): PEG 300 is common and accepted as vehicle
- Concentration in vehicle: 0, 8, 40, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): 1078164, 12004034, and 2034733
- Purity: n.a.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
nominal measured
8 mg/mL 105.3 %
30 mg/mL 105.3 %
100 mg/mL 112.8 %
Duration of treatment / exposure:
Test duration: 28 days / daily treatment
Frequency of treatment:
Dosing regime: daily on 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 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:
Male: 10 animals at 0 mg/kg bw/day (treatment+recovery)
Male: 5 animals at 40 mg/kg bw/day (treatment)
Male: 5 animals at 200 mg/kg bw/day (treatment)
Male: 10 animals at 1000 mg/kg bw/day (treatment+recovery)
Female: 10 animals at 0 mg/kg bw/day (treatment+recovery)
Female: 5 animals at 40 mg/kg bw/day (treatment)
Female: 5 animals at 200 mg/kg bw/day (treatment)
Female: 10 animals at 1000 mg/kg bw/day (treatment+recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
In this subacute toxicity study, the test material was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 40, 200 and 1000 mglkg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, PEG 300, only.

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 500 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 acclimatization, 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, liver and thyroid glands were examined to establish a no-effect level.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Observations/Measurements (Frequency)
MORTALITY / VIABILITY
Observations for mortality/viability were recorded twice daily.

GENERAL CAGESIDE OBSERVATIONS (DAILY)
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 (WEEKLY)
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.

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

BODY WEIGHTS (WEEKLY)
Body weights were recorded weekly during acclimatization, treatment and recovery and before each necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the computer.

FUNCTIONAL OBSERVATIONAL BATTERY
During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals.
The behavioral parameters of the functional observational battery are similar to those evaluated during weekly observations.

GRIP STRENGTH
Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.

LOCOMOTOR ACTIVITY
Locomotor (decreased or increased) activity was measured quantitatively with AMS Fohr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded.
Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.



CLINICAL LABORATORY INVESTIGATIONS
Blood samples for hematology and clinical biochemistry were collected from all animals under light isoflurane anesthesia. The animals were fasted in metabolic cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
Urine was collected during the 18-hour fasting period into a specimen vial.


The assays were performed under internal laboratory quality control conditions to assure reliable test results.

HEMATOLOGY
The following parameters have been investigated:
RBC, HB, HCT, MCV, MCH, MCHC, Platelets, Reticulocytes, HFR, MFR, LFR, nucleated erythrocytes, Heinz bodies, MET-HB, WBC, diff. WBC Count, red blood cell morphology, PT, APTT

CLINICAL BIOCHEMISTRY
Glucose, Urea, Creatinine, Uric acid, Bilirubin, Cholesterol, Triglycerides, Phospholipids, GOT, GPT, LDH, CK, ALP, GT, Ca, P, Na, K, Cl, Albumin, Protein, Globulin, A/G Ratio, Bile acids

URINALYSIS
Volume, specific gravity, Color, Appearance, pH, Nitrite, Protein, Glucose, Ketone, Urobilinogen, Bilirubin, Erythrocytes, Leucocytes
Sacrifice and pathology:
NECROPSY
after 4 weeks
after 6 weeks (post recovery)

All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by an experienced veterinary pathologist.

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, unless indicated otherwise, fixed in neutral phosphate buffered 4 % formaldehyde solution:

Adrenal glands
Aorta
Bone (sternum, femur including joint)
Bone marrow (sternum, 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
Lacrimal gland (exorbital)
Liver
Lungs (infused with formalin)
Lymph nodes (mesenteric, mandibular) Mammary gland area
Nasal cavity
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 Trachea
Urinary bladder (infused with formalin)
Uterus
Vagina
Gross lesions

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
The following organ weights were recorded on the scheduled dates of necropsy:

Brain Thymus Spleen Ovaries
Heart Kidneys Testes
Liver Adrenals Epididymides

The organ to terminal body weight ratios as well as organ to brain weight ratios were determined.
The determination of the terminal body weight was performed immediately prior to necropsy.

HISTOTECHNIQUE
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin.

HISTOPATHOLOGY
Slides of all organs and tissues listed in boldface type (see Necropsy, above) that were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist.
As test item-related morphologic changes were detected in the organs of high-dose animals, the same organs (liver and thyroid gland) from animals of the mid- and low-dose groups were examined.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, organ weights and ratios, as well as:
• 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) were applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
• Fisher's exact-test were applied to the macroscopic findings. 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 homogeneous according to Bartlett. Alternatively, if the variances are considered to be heterogenous (p50.05), 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% level and by Dunn's test in the case of a significant Kruskal-Wallis test

