MMCT

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:

30 Nov 2005 - 03 Mar 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

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 in 1995

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Deviations:

yes

Remarks:

no satellite group included, unclear if coagulating gland was examined

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 1996

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Rj:WI (IOPS HAN)

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% aqueous solution

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were checked twice daily for moribundity and mortality. Clinical signs were recorded at least once daily for all animals.
- Cage side observations included evident signs of toxicity, moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: On the first day of test subtance administration, afterwards at weekly intervals throughout the treatment period and before necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, the weight of food supplied and of that remaining at the end of the food consumption period was recorded weekly for all animals during the treatment period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule for examinations: during study Week 4
- Dose groups that were examined: all surviving animals from control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: yes (Isoflurane inhalation)
- Animals fasted: yes
- How many animals: all animals
- Parameters examined: red blood cell count, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocyte count, white blood cell count, differential count evaluation, platelet count and prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: yes (Isoflurane inhalation)
- Animals fasted: yes
- How many animals: all animals
- Parameters examined: total bilirubin, glucose, urea, creatinine, total protein, albumin, total cholesterol, triglycerides, chloride, sodium, potassium, calcium, inorganic phospherus, aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase

URINALYSIS: Yes
- Time schedule for collection of urine: on study Day 26
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: not specified
- Parameters examined: general appearance, volume, pH, urinary refractive index, glucose, bilirubin, ketone bodies, occult blood, protein and urobilinogen; microscopic examination of the urinary sediment, presence of red vlood cells, white blood cells, epithelial cells, bacteria, casts and crystals

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: during study Week 4
- Dose groups that were examined: all dose groups
- Battery of functions tested: motor activity, sensory reactivity, grip strength and open field observations (changes in gait, posture, clonic or tonic movements, stereotypic or bizarre behaviour)

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

Sacrifice:
On study Days 29 and 30, all animals from all groups were sacrificed by exsanguination under deep anesthesia (by inhalation of Isoflurane). Animals were diet fasted overnight prior to sacrifice. All animals were necropsied. The necropsy included the examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded.

Gross necropsy was performed on the following organs: Adrenal gland, aorta, articular surface (femorotibial joint), bone sternum, bone marrow sternum, brain, cecum, colon, duodenum, epididymis, esophagus, eye, exorbital lachrymal gland, Harderian
gland, heart, ileum, jejunum, kidney, larynx/pharynx, liver, lung, mesenteric lymph node, submaxillary lymph node, mammary gland, nasal cavities, optic nerve, ovary, pancreas, parathyroid gland, pituitary gland, prostate gland, rectum, submaxillary salivary gland, sciatic nerve, seminal vesicle, skeletal muscle, skin, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testis, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus and vagina

The following organs were weighed for all selected animals at necropsy: Adrenal gland, brain, epididymis, heart, kidney, liver, ovary, pituitary gland, prostate gland, spleen, testis, thymus, thyroid gland (including parathyroid) and uterus (including cervix) were weighed fresh at scheduled sacrifice only. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
All the above mentioned samples except exorbital gland, larynx/pharynx and nasal cavities were embedded in paraffin wax. Histological sections, stained with hematoxylin and eosin, were prepared from all the animals in control group and high dose group.
Kidney, liver, lung, thyroid gland and macroscopic findings were prepared from all animals in intermediate groups. Histopathological examinations were performed on all the animals. In the control group and high dose groups, all organs were examined. Liver, kidney, lung and thyroid gland were examined in the intermediate groups. Significant macroscopic findings were also examined in all dose groups.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day: increased salivation in 5/5 males and 5/5 females on most occassions between study Days 8 and 29 (treatment-related) and wasted appearance in 1/5 males between study Days 22 and 24 (not treatment-related)
200 mg/kg bw/day: increased salivation in 5/5 males and 5/5 females on several occassions between study Days 10 and 29
50 mg/kg bw/day: increased salivation in 1/5 males on study Day 29

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A transient body weight loss in 1/5 males of the 500 mg/kg bw group was noted, but the finding was considered as incidental. The transient reduction resulted in a lower mean body weight on study Day 22 (-8%, not statistically significant) and a reduced mean body weight gain per day between study Days 15 and 22 (-82%, statistically not significant). The change was correlated to a lower food consumption in this animal at that time point.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A transient lower food consumption was observed in 1/5 males between study Days 15 and 22. The finding was considered to be incidental. The transient reduction resulted in slightly reduced food consumption in males (-14%, not statistically significant) on study Day 22.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Platelet counts were statistically significantly increased in males at 500 mg/kg bw/day (+20%). In isolation, this slight variation was considered to be incidental.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg bw/day there was an increase in total cholesterol (+54% in males, +43% in females) and in triglycerides (+140% in males, +155% in females). In addition, a slightly higher albumin concentration (+11%) was noted in males of the high dose group.
At 200 mg/kg bw/day, there was an increase in total cholesterol (+38% in males, +36% in females) and in triglycerides (+92% in males, +172% in females).
All findings were considered dose- and treatment-related and to be of toxicological relevance.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

