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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 July 2007 to 11 May 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid - liquid: aqueous solution
Details on test material:
- Description: Clear colorless liquid
- Storage condition of test material: At room temperature (15-25 °C) away from direct sunlight
- Stability: Stable under storage conditions

Test animals

Species:
rat
Strain:
other: HanRcc: WIST
Details on species / strain selection:
Rat was recognized by international guidelines as a recommended rodent test system.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland.
- Age at study initiation: Ca. 7 weeks
- Weight at study initiation: Males: 155-181 g (mean 168 g); Females: 125-146 g (mean 135 g)
- Housing: Animals were housed in groups of three in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ Schill AG, 4132 Muttenz / Switzerland).
- Diet: Pelleted standard Kliba Nafag 3433 (batch no. 16/07) rat / mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Itingen was available ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 - 70%
- Air changes: Air-conditioned with 10 - 15 air changes per hour
- Photoperiod: 12-hour fluorescent light/12-hour dark cycle with music during the light period

IN-LIFE DATES: 04 July 2007 to 11 May 2008

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral ingestion is a possible route of human exposure.
Vehicle:
water
Remarks:
Bidistilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The dose formulations were prepared weekly to 100% to compensate for the purity. The appropriate volume of test substance was measured with syringes into a glass beaker and the vehicle added (volume:volume). The mixtures were prepared using a magnetic stirrer.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

DOSE VOLUME:
10 mL/kg bw/day

STABILITY OF DOSE FORMULATIONS:
- The test item formulations were found to be stable up to 7 days, according to information provided by the Sponsor (SEAC study number AH070081).

STORAGE OF DOSE FORMULATIONS:
The test item formulations were stored in the refrigerator (2-8 °C) in glass beakers. Aliquots were removed each day from the weekly supply of formulated test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
After experimental start, samples of the control group as well as three samples (top, middle and bottom) of 5 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Samples of 5 g of each concentration were taken on day 1 of treatment and during weeks 7 and 13 of treatment to confirm homogeneity and concentration.
Duration of treatment / exposure:
91/92 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
12 animals/sex/dose for 0, 100, 300 and 1000 mg/kg bw/day (treatment groups)
6 animals/sex/dose for 0 and 1000 mg/kg bw/day (recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats (RCC Study Number B19214).
- Rationale for animal assignment: Computer-generated random algorithm
- Post-exposure recovery period in satellite groups: 28 days
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
General cageside observations (daily): The animals were observed for clinical signs once before commencement of administration as well as twice daily on days 1 to 3, and once daily thereafter.
Viability / Mortality: Observations for viability / mortality were recorded twice daily.

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

FOOD CONSUMPTION:
- 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 RCC computer.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined:
Acclimatization Phase: Once in all animals (Satellite A and B);
Treatment Phase: During week 12: in control and high dose animals (Satellite A and B); As changes of toxicological relevance were not seen at the high dose level, the intermediate dose groups were not examined.
Recovery Phase: During week 16: in control and high dose animals (Satellite B).

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 13 Weeks (satellite A and B); After 17 Weeks (satellite B)
- Anaesthetic used for blood collection: Yes; Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia.
- Animals fasted: Yes; animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
- Parameters checked:
Haematology:
- Complete Blood Cell Count: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Reticulocyte count, Reticulocyte maturity index (low, medium, high fluorescence), Leukocyte count (total), Differential leukocyte count: Neutrophils, Eosinophils, Basophils, Lymphocytes, Monocytes, Large unstained cells, Platelet count
- Hemoglobin Derivatives: Methemoglobin, Heinz bodies
- Coagulation: Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time
Clinical chemistry: Glucose, Urea, Creatinine, Bilirubin total, Cholesterol total, Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Alkaline phosphatase, Gamma-glutamyl-transferase, Creatine kinase, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein total, Albumin, Globulin, Albumin/Globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: After 13 Weeks (satellite A and B); After 17 Weeks (satellite B)
- Animals fasted: Yes; Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolism cage.
- Parameters checked:
- Physical Examination: Urine volume (18 hour), Specific gravity (relative density), Color, Appearance
- Chemical Examination: pH value, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 12) thereafter.
Functional Observational Battery: During week 13, relevant parameters from a modified Irwin screen test were evaluated in all animals.
- Battery of functions tested: grip strength / motor activity

IMMUNOLOGY: No

OTHER:
- Estrus: The estrus cycle duration was determined for a two-week period in all control and treatment group females starting week 10 (Satellite A and B) and for a one-week period during week 15 (Satellite B).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

SACRIFICE:
After 13 Weeks: Satellite A
After 17 Weeks: Satellite B

All allocation A and B animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.

