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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
Study period:
11 May 2010 to 7 Jun 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
fertility, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 May 2010 to 7 Jun 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Related information:
Composition 1
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21 September 1998.
Deviations:
no
Qualifier:
according to
Guideline:
other: 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)
Remarks:
Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen, Switzerland
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Remarks:
RccHanTM: WIST(SPF)
Details on species / strain selection:
Recognized by international guidelines as a recommended test system.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
- Age: 7 weeks at delivery
- Weight at acclimatization: Males: 143.9 to 203.8 g, Females: 102.8 to 166.7 g
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK).
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 20/10) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: 10% ethanol in corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The dose formulations were prepared weekly. Based upon the results of dose formulation analyses performed during a non-GLP dose range finding study, the stability of the test item formulations was considered to be sufficient to justify weekly preparation.
- Prior to the preparation of the dose formulations, the test item was molten in the flask at 80 °C for 24 h to ensure homogeneity of the test item. Thereafter, the test item was allowed to cool down and solidify. Small flakes of test item were scraped from the block and weighed into a tared glass container on a suitable precision balance. Then ethanol was added (The volume of ethanol equalled 10% of the final volume of the dosing solution) and the mixture was stirred until the test item was completely dissolved. To ensure complete solution of the test item in ethanol, the dose formulation of group 5 with a concentration of 160 mg/mL was slightly heated. Then, corn oil was added to give the appropriate final concentration of the test item in the solution. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on a previous formulation trial conducted for the dose range finding study where 10% ethanol in corn oil was found to be the only vehicle suitable to achieve sufficient test item concentrations. Effects of ethanol administration to rats are well characterized, and 8 mL/kg of a 10% solution for 90 days are “well tolerated”.
- Concentration in vehicle: 16, 48 and 160 mg/mL dose solutions are corresponding to the doses of 80, 240 and 800 mg/kg bw/day
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- After treatment start and during weeks 6 and 13, the following samples of approximately 2 g were drawn in duplicate prior to dosing for analysis of homogeneity and concentration: one sample of the control group (group 1) as well as three samples (top, middle and bottom) of the vehicle group (group 2) and of each test item-treated group (groups 3, 4 and 5).
- Duplicate samples of about 2 g of each dose formulation of groups 2 to 5 were taken after treatment start to confirm stability (4 hours and 8 days at room temperature, 20 ± 5 °C).
- The test item was used as analytical standard.
- Acceptance criteria for dose formulation concentration, homogeneity and stability determinations were ±20% of nominal content.
Duration of treatment / exposure:
91/ 92 days
Frequency of treatment:
Daily
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Dose / conc.:
240 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15 females and 15 males in control, vehicle and top-dose group
10 females and 10 males in low and mid-dose group
Control animals:
yes, concurrent vehicle
other: corn oil only
Details on study design:
- Dose selection rationale:
The dose levels were selected based on a previous 14-day dose range-finding toxicity study in Wistar rats, using dose levels of 100, 300 and 1000 mg/kg/day. Body weight gain of males treated with 1000 mg/kg was reduced. In both sexes, liver and kidney weight was increased in animals treated with 1000 mg/kg. In females, increased liver weight was also observed in animals treated with 300 mg/kg, and increased adrenal weight was observed in females treated with 1000 mg/kg.

- Post-exposure recovery period:
28 days, for 5 additional animals in control, vehicle control and high dose group. After 91 days of treatment with ethanol-containing vehicle, rats may become addicted. To prevent ethanol-withdrawal symptoms, treatment with vehicle was continued in the vehicle group and the high-dose group throughout recovery. The control group was treated with corn oil during recovery.
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Observations for viability / mortality were recorded twice daily.

DETAILED CLINICAL OBSERVATIONS:
The animals were observed for clinical signs once before commencement of administration as well as twice daily during days 1 - 3 and daily on days 1 - 91/92 of the treatment period and once daily during days 1 - 28 of the recovery period.

BODY WEIGHT:
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

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.

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmoscopic examinations were performed once on all animals during acclimatization and on groups 1, 2 and 5 during week 13. The ophthalmoscopic examinations of both eyes of the animals were performed after the application of a mydriatic solution (Ciba Vision AG, 3172 Niederwangen / Switzerland) using a direct ophthalmoscope.

HAEMATOLOGY:
- Time schedule for collection of blood: After 13 and 17 (recovery) weeks
- Anaesthetic used for blood collection: Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia.
- Animals fasted: The 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. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
- 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 (slides were prepared but not evaluated)
- Coagulation: Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time

CLINICAL CHEMISTRY:
The following clinical biochemistry parameters were determined: Glucose, Urea, Creatinine, Bilirubin total, Cholesterol total, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Alkaline, phosphatase, Bile acids, Gamma-glutamyl-transferase, Creatine kinase, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein total, Albumin, Globulin, Albumin/Globulin ratio

URINALYSIS:
- Time schedule for collection of urine: After 13 and 17 (recovery) weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Urine was collected during the 18 hours fasting period into a specimen vial.
- Physical Examinations: Urine volume (18 hour), Specific gravity (relative density), Color, Appearance
- Chemical Examination: pH value, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes

