Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MGDN
- Substance type: powder
- Physical state: solid
- Analytical purity: 99.9 %
- Lot/batch No.: Betriebsware C404
- Stability under test conditions: stable until January 2007 (Study No. 06L00290) at room temperature
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
other: CRL:WI(HAN)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 33 +/- 1 day; males/females on day of receipt
- Weight at study initiation: males about 170 g (mean); females 135-138 g (The weight variation of the animals used did not exceed ±20
percent of the mean weight of each sex).
- Fasting period before study: At the end of the administration period the animals were sacrificed after a fasting period (withdrawal of food)
for at least 16 - 20 hours.
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: On the day of arrival the animals were subjected to an acclimatization period during which
they received ground diet and drinking water ad libitum.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): The animals were housed in a fully air-conditioned room.
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a suspension. To prepare the suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then the vehicle (0.5% carboxymethylcellulose in drinking water) was filled up to the desired volume, subsequently
mixed using a magnetic stirrer. During application the test substance preparations were kept homogeneous using a magnetic stirrer. The test
substance preparations were prepared twice a week.


VEHICLE
- Concentration in vehicle: 0.1, 0.3 and 0.45/0.6 g/100 ml
- Amount of vehicle (if gavage): 10 ml
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in the vehicle over a period of up to 96 hours was proven before the start of the study. Before the beginning of the administration period, 3 samples were taken from the lowest and highest concentration for homogeneity analysis; these samples were used as a concentration control at the same time. Only one sample was taken from the intermediate dose group for concentration control. A reserve sample of each sample was removed. The reserve samples were frozen until finalization of the report.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 30, 60(45) mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
By request of the sponsor, the following dose levels were selected for the present study:
60 mg/kg body weight/day: as high dose with expected toxic effects
30 mg/kg body weight/day: as mid dose
10 mg/kg body weight/day: as low dose
Due to severe clinical signs of toxicity, the high dose level was reduced to 45 mg/kg body
weight/day from study day 8 onwards.
The oral route was selected since this has been proven to be suitable for the detection of a
toxicological hazard.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
All animals were checked daily before, about 1 hour after and about 3 - 4 hours after application for any clinically abnormal signs. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Detailed clinical observations were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with sides of 25 cm high). The following parameters were examined:
1. abnormal behavior during “handling”
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmus
15. feces (appearance/consistency)
16. urine
17. pupil size

BODY WEIGHT: Yes
- Time schedule for examinations:
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the
respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION:
Individual food consumption was determined weekly over a period of 7 days and calculated as mean food consumption grams per animal and day.

FOOD EFFICIENCY:
Food efficiency (group means) was calculated based upon individual values for body weight and food consumption

WATER CONSUMPTION:
Individual water consumption was determined weekly over a period of 4 days and calculated as mean water consumption in grams per animal and day.

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On day 29 of the study: In the morning, blood was taken from the retro-orbital venous plexus from fasted animals.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: as specified in the guideline
- Parameters examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes, Prothrombin time (Hepato Quick’s test) (HQT), Furthermore differential blood smears were prepared and stained according to Wright without
being evaluated

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Furthermore differential blood smears were prepared and stained according to Wright without
being evaluated
- Animals fasted: Yes
- How many animals: as specified in the guideline
- Parameters examined: Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; EC 2.6.1.2.), Aspartate aminotransferase
(AST) (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.), Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase;
EC 3.1.3.1.), γ-Glutamyltransferase (GGT) (γ -glutamyl) peptide: aminoacid-γ- glutamyl-transferase; EC 2.3.2.2.), Sodium (NA), Potassium (K), Chloride
(CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin
(ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG)

URINALYSIS: Yes
- Time schedule for collection of urine: Study day 25: For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, Proteine, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery was performed in all animals at the end of the administration period starting at about 10.00 a.m.. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Home cage observations:
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:
1. posture
2. tremor
3. convulsions
4. abnormal movements
5. impairment of gait
6. other findings

Open field observations:
The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and observed for at least 2 minutes. Following parameters were examined:
1. behavior when removed from cage
2. fur
3. skin
4. salivation
5. nose discharge
6. lacrimation
7. eyes/pupil size
8. posture
9. palpebral closure
10. respiration
11. tremors
12. convulsions
13. abnormal movements
14. impairment of gait
15. activity/arousal level
16. feces (number of fecal pellets/appearance/consistency) within two minutes
17. urine (appearance/quantity) within two minutes
18. number of rearings within two minutes