There was a dose-related increase in urinary cellular casts in all treated males of all dose groups with increasing grade of severity (50 mg/kg bw/day: 1/4 males grade 1, 3/4 males grade 2; 200 mg/kg bw/day: 2/5 males grade 2, 2/5 males grade 3 and 1/5 males grade 4; 500 mg/kg bw/day: 4/5 males grade 3, 1/5 males grade 4). In addition, there was a statistically significant increase in mean urinary volume in males of the 200 (+79%) and the 500 mg/kg bw/day dose group (+79%). The findings were considered treatment-related and associated with kidney weight and histopathological findings.
At 500 and 200 mg/kg/day, a tendency towards lower mean urinary pH was noted in males (pH 6.0 and 6.1, respectively, compared to pH 6.7 in the control males, not statistically significant) and in females (pH 5.2 and 5.3, respectively, compared to pH 6.0 in the control females, statistically significant). Cellular casts were also noted (grade 1) in 1/5 females.

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):

At 500 and 200 mg/kg/day, mean absolute and relative liver weights were statistically significantly higher in males and females, when compared to controls. The increase in absolute liver weight in males was +17% at 200 mg/kg bw/day and +27% at 500 mg/kg bw/day; in the increase in females was +25% and +57% at 200 and 500 mg/kg bw/day, respectively. The mean liver to body weight ratio was +17% and +35% in males and +22% and +52% in females at 200 and 500 mg/kg bw/day; the mean liver to brain weight ratio was +17% and +25% in males and +25% and +56% in females at 200 and 500 mg/kg bw/day, respectively. These liver weight changes were dose-related and considered to be toxicologically relevant.
In addition, there was a dose-related and toxicologically relevant increase in the mean absolute and relative kidney weight of statistical significance observed in both sexes at 200 and 500 mg/kg bw/day. The increase in absolute kidney weight was +25% and +31% in males and +19% and +27% in females. The mean kidney to body weight ratio was +22% and +39% in males and +16% and +24% in females at 200 and 500 mg/kg bw/day; the mean kidney to brain weight ratio was +25% and +29% in males and +18% and +27% in females at 200 and 500 mg/kg bw/day, respectively. The changes in kidney weight were considered to be toxicologically relevant in both sexes.
A tendency towards higher mean absolute and relative adrenal gland weight was further observed in males of the high dose group (+17% and +27%, statistically not significant) and in females of the high dose group (+40% and +44%, statistically significant). Due to the absence of any histopathological abnormalities, the changes in adrenal gland weight were not considered to be toxicologically relevant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged livers were found in 2/5 males and 3/5 females at 200 mg/kg bw/day and in 5/5 males and 5/5 females at 500 mg/kg bw/day, respectively. In addition, pale kidneys were noted in 2/5 males and 0/5 females at 200 mg/kg bw/day and in 2/5 males and 2/5 females at 500 mg/kg bw/day. The findings were considered treatment-related and of toxicological relevance.

Neuropathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg/day, a tendency towards a lower overall mean exploratory locomotor activity was noted in males (-44%) and in females (-38%), in comparison to controls (not statistically significant). At 200 mg/kg/day, overall mean exploratory locomotor activity was also lower in males (-43%), in comparison to controls (not statistically significant). At 200 mg/kg/day in females and 50 mg/kg/day in both sexes, overall mean exploratory locomotor activity was comparable to control values. The findings were considered treatment-related and toxicologically relevant. There were no treatment-related findings in open field observations, sensory reactivity and grip strength. A slight decrease in hindlimb grip strength was observed in females at 500 mg/kg/day (-17%, not statistically significant) but considered to reflect inter-individual variations and not to be a treatment-related effect.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related histopathological changes were observed in the livers and kidneys of animals of both sexes. Minimal to moderate diffuse centrilobular hepatocellular hypertrophy was noted in 5/5 males and 3/5 females at 200 mg/kg bw/day and in 5/5 males and 5/5 females at 500 mg/kg bw/day. The effect was dose-related and correlated to a higher mean liver weight and gross necropsy observations. In addition, minimal to marked increases in the presence of hyaline droplets were noted in the proximal tubulues of the kindeys in males of all dose groups (5/5, 5/5 and 5/5 males at 50, 200 and 500 mg/kg bw/day). The effect was dose-related in incidence and severity and associated with the presence of granular casts in 3/5 and 3/5 males at 200 and 500 mg/kg bw/day. There were no histopathological changes in the kidneys of female animals.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

clinical signs

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

clinical signs

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, the NOAEL for systemic toxicity was < 50 mg/kg bw/day in male and 50 mg/kg bw/day in female rats.