ORGAN WEIGHTS
The following organs were weighed before fixation and recorded on the scheduled dates of necropsy. Paired organs were weighed separately. Relative organ weights were calculated on the basis of the body weight and brain weight. Brain, heart, liver, thyroids/parathyroids, thymus, kidneys, adrenals, uterus, spleen, testis, epididymis, ovaries
The terminal body weight was recorded immediately prior to necropsy and the organ to terminal body weight ratios as well as organ to brain weight ratios were determined.

HISTOPATHOLOGY: Yes
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution except for eyes with optic nerve and Harderian gland which were fixed in Davidson's solution or epididymides and testes which were fixed in Bouin’s solution:
Adrenal glands#, Aorta#, Bone (sternum, femur including joint), Bone marrow (femur)#, Brain – including section of medulla/pons, cerebral and cerebellar cortex#, Cecum#, Colon#, Duodenum#, Epididymides* (fixed in Bouin's solution)#, Esophagus#, Eyes w/optic nerve (fixed in Davidson's solution)#, Harderian gland (fixed in Davidson's solution), Heart including auricles#, Ileum, with Peyer's patches#, Jejunum with Peyer's patches#, Kidneys#, Larynx, Lacrimal gland (exorbital), Liver#, Lungs, filled w/formalin at necropsy#, Lymph nodes - mesenteric and mandibular#, Mammary gland area#, Nasal cavity, Ovaries#, Pancreas#, Pituitary gland#, Prostate gland incl. coagulating glands#, Rectum#, Salivary glands - mandibular, sublingual#, Sciatic nerve#, Seminal vesicles#, Skeletal muscle#, Skin, Spinal cord - cervical, midthoracic, lumbar#, Spleen#, Stomach#, Testis* (fixed in Bouin's solution)#, Thymus#, Thyroid (incl. parathyroid gland, if possible)#, Tongue, Trachea#, Urinary bladder (filled w/formalin at necropsy)#, Uterus#, Vagina#, Gross lesions#
*Right organ only. Contralateral organ used for seminology.
Histotechnique: All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers and stained with hematoxylin and eosin.
Histopathology: Slides of all organs and tissues listed above (#) collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist.
Other examinations:
Seminology
Seminology and Spermatid Count: Sperm count, motility and morphology were assessed at necropsy in all control and treatment group males after 13 weeks (Satellite A) and 17 weeks (Satellite B).
Motility: At necropsy, an epididymal sperm sample was obtained from the left caudal epididymidis of each male. Each sample was diluted with a prewarmed (about 35°C) physiological medium, and immediately after being sampling, one hundred sperm were counted microscopically for determination of percentage of not motile, stationary motile and progressively motile sperm.
Morphology: A sperm sample from the caudal epididymidis was used for morphological assessment after fixation and Eosin staining. At least 500 sperm per sample were evaluated microscopically and classified.
Sperm, Spermatid Count: The left caudal epididymis and left testis were taken for determination of homogenization resistant spermatids and caudal epididymal sperm reserve. These tissues were frozen at -20 ± 5 °C pending evaluation. For evaluation the weighed tissues were placed in Triton-X-100 solution and homogenized with a blender (Ultra Turrax) and an ultrasonic water bath. Sperm or spermatid heads were counted microscopically using a Neubauer chamber.
Statistics:
The following statistical methods were used to analyze food consumption, body weight, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
General Cageside Observations (Daily):
Treatment (weeks 1-13)
- At 1000 mg/kg bw/day, soft feces were noted in males and females from day 53 of treatment onwards. Minor dermal findings (scabs or ear fissures) were noted in males only.
- At 300 mg/kg bw/day, soft feces were noted in females from day 76 of treatment onwards. Minor dermal findings were seen in one male (wounds) from days 75-79 of treatment.
- At 100 mg/kg bw/day, minor dermal changes (slight to moderate alopecia, sores, scabs and/or wounds) were noted in males at various time points throughout the treatment period. There were no findings evident in the females.
- Control animals showed minor dermal findings (alopecia, scabs) or ocular discharge (clear or reddish).
Recovery (weeks 14-17)
- Males previously treated with 1000 mg/kg bw/day showed soft feces on day 2 of recovery only.
- Minor dermal findings (alopecia, scabs and/or sores) were noted in the males during the recovery period. No findings were noted in females.