Sensorimotor Tests/Reflexes:
The animals were removed from the open field and subjected to following sensorimotor or reflex tests:
1. approach response
2. touch response
3. vision ("visual placing response")
4. pupillary reflex
5. pinna reflex
6. audition ("startle response")
7. coordination of movements ("righting response")
8. behavior during "handling"
9. vocalization
10. pain perception ("tail pinch")
11. grip strength of forelimbs
12. grip strength of hindlimbs
13. landing foot-splay test
14. other findings

Motor activity was measured on the same day as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The animals were put into the cages in a randomized order. The measurements started at about 2.00 p.m. The number of beam
interrupts was counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes:
The surviving animals were sacrificed by decapitation under Isoba® (Essex GmbH Munich, Germany) anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology. Animals that died intercurrently were necropsied as soon as possible after their death and assessed by gross pathology.

The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Liver
3. Kidneys
4. Adrenals
5. Testes
6. Epididymides
7. Ovaries
8. Uterus
9. Spleen
10. Brain
11. Heart
12. Thymus
13. Thyroid glands

The following organs or tissues were fixed in 4% formaldehyde solution:
1. All gross lesions
2. Brain
3. Pituitary
4. Thyroids (with Parathyroids)
5. Thymus
6. Oesophagus
7. Salivary glands (mandibular and sublingual glands)
8. Trachea
9. Lungs
10. Pharynx
11. Larynx
12. Nose (nasal cavity)
13. Aorta
14. Heart
15. Liver
16. Pancreas
17. Spleen
18. Kidneys
19. Adrenals
20. Testes
21. Ovaries with Oviducts
22. Uterus and Vagina
23. Epididymides, prostate and seminal vesicle
24. Stomach (forestomach and glandular stomach)
25. Duodenum, jejunum and ileum
26. Cecum, colon and rectum
27. Urinary bladder
28. Lymph nodes (mesenteric and axillary lymph nodes)
29. Sciatic nerve
30. Bone marrow (femur)
31. Eyes
32. Extraorbital lacrimal glands
33. Skin
34. Female mammary gland
35. Spinal cord (cervical, thoracic and lumbar cords)
36. Sternum with marrow
37. Femur with knee joint
38. Skeletal muscle
From the liver, each one slices of the Lobus dexter lateralis and the Lobus sinister lateralis were fixed in Carnoy´s solution and embedded in paraplast.

HISTOPATHOLOGY: Yes

Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings:

1. All gross lesions in all effected animals from all dose groups
The following examinations were conducted in all animals from the control and high dose groups:
2. Brain
3. Thyroids (with parathyroids)
4. Thymus
5. Trachea
6. Lung
7. Heart
8. Liver
9. Spleen
10. Kidneys
11. Adrenals
12. Testes
13. Ovaries (with oviducts)
14. Uterus
15. Vagina
16. Epidymides
17. Prostate
18. Seminal vesicle
19. Stomach (forestomach and glandular stomach)
20. Duodenum and ileum
21. Jejunum (with Peyer’s plaques)
22. Cecum, colon and rectum
23. Urinary bladder
24. Lymph nodes (mesenteric and axillary lymph nodes)
25. Sciatic nerve
26. Bone marrow (femur)
27. Spinal cord (cervical, thoracic and lumbar cords)

Animals that died or were sacrificed in a moribund state were processed histotechnically and assessed like control animals.

The immunorelevant organs and tissues were evaluated according to the following parameters:

Thymus:
Increased/decreased grade of cortico-medullar ratio (related only to area)
Increase of starry sky cells
Changes of cellular density in the cortex
Changes of cellular density in the medulla

Spleen:
Changes of the cellularity of PALS, lymphoid follicles, marginal zone, red pulp
Altered cellular composition of follicles
Altered number of germinal centers

Lymph nodes (mesenteric and axillar lymph nodes):
Changes in the cellularity of follicles, interfollicular area, paracortical area, medulla
Altered cellular composition of paracortex
Altered number of germinal centers
Hyperplasia of high endothelial venules
Apoptotic necrosis increased

Peyer's patches (of the jejunum):
Changes of the cellularity of follicles (including mantle zone and germinal centers)
Changes of the cellularity of interfollicular area
Apoptotic necrosis increased

