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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 21 September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
30 September 1996 (including additional testing for Neurotoxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Description white powder
Purity 100.3%

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is recognized by international guidelines as the recommended test system for repeat dose toxicity studies.
Strain used: HanRcc:WIST (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, Fuellinsdorf, Switzerland
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks (49 days)
- Weight at study initiation: Males: 161.2 - 185.3 g (mean: 171.2 g)
Females: 130.9 - 153.7 g (mean: 144.0 g)
- Fasting period before study: no
- Housing: Groups of 5 in Makrolon type-4 cages
- Diet: pelleted standard Kliba Nafag 3433 ad libitum
- Water: tap-water ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The absence of contaminants in food and water was confirmed and certified.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3°C
- Humidity: 30-70%
- Air changes: 10 - 15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light (Music during light cycle)


IN-LIFE DATES: From: 2007-02-28 To: 2007-06-27

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Frequency of Administration: Once daily
Dose Volume: 10 mL/kg body weight
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: Group 1 (Control): 0 mg/mL
Group 2 (250 mg/kg bw/day): 25 mg/mL
Group 3 (500 mg/kg bw/day): 50 mg/mL
Group 4 (1000 mg/kg bw/day): 100 mg/mL

- Amount of vehicle (if gavage): 10 mL/kg bw
- Frequency of preparation: Daily
- Administration within 4 hours after preparation of dose formulations.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical verification (identity, concentration, homogeneity, stability) was performed during the course of the study under GLP regulation.
Samples of dose formulations (top, middle, bottom) were taken from each group at 4 different treatment days. The test item concentrations were determined by HPLC coupled to an UV detector and quantified with the area under the peak.
The identity of the test item was confirmed. The test material concentration in all samples were found to be in the predefined acceptance range of +/-20% of the nominal content. The homogenous distribution of the test item in the dose formulations was demonstrated.
In conclusion, the results obtained during formulation analysis confirmed the correct preparation and storage of application formulations during the conduct of this study.
Duration of treatment / exposure:
91 or 92-days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
15 males; 15 females (Groups 1 and 4)
10 males, 10 females (Groups 2 and 3)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 14-day GLP dose-range-finding study in rats (oral), where no adverse effects were observed.
- Rationale for animal assignment: Computer-generated random algorithm
- Fasting period before blood sampling for clinical biochemistry: 18 hours
- Rationale for selecting satellite groups: Recovery animals for control and highest dose group as proposed in Guideline
- Post-exposure recovery period in satellite groups: 27 days
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during pretest, treatment and recovery and before necropsy

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

Body weight gain: Yes
- Body weight gain calculated from body weights

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Acclimatitation period, week 13, week 17 (recovery)
- Dose groups that were examined: All groups at acclimatization; Control and high dose group at week 13 and week 17.

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Time schedule for examinations: week 13 and week 17
- Dose groups that were examined: All dose groups at week 13 and all recovery animals at week 17(control and high dose)
- Parameters investigated: Grip strenght; Locomotor activity;

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after week 13 and week 17
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (18 hours)
- How many animals: all animals
- Parameters examined:
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Platelet (thrombocyte) count
Reticulocyte count
Reticulocyte maturity index
Methemoglobin
Heinz bodies (slides were prepared and evaluated due to slight changes seen in methemoglobin levels)
Total leukocyte count
Differential leukocyte count
Coagulation:
Thromboplastin time
Activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after week 13 and week 17
- Animals fasted: Yes (18 hours)
- How many animals: all animals
- Parameters examined:
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Aspartate aminotransferase
Alanine aminotransferase
Lactate dehydrogenase
Glutamate dehydrogenase
Creatine kinase
Alkaline phosphatase
Gamma-glutamyl-transferase
Sodium
Potassium
Chloride
Calcium
Phosphorus inorganic
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: after week 13 and week 17
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Urine was collected during the 18-hour fasting period
- Parameters examined.:
Volume (18 hours)
Specific gravity (relative density)
Osmolality
Color
Appearance
pH
Nitrite
Protein
Glucose
Ketones
Urobilinogen
Bilirubin
Erythrocytes
Leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 13 and week 17
- Dose groups that were examined: All dose groups at week 13 and all recovery animals at week 17(control and high dose)
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see any other information)