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
transient salivation (primarily during the second half of the treatment period) and rales in males and females; @1000 mg/kg bw
As these findings were noted primarily du ring the second half of the study, they were considered to be a possible indication of irritation rather that findlngs related to systemic toxicity.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two unscheduled deaths occurred amongst females treated with 1000 mg/kg/day. One female treated with 1000 mg/kg/day died spontaneausly on treatment day 13. A second female was selected for ethical sacrifice on treatment day 24 but died before being necropsied. All other animals survived untll scheduled necropsy.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
differences in the clinical biochemistry parameters of males and females treated with 1000 mg/kg/day
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
reductions of mean absolute and relative spleen weights in males, and a slight increase in the mean absolute liver weight In females, indicating a probable adaptive response@1000 mg/kg
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
@1000mg/kg: an increased incidence and severity of cytoplasmic vacuolation of the hepatocytes in males and females, lymphoid depletion in the spleen of males and in the thymus of both males and females.complete recovery observed after 2 weeks
Histopathological findings: neoplastic:
no effects observed
Details on results:
Oral administration of the test item to Wistar rats at doses of 40, 200 and 1000 mg/kg/day, for 28 days and 14 days recovery, resulted in two mortalities (one of which was considered to be a possible dosing error), no clinical signs of toxicological relevance during weekly observations (weeks 1-3) or during functional observational battery (week 4), no effects on the locomotor activity or fore- and hindlimb grip strength, no effects on food consumption or body weight and no effects of toxicological relevance on the hematological or urinalysis parameters, and no macroscopical findings of toxicological relevance.

Test item-related findings were generally restricted to rats treated with 1000 mg/kg/day and included

• transient salivation (primarily during the second half of the treatment period) and rales in males and females;

• differences in the clinical biochemistry parameters of males and females treated with 1000 mg/kg/day for four weeks (elevated cholesterol, triglyceride and phospholipid levels, and reduced bile acids, indicating that the liver was a target organ. Elevated sodium levels noted after four weeks treatment in males and females were considered to be a test item related change);

• differences in organ weights (reductions of mean absolute and relative spleen weights in males, and a slight increase in the mean absolute liver weight In females, indicating a probable adaptive response);

• microscopic changes in the liver, spleen and thymus after four weeks' treatment, consisting of an increased incidence and severity of cytoplasmic vacuolation of the hepatocytes in males and females, lymphoid depletion in the spleen of males and in the thymus of both males and females. After two weeks of recovery period, there was a complete recovery from these changes.

All differences in the clinical biochemistry parameters, organ weights and microscopical changes were reversible after two weeks recovery.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: All changes were reversible after two weeks recovery.
Key result
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, 200 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL). Insofar as all observed changes were reversible after the recovery period, the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg/day.
Executive summary:

Study design

The test material was administered orally by gavage, once daily, 7 times a week for 4 weeks to 3 groups of SPF-bred Wistar rats kept under conventional conditions at the test facility. A similarly constituted group received the vehicle, PEG-300, and served to generate contemporary control data. 5 rats per sex of the control group and of group 4 were scheduled for a 2 week treatment-free follow-up period.

Daily dose levels and number of rats used:

Group Doses Number of animals
(mg/kg) Main Recovery
1 Control 0 5M/5F 5M/5F
2 Test Group 40 5M/5F
3 Test Group 200 5M/5F
4 Test Group 1000 5M/5F 5M/5F


The total number of rats was 60 (30M, 30F).
Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during acclimatization, 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, liver and thyroid glands were examined to establish a no-effect level.

Results

Oral administration of the test item to Wistar rats at doses of 40, 200 and 1000 mg/kg/day, for 28 days and 14 days recovery, resulted in two mortalities (one of which was considered to be a possible dosing error), no clinical signs of toxicological relevance during weekly observations (weeks 1-3) or during functional observational battery (week 4), no effects on the locomotor activity or fore- and hindlimb grip strength, no effects on food consumption or body weight and no effects of toxicological relevance on the hematological or urinalysis parameters, and no macroscopical findings of toxicological relevance. Test item-related findings were generally restricted to rats treated with 1000 mg/kg/day and included: • transient salivation (primarily during the second half of the treatment period) and rales in males and females; • differences in the clinical biochemistry parameters of males and females treated with 1000 mg/kg/day for four weeks (elevated cholesterol, triglyceride and phospholipid levels, and reduced bile acids, indicating that the liver was a target organ. Elevated sodium levels noted after four weeks treatment in males and females were considered to be a test item related change); • differences in organ weights (reductions of mean absolute and relative spleen weights in males, and a slight increase in the mean absolute liver weight In females, indicating a probable adaptive response); • microscopic changes in the liver, spleen and thymus after four weeks' treatment, consisting of an increased incidence and severity of cytoplasmic vacuolation of the hepatocytes in males and females, lymphoid depletion in the spleen of males and in the thymus of both males and females. After two weeks of recovery period, there was a complete recovery from these changes. All differences in the clinical biochemistry parameters, organ weights and microscopical changes were reversible after two weeks recovery.

Conclusion

Based on the results of this study, 200 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL). Insofar as all observed changes were reversible after the recovery period, the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg/day.