Bone marrow:
Changes of the cellularity
Changes of the myeloid/erythropoid ratio

Whenever the histopathologic evaluation of the immunorelevant organs and tissues did not reveal a morphologic alteration of these items and/or
whenever no other pathologic finding was noted, these organs were diagnosed as "no abnormalities detected”.
Statistics:
Detailed statistical analyses were performed

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
1 male and 1 female of group 3 (60 mg/kg bw/day) died prematurely on day 6 and on day 7, respectively; 1 female of group 3 (45 mg/kg bw/day) was sacrificed moribund on day 11. These cases of deaths were assessed as related to the test-substance.

High dose (60 mg/kg body weight/day; from day 0 until day 7):
• Convulsions, clonic were observed moderate in 4 females, severe in 2 females, epileptoid slight in 1 male, moderate in 1 female, severe in 2 males and 1 female, tonic slight in 1 female and severe in 1 male and 2 females
• Hyperesthesia was observed slight in 2 males and 5 females, moderate in 4 females and severe in 1 male and 2 females
• Hyperexcitability was observed slight in 2 males and 5 females, moderate in 4 females and severe in 1 male and 2 females
• Nose, discharge, red was observed moderate in 1 female
• Piloerection was observed in 2 females
• Salivation was observed slight in 2 females, moderate in 1 male and 4 females and severe in 1 male and female
• Skin, paleness was observed moderate in 2 females
• Twitching was observed slight in 3 males and 5 females, moderate in 2 males and 3 females and severe in 1 male and 3 females

High dose (45 mg/kg body weight/day; from day 8 until the end of the administration period):
• Anogenital region, smeared with urine was observed severe in 1 female
• Convulsions, tonic were observed slight, moderate and severe in each 1 female
• Hyperactivity was observed in 1 female
• Hyperesthesia was observed slight in 2 males and 1 female
• Hyperexcitability was observed slight in 4 males and 3 females, moderate in 4 males and females and severe in 1 male and 2 females
• Lateral position was observed in 1 female
• Nose, discharge, red was observed slight in 1 female and severe in 2 females
• Piloerection was observed in 2 males and 2 females
• Reduced general condition was observed slight in 1 female and severe in 2 females
• Salivation was observed slight in 1 female, moderate in 2 females and severe in 1 female
• Twitching was observed slight in 4 males and 3 females, moderate in 1 male and 4 females and severe in 2 females

Mid dose (30 mg/kg body weight/day):
• Convulsions, tonic were observed moderate in 1 male
• Hyperactivity was observed in 1 female
• Hyperesthesia was observed slight in 1 male and 5 females
• Hyperexcitability was observed slight in 5 males and 5 females, moderate in 3 males and 4 females and severe in 1 female
• Piloerection was observed in 1 male
• Twitching was observed slight in 5 males and females, moderate in 4 females and severe in 1 female
Most of the above-mentioned findings were observed on different days of the study, starting on day 0 after treatment and were present most of the time within the whole study period, alternating in occurrence in each individual animal. The findings were more pronounced in females.
Due to a clear dose-response relationship, these findings were assessed as related to the test-substance.


BODY WEIGHT AND WEIGHT GAIN
Body weight was statistically significantly decreased of -13.9% in males and not statistically significantly decreased of -6.5% in females of group 3 (60 mg/kg bw/day) on day 7; it was also decreased during the administration period, up to -8.6% in males and -4.4% in females of group 3 (45 mg/kg bw/day) on day 14, up to -7.7% in males on days 7 and 21 and -6.3% in females of group 2 (30 mg/kg bw/day) on day 7 and up to -4.8% in males on days 7 and 28 and -4.3% in females of group 1 (10 mg/kg bw/day) on day 7.
In conclusion it can be stated that decreased body weight is more pronounced in males than in females.
All above-mentioned body weight deviations to control groups were assessed to be substance-related.

Body weight change was statistically significantly decreased of -60.8% in males and -50.9% in females of group 3 (60 mg/kg bw/day) on day 7; it was also decreased during the administration period in all dosed animals, up to -24.4% in males and -20.5% in females of group 3 (45 mg/kg bw/day) on day 14, statistically significantly up to -37.7% in males and -48.7% in females of group 2 (30 mg/kg bw/day) on day 7 and -20.9% in males and statistically significantly of -35.3 in females of group 1 (10 mg/kg bw/day) on day 7.
All above-mentioned body weight change deviations to control groups were assessed to be substance-related.