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

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Minor clinical signs such as hair loss, scabs, fissures, or reddish sores were noted for different time periods during acclimatization and treatment in single males of all groups and in single females treated with 1000 or 250 mg/kg/day or of the control group.
The same clinical signs as in general daily cageside observation (hair loss, crusts, fissures) were noted in detailed weekly observations performed during weeks 1-12 and 14-16. Additionally, vocalization was noted in one control male (no. 13) in week 9.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male treated with 1000 mg/kg/day (No. 45) was found dead on day 66 without any prior clinical signs. Macroscopically, dark red discoloration was found in the lung associated with lung congestion at microscopical examination. These findings may be related to a gavage error. This death is considered to be not test item-related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item-related effects on body weights and body weight gain were noted.
(Details see any other information on results incl. tables)
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test item-related effect on food consumption was noted.
(Details see any other information on results incl. tables)
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No test item-related changes were noted during the ophthalmoscopic examinations.
(Details see any other information on results incl. tables)
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related changes in hematology parameters were noted.
(Details see any other information on results incl. tables)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in clinical biochemistry parameters were noted.
(Details see any other information on results incl. tables)
Urinalysis findings:
no effects observed
Description (incidence and severity):
No findings in urinalysis parameters were noted.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test item-related effects on behaviour were noted.
(Details see any other information on results incl. tables)
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item related changes in organ weights were noted.
(Details see any other information on results incl. tables)
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic lesions were similar in nature and incidence between test item-treated and control groups at the end of treatment and recovery periods.
(Details see any other information on results incl. tables)
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopically, no lesions that distinguished controls from test item-treated animals were detected.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

GENERAL CAGESIDE OBSERVATIONS

No test item- related clinical signs were noted. Minor clinical signs such as hair loss, scabs, fissures, or reddish sores were noted for different time periods during acclimatization and treatment in single males of all groups and in single females treated with 1000 or 250 mg/kg/day or of the control group.

DETAILED CLINICAL OBSERVATIONS (WEEKLY)

The same clinical signs as in general daily cageside observation (hair loss, crusts, fissures) were noted in detailed weekly observations performed during weeks 1- 12 and 14- 16. Additionally, vocalization was noted in one control male (no. 13) in week 9.

FUNCTIONAL OBSERVATIONAL BATTERY

During detailed observations in week 13, dyspnea was noted in one control female (no. 60), and one female treated with 1000 mg/kg/day (no. 96) had a fissure of the right ear from week 13 of treatment throughout recovery. Grip Strength No differences were noted in the mean grip strength between Dihydroxyacetone- treated and control groups. Locomotor Activity No test item- related differences in locomotor activity were noted. In week 13 of treatment in males treated with 500 or 250 mg/kg/day, the total locomotor activity and in males treated with 250 m/kg/day the locomotor activity at 20- 30 minutes were lower than in the respective controls. The differences were statistically significant but without dose- relation. There were no corresponding findings in females. After recovery, locomotor activity of females treated with 1000 mg/kg/day at 20- 30 and 50- 60 minutes and in total was slightly decreased with statistical significance. However, there was no corresponding finding in week 13 of treatment. Thus, the above mentioned findings are considered not to be test item- related.

OPHTHALMOSCOPY

No test item- related changes were noted during the ophthalmoscopic examinations.

Typical background findings - at incidences similar to those observed spontaneously in untreated rats of this strain and age- (corneal opacity, persistent pupillary membrane, persistent hyaloid vessel in vitreous body and hemorrhage in the vitreous body) were noted in males and females without dose relation at acclimatization, treatment and recovery examination. On day 85 of treatment, corneal opacity was noted in 6 of 14 males treated with 1000 mg/kg/day as compared to 1 of 15 control males. The difference reached statistical significance. Corneal opacity was also found in 7 of 15 females treated with 1000 mg/kg/day. However, it was additionally found in 7 of 15 females of the control group, and corneal opacity was mostly uni- lateral (bilateral in 2 males and 1 female treated with 1000 mg/kg/day and 2 control females). Thus, this finding is considered to be incidental.

FOOD CONSUMPTION

No test item- related effect on food consumption was noted. In males treated with 500 mg/kg/day, the absolute food consumption was very slightly lower than in controls over the complete treatment period. The difference did not reach statistical significance. In all other test item- treated males and females, the absolute food consumption during treatment was comparable to that of the respective controls. The relative food consumption of the test item- treated animals during treatment was comparable to that of their respective controls. During recovery, the absolute food consumption of males treated with 1000 mg/kg/day was very slightly lower than that of the respective controls. However, relative food consumption in all animals and absolute food consumption in females treated with 1000 mg/kg/day were similar to the respective controls.