Detailed Clinical Observations (Weekly):
Treatment (weeks 1-12)
- At 1000 mg/kg bw/day, minor dermal findings (ear fissures) were noted in one male. No findings were evident in females.
- At 300 mg/kg bw/day, the iridic light reflex was absent in the right eye of a single male during the first treatment week only. No findings were evident in females.
- At 100 mg/kg bw/day, there were no findings recorded in any male or female.
- Control females showed minor dermal findings (scabs) or ocular discoloration (dark).
Recovery (weeks 14-16)
- No findings were evident in any animal previously treated with the test item.
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled necropsy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment (weeks 1-13)
- The mean absolute body weight and mean body weight gain of males and females compared favorably with the respective control values throughout the treatment period.
Recovery (weeks 14-17)
- The mean absolute body weight of males compared favorably with the controls throughout the recovery period. In general, the mean absolute body weight of the females previously treated with 1000 mg/kg bw/day was marginally lower than that of the control females. On day 15 of the recovery period, the difference attained statistical significance (p<0.05).
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Treatment (weeks 1-13)
- The mean food consumption values of the test item-treated males and females were generally similar to those of the respective controls.
Recovery (weeks 14-17)
- The mean daily food consumption of the males previously treated with 1000 mg/kg bw/day was marginally elevated throughout the recovery period.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment (week 12)
- At 1000 mg/kg/day, uni- or bilateral corneal opacities were noted. Persistent pupillary membrane or persistent hyaloid vessels were noted in males whereas only persistent hyaloid vessels were seen in females.
- In control animals, uni- or bilateral corneal opacities were noted. Persistent pupillary membrane or persistent hyaloid vessels were noted in males whereas only persistent hyaloid vessels were noted in females.
Recovery (week 16)
- At rats previously treated with 1000 mg/kg bw/day, uni- or bilateral corneal opacities were noted. Persistent pupillary membrane was noted in males. No further differences were noted in females.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment (weeks 1-13)
- In males treated with 1000 mg/kg bw/day, significantly elevated mean hemoglobin levels (p<0.05) were noted, and the mean thromboplastin time was significantly elevated (p<0.05) when compared with the controls. Females treated with 1000 mg/kg bw/day showed significantly lower mean corpuscular hemoglobin concentration (p<0.05), and, as in males, significantly elevated (p<0.01) elevated mean thromboplastin time.
- At 300 mg/kg bw/day, the mean relative basophil count of males was significantly elevated (p<0.01). The mean relative basophil count of females was unaffected, but the relative count of “large unstained cells” was significantly elevated (<0.05) when compared with controls.
- At 100 mg/kg bw/day, the mean relative and absolute basophil counts of males were significantly elevated (p<0.01 and p<0.05, respectively). Females were unaffected.
- All other parameters compared favorably with those of the respective control animals.
Recovery (weeks 14-17)
- In males treated previously with 1000 mg/kg bw/day, a significantly elevated mean relative basophil count (p<0.05) was noted, as was a significantly reduced mean absolute lymphocyte count (p<0.05) and a significantly reduced mean absolute monocyte count (p<0.05). The mean methemoglobin level was significantly higher in males (p<0.05) after the recovery period.
- After the recovery period, the females showed significantly reduced hemoglobin (p<0.05), significantly increased mean red cell distribution width (p<0.05), and significantly reduced mean absolute neutrophil count (p<0.05).
- All other parameters compared favorably with those of the respective control animals.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment (weeks 1-13)
- Due to insufficient sample volume, glutamate dehydrogenase was not measured in several rats after the end of the treatment period. This was considered to have had no impact on the interpretion of the clinical biochemistry results.
- Males treated with 1000 mg/kg bw/day showed significantly reduced creatinine levels (p<0.01) when compared with controls. The mean activity for alkaline phosphatase was significantly elevated (p<0.05). Potassium, phosphorus and protein levels were significantly elevated in males (p<0.05, p<0.01 and p<0.05, respectively). In females treated with 1000 mg/kg bw/day, the mean activity of lactate dehydrogenase was significantly elevated (p<0.05) when compared with controls. Significant differences noted in females treated with 1000 mg/kg bw/day included elevations in sodium (p<0.01), chloride (p<0.01), calcium (p<0.01), phosphorus (p<0.01), protein (p<0.01) and globulin (p<0.01).
- At 300 mg/kg bw/day, females showed elevations in sodium (p<0.01), potassium (p<0.05), chloride (p<0.01), calcium (p<0.05), phosphorus (p<0.01), protein (p<0.05) and globulin (p<0.05). The clinical biochemistry parameters of the males varied only slightly from those of the control males and were considered to be within the range of typical background variation.
- At 100 mg/kg bw/day, males showed significantly reduced creatinine levels (p<0.05) when compared with controls. Females showed elevations in sodium (p<0.01), and phosphorus (p<0.01) when compared with the controls.
- All other parameters compared favorably with those of the respective control animals.
Recovery (weeks 14-17)
- None of the findings seen after 13 weeks in the males previously treated with 1000 mg/kg bw/day persisted. In females previously treated with 1000 mg/kg bw/day, significantly reduced total bilirubin (p<0.01) was noted, as was significantly reduced creatine kinase (p<0.05).
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment (weeks 1-13)
- At 1000 mg/kg bw/day, the pH of the urine was slightly acidic. In males, the pH was 6.0 (p<0.01), whereas in females it was 5.9 (p<0.05). A significant number of erythrocytes were detected in the urine of males only (p<0.05).
- At 300 mg/kg bw/day, the pH of the urine was slightly acidic. In males, urine pH was 6.1 (p<0.01), whereas in females it was 6.0 (without statistical significance).
- No other differences to the control values were noted after 13 weeks’ treatment.
Recovery (weeks 14-17)
- No statistically significant differences in the urinalysis parameters were noted after the recovery period.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional Observational Battery (weeks 13 and 17)
Treatment (week 13)
- At 1000 mg/kg bw/day, minor dermal findings (ear fissures) were noted in one male. No findings were evident in females.