FOOD CONSUMPTION
Food consumption was reduced in all dosed animals; statistically significantly of -29.4% in males and -28.2% in females of group 3 (60 mg/kg bw/day) on day 7; up to -11.9% in males and statistically significantly of -15.3% in females of group 3 (45 mg/kg bw/day) on day 14; up to -9.8% in males and statistically significantly of -18.0% in females of group 2 (30 mg/kg bw/day) on day 7 and up to -6.1% in males and statistically significantly of -12.3% in females of group 1 (10 mg/kg bw/day) on day 14.
Due to a clear dose-response relationship, the influence on food consumption was assessed to be substance-related.


FOOD EFFICIENCY
Food efficiency was statistically significantly decreased in both sexes of group 3 (60 mg/kg bw/day) on day 7, statistically significantly increased in males of group 3 (45 mg/kg bw/day) on day 14 and not statistically increased in both sexes during the administration period; food
efficiency was also statistically significantly decreased in both sexes of group 2 (30 mg/kg bw/day) and in females of group 1 (10 mg/kg bw/day) on day 7.
Because of a clear relationship to other clinical findings, like body weight data and food consumption, this finding was assessed as substance-related.

On several occasions, food efficiency differed (increased/decreased values) in treatment groups 2 and 1 from controls in males and females on some days of the study. These findings were spontaneous in nature and therefore not substance-related.

WATER CONSUMPTION
Water consumption was predominantly increased in all dosed animals, more pronounced in females; viz of +13.9% in males and statistically significantly of +36.9% in females of group 3 (60 mg/kg bw/day) on day 7; up to +8.1% in males on day 28 and +24.7% in females of group 3 (45 mg/kg bw/day) on day 14; up to +6.3% in males on day 21, +26.4% in females on day 14 and statistically significantly of +24.6% in females of group 2 (30 mg/kg bw/day) on day 28 and up to +14.0% in males on day 21 and +27.3% in females of group 1 (10 mg/kg bw/day) on day 14.
Because of the high toxic effect of the test-substance in all dosed animals, this was assessed as substance-related.
The decreased values of -8.5% in males group 3 (45 mg/kg bw/day) and -13.6% in males of group 2 (30 mg/kg bw/day) on day 14, were assessed as outliers and therefore not substance-related.

OPHTHALMOSCOPIC EXAMINATION
No data

HAEMATOLOGY
After administration of the test substance for 4 weeks there was noticed a statistically weakly significant decrease of the haemoglobin concentration in the male 10 mg/kg bw/d as well as the 45 mg/kw bw/d dose group. This decrease of the heamoglobin levels was not accompanied by a deviation of any other red blood cell marker. Compared to the historical control data (min value: 9.1 mmol/L, max. value 9.8 mmol/L), the median of the control rats in this study was slightly higher than the historical controls, whereas the value of the 10 mg/kg bw/d as well as the 45 mg/kg bw/d dose groups were within the historical controls.
Therefore, this decrease of the heamoglobin concentration is not regarded as substance-related.

CLINICAL CHEMISTRY
In the male rats there was observed a decrease of the median of the total protein level beginning in the 30 mg/kg bw/d dose group. This decrease was reflected in the decrease of the globulin fraction in the 45 mg/kg bw/d dose group.
Even in the female rats of the 45 mg/kg bw/d dose group the total protein as well as the albumin and globulin fraction were decreased even not statistically significant. This decrease is accompanied by a slight but not statistically significant decrease of the serum glucose and triglyceride values in the dosed animals of both sexes.

Additionally, there was found a decreased median of the potassium levels in the 10 mg/kg bw/d as well as 30 mg/kg bw/d dose group of the female rats. This deviation was inconsistent, when compared with the other sex, and lack dose-response relationship. Accordingly, this finding is considered to be of no toxicological significance.

URINALYSIS
There were no treatment-related changes in the urinalyses parameters measured.

NEUROBEHAVIOUR
Functional observational battery.
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.
Beside this, the following findings were observed and have to be assessed individually:

Home cage observations:
“Twitching” was observed in 3/4 males and 3/3 females of group 3 (45 mg/kg bw/day). This was assessed as substance-related, because of the systemic toxicity of the test article.