BODY WEIGHTS

No test item- related effects on body weights and body weight gain were noted. The mean body weight of test item- treated females at all dose levels and controls was comparable during treatment and recovery. In test item- treated males at all dose levels, the mean body weight during acclimatization, treatment, and recovery was slightly lower than in controls. The difference was very minor in those treated with 1000 mg/kg/day (reaching statistical significance on days 2, 7, and 14 of recovery) and slight in those treated with 250 mg/kg/day (reaching statistical significance once on day 1 of treatment). In males treated with 500 mg/kg/day, the difference was clear and statistically significant at all time points measured except day 1 of acclimatization and day 43 of treatment. Hence, this finding showed no dose relation and was inconsistent across sexes. It is considered not to be test item- related. Starting in week 3 to the end of treatment, the body weight gain of test item- treated females of all dose groups was very slightly higher than that of the controls without statistical significance. In males treated with 500 mg/kg/day, the mean body weight gain was slightly lower than in controls from week 2 to the end of treatment. The difference reached statistical significance once on day 78. These differences were minor and without dose- relation. Thus, they are considered to be incidental. Body weight gain during recovery generally showed no difference between test item- treated animals and controls. In females treated with 1000 mg/kg/day, mean body weight gain during recovery was lower than in the respective controls with statistical significance once on day 14. This difference was isolated and was considered not to be related to the test item.

CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

No test item- related changes in hematology parameters were noted. After 13 weeks of treatment, red cell volume distribution width was slightly decreased in males (- 6%) and females (- 9%) treated with 1000 mg/kg/day and females (- 9%) treated with 250 mg/kg/day. Mean corpuscular hemoglobin concentration was very slightly decreased with statistical significance in males (- 2%) and females (- 1%) treated with 1000 mg/kg/day. Reticulocyte count (absolute) was slightly but significantly (statistically) decreased in males treated with 250 mg/kg/day (- 12%). A very slight but statistically significant decrease was noted in methemoglobin in females treated with 250 or 500 mg/kg/day (- 12%). No difference in Heinz bodies, which anyway generally occurs only as a consequence of increased methemoglobin, was seen. Additionally, a slight and statistically significant increase in prothrombin time was seen in males treated with 1000 mg/kg/day (+ 5%). After recovery, red cell volume distribution width was still slightly decreased in males (- 6%) and females (- 26%) treated with 1000 mg/kg/day. The difference did not attain statistical significance. These and all other changes noted were within the normal range of historical data for rats of this strain and age. As these changes were without corresponding changes in histopathology or other investigated parameters, they are considered to be incidental.

CLINICAL BIOCHEMISTRY

After 13 weeks of treatment, decreases in lactate dehydrogenase were noted in males treated with 250 or 500 mg/kg/day (- 34% and –38%). Decreased glutamate dehydrogenase was noted in males at all dose levels (- 14%, - 14%, and –22%) and in females treated with 250 or 500 mg/kg/day (- 40%, - 45%). Creatine kinase was slightly decreased in males (- 14%, - 31%, - 24%) and females (- 20%, - 14%, - 6%) at all dose levels. These changes were without statistical or biological significance and without dose- relationship. Thus, they are considered to be incidental. Slightly increased sodium and chloride levels were noted in males at all dose levels (+ 1%, + 1%, + 2% for both) and females treated with 500 or 1000 mg/kg/day (+ 1%, + 2% for sodium, + 2%, + 2% for chloride). The difference attained statistical significance in males at all dose levels and in females treated with 1000 mg/kg/day. However, the low magnitude of these changes was within the normal range of historical data for rats of this strain and age and in the absence of changes in other electrolytes, these findings are not considered to be test item- related. After the recovery period, lactate dehydrogenase and creatine kinase were still decreased in males treated with 1000 mg/kg/day but increased in females treated with 1000 mg/kg/day. A slight statistically significant decrease was noted in phosphorus and in total protein in males treated with 1000 mg/kg/day only. These and all other changes were within the normal range of historical data for rats of this strain and age and were considered to be incidental.

URINALYSIS

No findings in urinalysis parameters were noted.

PATHOLOGY

ORGAN WEIGHTS

No test item related changes in organ weights were noted.

After 13 Weeks: In males treated with 1000 mg/kg/day, absolute and relative thyroid weights and absolute and relative liver weights were slightly increased while absolute and relative thymus weights were decreased. In females treated with 1000 mg/kg/day, increased absolute and relative thyroid, spleen, and ovarian weights were noted. None of these differences reached statistical significance.

After 17 Weeks: In males and females treated with 1000 mg/kg/day after recovery, decreases were noted in several absolute organ weights and brain to organ weight ratios, e.g. in thyroids, liver, and kidneys. These differences partly reached statistical significance. However, the changes were in opposite direction to changes directly after treatment or not present after treatment and are considered not to be test item- related (even if reaching statistical significance in males).