Grip Strength (week 13)
- The mean fore- and hind limb grip strength values of the test item-treated rats were not affected by treatment.
Locomotor Activity (week 13)
- There were no consistent changes in locomotor activity.
- At 1000 mg/kg bw/day, the locomotor activity of males was significantly elevated during 0-10 minutes (p<0.05), and significantly reduced during 30-40 minutes (p<0.01) and 50-60 minutes (p<0.05). In females, significantly reduced locomotor activity was recorded during 20-30 minutes (p<0.05).
- At 300 mg/kg bw/day, significantly reduced locomotor activity of males was noted at 30-40 minutes and 40-50 minutes (both p<0.01), and the overall locomotor activity (0-60 minutes) was significantly reduced (p<0.05). Females at this dose level showed reduced mean locomotor activity during 0-10 minutes (p<0.05), 20-30 minutes (p<0.01), 30-40 minutes (p<0.05) and 40-50 minutes (p<0.01), and the overall locomotor activity 0-60 minutes was reduced (p<0.01).
- At 100 mg/kg bw/day, only the males differed significantly from the control values, showing significantly increased locomotor activity during 0-10 minutes (p<0.05).

Recovery (week 17)
No test item-related changes were noted during week 17.
Grip Strength (week 17)
- The mean fore- and hind limb grip strength values of the test item-treated rats were not affected.
Locomotor Activity (week 17)
- In females treated previously with 1000 mg/kg bw/day, the mean locomotor activity was reduced during 40-50 minutes (p<0.05). Males were unaffected.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment (after week 13)
- At 1000 mg/kg bw/day, the mean absolute and relative organ weights of males were generally similar to those of the control males. In females, slightly elevated mean absolute liver weights and mean absolute kidney weights were noted. The latter difference attained statistical significance (p<0.05) when compared with the controls. The mean absolute adrenal weights of females were slightly elevated, as were mean absolute uterus weights. The mean organ-to-body weight ratios of females treated with 1000 mg/kg bw/day showed a statistically significant increase (p<0.05) in the mean liver-to-body weight ratio. Significantly elevated mean organ-to-brain weight ratios of the heart (p<0.05), liver (p<0.05) and kidneys (p<0.01) were noted in females.
- At 300 mg/kg bw/day, the mean absolute and relative organ weights were generally similar to those of the controls.
- At 100 mg/kg bw/day, the mean absolute and relative organ weights were generally similar to those of the controls.