Open field observations:
No substance-related findings were observed.

Sensorimotor tests/reflexes:
“Twitching” was observed in 2/4 males and 2/3 females of group 3 (45 mg/kg bw/day)
This was assessed as substance-related, because of the systemic toxicity of the test article.

Quantitative parameters:
“Rearing” was increased of +48.0% in males and +63.7% females of group 3 (45 mg/kg bw/day), of +26.3% in males and +21.4% in females of group 2 (30 mg/kg bw/day) and +10.5% in males and +14.3% in females of group 1 (10 mg/kg bw/day).
Due to a clear dose-response relationship, this was assessed as substance-related.

“Grip strength forelimbs” was reduced of -9.2% in males and -9.9% females of group 3 (45 mg/kg bw/day), of -39.9% in females of group 2 (30 mg/kg bw/day) and -8.8% in females of group 1 (10 mg/kg bw/day).
“Grip strength hindlimbs” was reduced of -16.8% in males and -5.9% females of group 3 (45 mg/kg bw/day), of -24.4% in males and -26.2% in females of group 2 (30 mg/kg bw/day) and -17.9% in males group 1 (10 mg/kg bw/day).
This was assessed as substance-related, because of the systemic toxicity of the test article.

Motor activity measurement
Regarding the overall motor activity statistically significantly increased deviations were measured in males and females of group 3 (45 mg/kg bw/day), 2 (30 mg/kg bw/day) and 1 (10 mg/kg bw/day).
This was assessed as being substance-related, caused by systemic toxicity.

Comparing the single intervals with the control groups, interval 4 in all dosed males and interval 12 in group 3 male animals showed a statistically significantly increase.
These findings were clearly associated to the treatment with the test article and the abovementioned systemic toxicity.


ORGAN WEIGHTS
There were no treatment related weight deviations recorded (absolute and relative organ weights)

The significant weight decrease of the thymus and the significant weight increase of the brain in the mid dose group females is regarded to be incidental and of no biological relevance (no dose-response relationship, no substance-induced histopathological lesions in the high dose)

GROSS PATHOLOGY
There were no treatment-related findings noted.

HISTOPATHOLOGY: NON-NEOPLASTIC
All findings noted are spontaneous or incidental in nature and not related to treatment

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic toxicity/neurotoxicity
Effect level:
< 10 mg/kg bw/day (actual dose received)
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: body weight; food consumption; water consumption; motoneural effects and motor activity

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The following test substance-related adverse findings were noted:

60 mg/kg body weight/day (from day 0 until day 7)

• 1 male and 1 female were found dead on day 6 and on day 7, respectively

• Convulsions, clonic were observed moderate in 4 females, severe in 2 females, epileptoid

slight in 1 male, moderate in 1 female, severe in 2 males and 1 female, tonic slight in 1

female and severe in 1 male and 2 females

• Hyperesthesia was observed slight in 2 males and 5 females, moderate in 4 females and

severe in 1 male and 2 females

• Hyperexcitability was observed slight in 2 males and 5 females, moderate in 4 females and

severe in 1 male and 2 females

• Nose, discharge, red was observed moderate in 1 female

• Piloerection was observed in 2 females

• Salivation was observed slight in 2 females, moderate in 1 male and 4 females and severe

in 1 male and female

• Skin, paleness was observed moderate in 2 females

• Twitching was observed slight in 3 males and 5 females, moderate in 2 males and 3

females and severe in 1 male and 3 females

• Food consumption in both sexes was statistically significantly reduced of -29.4% in males

and -28.2% in females on day 7

• Water consumption in both sexes was increased of +13.9% in males and statistically

significantly increased of +36.9% in females on day 7

• Body weight was statistically significantly decreased of -13.9% in males and not

statistically significantly decreased of -6.5% in females on day 7

• Body weight change in both sexes was statistically significantly decreased of -60.8% in

males and -50.9% in females on day 7

• Food efficiency in both sexes was statistically significantly decreased on day 7

45 mg/kg body weight/day (from day 8 until the end of the administration period)