NEUROBEHAVIOURAL EXAMINATION:
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 13) thereafter. Summary of parameters: appearance, motor, behavior, respiration, reflexes, miscellaneous (Lacrimation, Limbs cyanotic, Mydriasis, Miosis, Exophthalmos, Reduced muscle tone), grip Strength, locomotor Activity.
Estrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
Not applicable
Postmortem examinations (parental animals):
GROSS PATHOLOGY:
- Sacrifice: after 13 or 17 (recovery) weeks
- Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.
- Organs and tissues collected and examined: 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, Esophagus, Eyes w/optic nerve, Harderian gland, 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, Pharynx, 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, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland), Tongue, Trachea, Ureter, Urinary bladder filled w/formalin at necropsy, Uterus (w/ cervix, vagina and oviducts), All gross lesions.
- Weights were determined of; Adrenal glands, brain including section of medulla/pons, cerebral and cerebellar cortex, epididymides, heart including auricles, kidneys, liver, ovaries, spleen, testes, thymus, Uterus (w/ cervix, vagina and oviducts). Oregans were weighed before fixation and the weights were recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight and brain weight.

HISTOPATHOLOGY:
-Slides of all collected organs were 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. Organ and tissue samples taken from animalswhich died spontaneously or which were killed in extremis were evaluated similarly to those organs taken from animals of the high-dose group. From the animals of the low and mid dose groups, liver, kidneys, stomach, prostate gland, nasal cavities level I (males only), nasal cavities levels II, III and IV and pharynx were examined to establish a no-effect level.
Postmortem examinations (offspring):
Not applicable
Statistics:
The following statistical methods were used to analyze body weight, grip strength, locomotor activity, 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.
Reproductive indices:
Reproductive organ weight and examinations
Offspring viability indices:
Not applicable
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- No clinical signs were recorded in any animal during the acclimatization period and during the recovery period. During the treatment period, no clinical signs were recorded in animals treated with corn oil (group 1) and with vehicle (10% ethanol in corn oil, group 2) as well as in females treated with 80 and 240 mg/kg/day.
- One male treated with 80 mg/kg/day was noted with moderate salivation on a single day during week 13, this was probably caused by accidental regurgitation of the dosing solution and is thus related to the treatment procedure. No test item-related clinical signs were noted in males treated with 80 mg/kg/day.
- One male treated with 240 mg/kg/day was noted with slight dyspnea and slight to moderate salivation on two consecutive days during week 8. No other test item-related clinical signs were noted in males treated with 240 mg/kg/day.
- In males and females treated with 800 mg/kg/day, slight to moderate dyspnea and slight to moderated salivation were observed in single animals throughout the treatment period. These clinical signs were noted in various individuals and were mostly observed for a short period of several days only, never affecting more than 30% of the group. These clinical signs are considered test item-related.
- Any other findings recorded in single animals such as fissures, scabs and wounds are considered incidental.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- No premature deaths occurred during the acclimatization period and during the recovery period.
- Four animals died prematurely during the treatment period. One female treated with 240 mg/kg/day and one female treated with 800 mg/kg/day were found dead during week 6. One male treated with 800 mg/kg/day spontaneously died during week 11 and one male treated with vehicle, 10% ethanol in corn oil, spontaneously died after treatment during week 13. The death of the female with the dose of 240 mg/kg/day was caused by renal failure; the cause of death of the other decedents could not be established.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Compared to vehicle-treated animals, absolute body weight and body weight gain were statistically significantly reduced in males treated with 800 mg/kg/day from the second week and first week onwards, respectively, until the end of the treatment period.
- In females treated with 800 mg/kg/day, statistically significantly lower absolute body weight and body weight gain was observed during weeks 1-4, 6 and 8 of the treatment period compared to vehicle-treated animals.
- In females treated with 240 mg/kg/day, body weight gain was slightly reduced during the first half of the treatment period compared to vehicle-treated animals, with the difference attaining statistical significance during week 4; however, the animals recovered towards the end of the treatment period.
- During the 4-week recovery period, no statistically significant differences in absolute body weight between animals treated with corn oil, vehicle and 800 mg/kg/day were observed. Body weight gain of females treated with 800 mg/kg/day was increased in week 4 of the recovery period and body weight gain of males treated with 800 mg/kg/day was increased in weeks 2 to 4 of the recovery period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected by treatment.
Food efficiency:
not examined
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 observed. Findings such as slight corneal opacity, persistent pupillary membranes or persistent hyaloid vessels observed in all groups were within the normal range of background lesions observed for animals of this strain and age.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of treatment, the following changes in hematology parameters observed in animals treated with the test substance compared to animals treated with vehicle only were considered test item-related:
- Increased prothrombin time (PT) and decreased partial thromboplastin time (PTT) observed in animals treated with 800 mg/kg/day.
- A shift in the reticulocyte count in females treated with 800 mg/kg/day characterized by an increase in low-fluorescence reticulocytes and a decrease in medium and highfluorescence reticulocytes. This shift was also observed in males treated with 800 mg/kg/day; however, only the decrease in medium-fluorescence reticulocytes attained statistical significance.
All values were within the range of historical control data.