Recovery (after week 17)
- Rats treated previously with 1000 mg/kg bw/day showed several differences to the control values. The significantly elevated mean absolute heart (p<0.05), liver (p<0.05), kidney (p<0.01) and adrenal (p<0.05) weights were noted in males. The mean absolute organ weights of females were unaffected.
- The mean organ-to-body weight ratios of males and females previously treated with 1000 mg/kg bw/day were similar to those of the respective controls.
- The following mean organ-to-brain weight ratios noted in males attained statistical significance: heart (p<0.05), liver (p<0.05), kidneys (p<0.01), adrenals (p<0.05). The mean organ-to-brain weight ratios of females previously treated with 1000 mg/kg bw/day were similar to those of the controls.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
After 13 weeks
The following macroscopical findings were noted after 13 weeks’ treatment.
- In rats treated with 1000 mg/kg bw/day, discoloration or foci on the thymus (male nos. 43, 48, female no. 105), dark red discoloration of the pancreas (male no. 51), dark red lung foci (male no. 44), liver enlargement (male no. 54), left exorbital lacrimal glands missing (male no. 52), uterine horn discoloration (female no. 105) were noted.
- In rats treated with 300 mg/kg bw/day, discoloration of and/or foci on the thymus (male nos. 34, 35, 40, 41, female nos. 98 and100), dark red discoloration of the pancreas (male nos. 36, 38, 42), nodule on the rectal wall (female no. 93), enlargement of the liver (male no. 40), and reddish discoloration uterine horns (female no. 100) were noted.
- In rats treated with 100 mg/kg bw/day, red foci or discoloration of the thymus (male nos. 22, and 27 female nos. 79, 83 and 87), dark red pancreas discoloration or foci (male nos. 23 and 28), a minor dermal injury (male no. 27), alopecia of the cheek region (male no 28) and a nodule in uterine adipose tissue (female no. 82) were noted.
- In control animals, a small number of macroscopical changes were noted after 13 weeks’ treatment. These changes included enlargement of the liver (male no. 9), renal pelvis dilation (male no. 8 and female no. 66), nodules in the pancreatic adipose tissue (male nos. 1 and 9), urinary bladder distension (male no. 5), foci on the thymus (male no. 8 and female nos. 66, 70, 71), discoloration of the mesenteric lymph nodes (male no. 5), foci or discoloration of the uterus (female no. 65 and 67, respectively), discoloration of the ovaries (female no. 67). These findings were considered to be within the range of normal background lesions that may be seen in rats of this strain and age in 13-week studies.

After 17 weeks
- In animals previously treated with the test item with 1000 mg/kg bw/day, unilateral foci on the seminal vesicles were recorded in two males (nos. 56 and 59), and reddish lung foci were also recorded on the latter male. Liver enlargement was noted in one male (no. 58) and reddish discoloration of the pancreas was noted one male (no. 60). No macroscopical findings were noted in females previously treated with the test item at 1000 mg/kg bw/day.
- In control animals, a small number of macroscopical changes were noted after 4 weeks’ recovery. These changes included dark red foci on the seminal vesicles (male nos. 56 and 59), enlargement of the liver (male no. 58), reddish lung foci (male no. 59), and discoloration of the pancreas (male no. 60), uterine cyst (female no. 77), and were considered to be within the range of normal background lesions that may be seen in rats of this strain and age in 13-week studies.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- No treatment-related microscopic changes were observed at the end of the treatment and recovery periods.
- The nodule reported in the body cavity of two control males correlated microscopically with inflammation in one case and with fat necrosis in the other. The nodule reported in the rectum of one female (no. 93) at 300 mg/kg bw/day corresponded to a well-differentiated carcinoma, however considered incidental.
- The minimal or slight tubular atrophy of the testes seen in two recovery males at 1000 mg/kg bw/day, and associated to cellular debris in the corresponding epididymides, was considered incidental and not related to the treatment with the test item. This was also supported by the absence of such changes at the end of the treatment period in males treated with 1000 mg/kg bw/day.
- The few other microscopic findings such as mixed cell infiltration in the kidneys, heart and in the liver, thymic congestion or uterine dilation sparsely distributed among the control and treated groups, were within the range and severity of spontaneous background lesions which may be observed in rats of this strain and age in this laboratory and considered to be of no toxicological significance.
Details on results:
Seminology:
- The comparison of sperm motility and sperm morphology did not show evidence of any test item-related changes.
- In males treated with 1000 mg/kg bw/day, the mean epididymal sperm count was significantly (p<0.01) lower than that of the control males. A slightly lower value was recorded in males treated with 300 mg/kg bw/day, but the difference did not attain statistical significance. These differences were considered to be unrelated to the test item, as they were still higher than the respective epididymal sperm counts registered in control males of reproduction toxicity studies.
- In the testicular sperm count, a very slight reduction was noted in males treated with 1000 mg/kg bw/day or 300 mg/kg bw/day. These differences were also considered to be fortuitous, as similar values have been observed in the control males of the reproduction studies referenced above.
- The mean epididymal and testicular sperm counts of males treated with 100 mg/kg bw/day were unaffected.