• 1 female was prematurely sacrificed moribund on day 11

• Anogenital region, smeared with urine was observed severe in 1 female

• Convulsions, tonic were observed slight, moderate and severe in each 1 female

• Hyperactivity was observed in 1 female

• Hyperesthesia was observed slight in 2 males and 1 female

• Hyperexcitability was observed slight in 4 males and 3 females, moderate in 4 males and

females and severe in 1 male and 2 females

• Lateral position was observed in 1 female

• Nose, discharge, red was observed slight in 1 female and severe in 2 females

• Piloerection was observed in 2 males and 2 females

• Reduced general condition was observed slight in 1 female and severe in 2 females

• Salivation was observed slight in 1 female, moderate in 2 females and severe in 1 female

• Twitching was observed slight in 4 males and 3 females, moderate in 1 male and 4

females and severe in 2 females

• Food consumption in both sexes was reduced during the administration period, with a

maximum of -11.9% in males and statistically significantly of -15.3% in females on day 14

• Water consumption in both sexes was increased during the administration period, with the

exception of day 14 (-8.5%) in males and a maximum of +8.1% in males on day 28 and

+24.7% in females on day 14

• Body weight in both sexes was decreased during the administration period, with a

maximum of -8.6% in males and -4.4% in females on day 14

• Body weight change in both sexes was decreased during the administration period, with a

maximum of -24.4% in males and -20.5% in females on day 14

• Food efficiency in males was statistically significantly increased on day 14

• Twitching was observed during FOB (home cage observation) in 3/4 males on day 26 and

3/3 females on day 27

• Twitching was observed during FOB (sensorimotor tests/reflexes) in 2/4 males on day 26

and 2/3 females on day 27

• Rearing during FOB (quantitative parameters) was increased of +48.0% in males and

+63.7% in females

• Grip strength forelimbs and grip strength hindlimbs during FOB (quantitative parameters)

was reduced of -9.2% and -16.8% in males and -9.9% and -5.9% in females

• Motor activity was statistically significantly increased in both sexes

• Decreased total protein and globulin levels in both sexes

• Decreased albumin levels in the females

30 mg/kg body weight/day (during the entire administration period)

• Convulsions, tonic were observed moderate in 1 male

• Hyperactivity was observed in 1 female

• Hyperesthesia was observed slight in 1 male and 5 females

• Hyperexcitability was observed slight in 5 males and 5 females, moderate in 3 males and 4

females and severe in 1 female

• Piloerection was observed in 1 male

• Twitching was observed slight in 5 males and females, moderate in 4 females and severe

in 1 female

• Food consumption in both sexes was reduced during the administration period, with a

maximum of -9.8% in males and statistically significantly of -18.0% in females on day 7

• Water consumption in both sexes was increased during the administration period, with the

exception of day 14 (-13.6%) in males and a maximum of +6.3% in males on day 21, a

maximum of +26.4% in females on day 14 and statistically significantly of +24.6% in

females on day 28

• Body weight in both sexes was decreased during the administration period, with a

maximum of -7.7% in males on days 7 and 21 and -6.3% in females on day 7

• Body weight change in both sexes was decreased during the administration period, with a

statistically significantly maximum of -37.7% in males and -48.7% in females on day 7

• Food efficiency in both sexes was statistically significantly decreased on day 7

• Rearing during FOB (quantitative parameters) was increased of +26.3% in males and

+21.4% in females

• Grip strength hindlimbs during FOB (quantitative parameters) was reduced of -24.4% in

males and in forelimbs and hindlimbs of -39.9% and -26.2% in females

• Motor activity was statistically significantly increased in both sexes

• Decreased total protein level in the males

10 mg/kg body weight/day (during the entire administration period)

• Food consumption in both sexes was reduced during the administration period, with a

maximum of -6.1% in males and statistically significantly of -12.3% in females on day 14

• Water consumption in both sexes was increased during the administration period, with a

maximum of +14.0% in males on day 21 and +27.3% in females on day 14

• Body weight in both sexes was decreased during the administration period, with a

maximum of -4.8% in males on days 7 and 28 and -4.3% in females on day 7

• Body weight change in both sexes was decreased during the administration period, with a

maximum of -20.9% in males and statistically significantly of -35.3% in females on day 7

• Food efficiency in females was statistically significantly decreased on day 7

• Rearing during FOB (quantitative parameters) was increased of +10.5% in males and

+14.3% in females

• Grip strength hindlimbs during FOB (quantitative parameters) was reduced of -17.9% in

males and in forelimbs of -8.8% in females

• Motor activity was statistically significantly increased in both sexes

Applicant's summary and conclusion