MACROSCOPIC FINDINGS

Macroscopic lesions were similar in nature and incidence between test item-treated and control groups at the end of treatment and recovery periods.

After 13 weeks of treatment, foci were found in the stomach of 1 male and female of the control group, 2 males treated with 250 mg/kg/day and 1 female treated with 500 mg/kg/day. One male treated with 250 mg/kg/day had nodules in the stomach. Foci in the thymus were noted in 1 male and 1 female treated with 500 mg/kg/day and 1 male treated with 1000 mg/kg/day. One male treated with 1000 mg/kg/day had foci in the mandibular lymph node and 2 males treated with 500 mg/kg/day had foci in the lacrimal glands. Nodules in the body cavities were found in 1 control male and 1 female treated with 1000 mg/kg/day. One male each, treated with 500 or 1000 mg/kg/day showed renal pelvic dilation and uterine dilation was noted in 1 female treated with 500 mg/kg/day. One female treated with 1000 mg/kg/day showed alopecia. One control male had a ruptured auricle.

These isolated findings were considered to be incidental.

Dark red discoloration of the lung was found in the 1 male treated with 1000 mg/kg/day which had spontaneously died (No. 45). This finding may be related to the procedure of gavage. After recovery, typical background findings, such as foci in the stomach mucosa, distended colon and accentuated lobular pattern of the liver in single control males, foci in the thymus of one female treated with 1000 mg/kg/day, and uterine dilation, watery cysts in the oviducts, and a watery cyst in the kidney in single control females were noted.

MICROSCOPIC FINDINGS

Microscopically, no lesions that distinguished controls from test item- treated animals were detected.

Applicant's summary and conclusion

Conclusions:
Oral administration of the test material to Wistar rats at doses of 250, 500 and 1000 mg/kg bw/day, for 13 weeks resulted in no test item-related mortality. There were no test item-related findings in clinical signs, functional observational battery, ophthalmoscopy, food consumption, body weights, hematology, clinical chemistry, or urinalysis. At necropsy, no test item-related changes in organ weights or macroscopic findings were noted. Microscopically, no lesions that distinguished controls from test item-treated animals were detected. Based on the results of this study, the dose level of 1000 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL).
Executive summary:

GENERAL

In this subchronic toxicity study, the test item was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 250, 500 and 1000 mg/kg body weight/day for a period of 13 weeks. A control group was treated similarly with the vehicle, bidistilled water, only.

The groups comprised 10 animals per sex, which were sacrificed after 13 weeks of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg bw/day. These animals were treated for 13 weeks and then allowed a 4-week 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 period. Ophthalmoscopic examinations were performed at pretest, the end of the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 13 and week 17.

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.

DOSE FORMULATION ANALYSIS

The results indicate the accurate use of the test item and bi-distilled water as vehicle during this study. Application formulations were found to be homogenously prepared and sufficient formulation stability under storage conditions was approved.

MORTALITY / VIABILITY

There were no deaths attributed to the test item.

CLINICAL SIGNS

No test item-related clinical signs were noted.

FUNCTIONAL OBSERVATIONAL BATTERY

No test item-related clinical signs were noted during detailed observation in weeks 13 and 17.

Grip Strength

No differences were noted in the mean grip strength when compared with the controls.

Locomotor activity

No test item-related differences in locomotor activity were noted.

OPHTHALMOSCOPY

No test item-related changes were noted during the ophthalmoscopic examinations.

FOOD CONSUMPTION

No test item related-effect on food consumption was noted.

BODY WEIGHT

No test item-related effects on body weights and body weight gain were noted.

CLINICAL LABORATORY INVESTIGATIONS

Hematology

No test item-related changes in hematology parameters were noted.

Clinical Biochemistry

No test item-related changes in clinical chemistry parameters were noted.

Urinalysis

No test item-related changes in urinalysis parameters were noted.

ORGAN WEIGHTS

No test item-related changes in organ weights were noted.

MACROSCOPIC / MICROSCOPIC FINDINGS

There were no macroscopic or microscopic findings that were considered to be related to treatment with the test item.

ASSESSMENT

Oral administration of the test item to Wistar rats at doses of 250, 500 and 1000 mg/kg bw/day, for 13 weeks resulted in no test item-related mortality.

There were no test item-related findings in clinical signs, functional observational battery, ophthalmoscopy, food consumption, body weights, hematology, clinical chemistry, or urinalysis. At necropsy, no test item-related changes in organ weights or macroscopic findings were noted.

Microscopically, no lesions that distinguished controls from test item-treated animals were detected.

Based on the results of this study, the dose level of 1000 mg/kg bw/day of the test item was established as the no-observed-effect-level (NOEL).