Estrus:
Treatment (weeks 1-13)
- After 13 weeks’ treatment, similar numbers of regular estrus cycles were recorded in the control and test item-treated groups. The number of irregular or prolonged cycles showed no relationship to dose.
Recovery (weeks 14-17)
- After 4 weeks’ recovery, similar numbers of regular estrus cycles were recorded in the control and test item-treated groups. The number of irregular or prolonged cycles showed no relationship to dose.

Effect levels

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
mortality
body weight and weight gain
food consumption and compound intake
ophthalmological examination
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
other: See 'Remarks'

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Analysis of Dose Formulations

The levels of test substance were measured in the samples from Day 1 and Weeks 7 and 13. The levels ranged from 97.1% to 101.9% of the nominal concentrations and the coefficients of variation for each set of dose solutions ranged from 0.21 – 1.52%. Test substance was not detected in the samples of dose vehicle.

The results obtained confirmed that the dosing solutions contained levels of test substance within acceptable ranges of the nominal values and were of satisfactory homogeneity.

Applicant's summary and conclusion

Conclusions:
Under these test conditions, the NOAEL for test substance was considered to be 1000 mg/kg bw/day in rats.
Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 408 and in compliance with GLP, test substance was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg bw/day for a period of at least 90 days. A control group was treated similarly with the vehicle, bidistilled water, only. The groups comprised 12 animals per sex which were sacrificed after 91/92 days of treatment. Additional 6 rats per sex and group were used at 0 and 1000 mg/kg bw/day. These animals were treated as those described above and then allowed a 28-day treatment-free recovery period after which they were sacrificed. During the study, clinical condition, detailed physical and arena observations, grip strength, motor activity, body weight, food consumption, ophthalmoscopy, hematology, blood chemistry, urinalysis, organ weight, sperm analysis, estrus, macropathology and histopathology investigations were undertaken.

 

The following parameters were considered to be unaffected by the test item: mortality, food consumption, body weights, weekly observations (weeks 1-12, 14-16), functional observational battery (including grip strength and locomotor activity at weeks 13 and 17), ophthalmoscopy, mean testicular sperm count and mean epididymal sperm count, duration of estrus, organ weights and ratios, macroscopical and microscopical evaluations.

 

Test item-related changes were restricted to:

Clinical Signs: Soft feces was noted in males and females treated with 300 and 1000 mg/kg bw/day. This finding was considered to be test item-related but was reversible during the recovery period.

Hematology: In males and females treated with 1000 mg/kg bw/day, the mean relative thromboplastin time was elevated when compared with the controls, and the differences exceeded the ranges of the historical control data.

Clinical Biochemistry: Although most remained within the historical control ranges, the differences noted in some parameters (sodium in all test item-treated females; protein in females at 1000 mg/kg bw/day and 300 mg/kg bw/day, globulin in females at 1000 mg/kg bw/day), exceeded the upper ranges, and were considered likely to represent functional effects of the test item.

Urinalysis: At 300 and 1000 mg/kg bw/day, the pH of the urine was slightly acidic in males and females at the end of the treatment period, and considered to be test item-related. This finding was not seen after recovery.

 

Under these test conditions, the NOAEL for test substance was considered to be 1000 mg/kg bw/day in rats.