After the 4-week recovery period, no statistically significant differences between animals treated with corn oil, vehicle and 800 mg/kg/day were observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of treatment, the following changes in clinical biochemistry parameters observed in animals treated with the test substance compared to animals treated with vehicle only are considered test item-related:
- Decreased glucose levels in males and females treated with 800 mg/kg/day
- Increased urea levels in males treated with 800 mg/kg/day and females treated with 240 and 800 mg/kg/day.
- Increased creatinine levels in males and females treated with 800 mg/kg/day.
- Increased cholesterol levels in males and females treated with 800 mg/kg/day.
- Increased triglyceride levels in males and females treated with 800 mg/kg/day.
- Increased phospholipid levels in males and females treated with 800 mg/kg/day.
- Increased lactate dehydrogenase levels (LDH) in males treated with 800 mg/kg/day.
- Increased alkaline phosphatase levels (ALP) in females treated with 800 mg/kg/day.
- Increased potassium levels in females treated with 240 and 800 mg/kg/day.
- Increased calcium levels in males treated with 800 mg/kg/day and in females treated with 80 and 800 mg/kg/day.
- Increased phosphorus levels in males treated with 800 mg/kg/day and in females treated with 80, 240 and 800 mg/kg/day.
- Increased protein levels in males treated with 800 mg/kg/day.
- Increased globulin concentration and decreased albumin/globulin ratio (A/G ratio) in males and females treated with 800 mg/kg/day.

All values, except for triglyceride levels in females treated with 800 mg/kg/day and phospholipids and globulin levels in males treated with 800 mg/kg/day, were within the range of historical control data.

After the 4-week recovery period, no test item-related differences between animals treated with corn oil, vehicle and 800 mg/kg/day were observed.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following changes in urinalysis parameters observed in animals treated with test substance compared to animals treated with vehicle only are considered test item-related:
- Increased urinary volume in males treated with 800 mg/kg/day
- Increased protein in males and females treated with 800 mg/kg/day
All values were within the range of historical control data.

After the 4-week recovery period, no statistically significant differences between animals treated with corn oil, vehicle and 800 mg/kg/day were observed.

Considering that all clinical laboratory values, except for triglyceride levels in females treated with 800 mg/kg/day and phospholipids and globulin levels in males treated with 800 mg/kg/day, were within the range of historical control data, and that all changes observed were reversible within the 4 week recovery period, the few changes in clinical laboratory values observed in females treated with 240 mg/kg/day are regarded to be adaptive rather than adverse.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Weekly Behavioral Observations: No test item-related findings were recorded during the acclimatization, treatment and recovery period.
Slightly decreased activity was recorded in one male treated with corn oil and one male treated with 240 mg/kg/day during week 13 as well as in two females treated with 240 mg/kg/day during weeks 1 and 5, respectively. These findings are considered incidental.
- Functional Observational Battery: No test item-related findings were recorded. Grip strength was not affected by treatment.Total locomotor activity was not affected by treatment A statistically significant increase of locomotor activity observed in females treated with 800 mg/kg/day in the second 10-minute recording interval was attributed to the low value of the vehicle-treated group in this interval and is therefore considered incidental. Similarly, statistically significantly decreased locomotor activity observed in test item-treated males of all dose groups observed in the first 10-minute recording interval was attributed to the exceptionally high value of the vehicle-treated group in this interval and is therefore considered incidental.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of treatment, no test item-related changes in absolute organ weight, organ/body weight ratio or organ/brain weight ratio was observed in females of any dose group, except for an increase in organ/body weight ratio of the liver of females treated with 800 mg/kg/day. In males treated with 240 and 800 mg/kg/day, absolute organ weight, organ/body weight ratio and organ/brain weight ratio of liver and kidneys was increased. Absolute organ weight, organ/body weight ratio and organ/brain weigh ratio of kidneys was also increased in males treated with 80 mg/kg/day.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- In the liver of two males treated with 240 mg/kg/day and of three males treated with 800 mg/kg/day, accentuated lobular pattern was noted. This finding correlated with hepatocellular hypertrophy. Discoloration of the kidneys was observed in one male treated with 240 mg/kg/day and in two males treated with 800 mg/kg/day. These findings were considered test item-related.
- All other findings were recorded in one or two animals only, did not show any dose-dependency and were within the range of background lesions expected for animals of this strain and age. These findings were considered incidental.
- After the 4-week recovery period, no test item-related findings were recorded in any animal. All findings observed were considered incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- The primary target organ for the test item is considered to be the kidney. Lesions were recorded predominantly in animals treated with 800 mg/kg/day and affected more males than females. They consisted of ncreased incidence or incidence and severity of hyaline droplets, tubular basophilia and mononuclear cell foci along with granular casts and tubular dilation in males treated with 80, 240 and 800 mg/kg/day. In addition, hyaline casts were increased in incidence animals treated with 240 and 800 mg/kg/day, and tubular cysts were recorded in animals treated with 800 mg/kg/day. In high dose group females, an increase in incidence or incidence and severity of tubular basophilia along with minimal granular casts, tubular dilation and tubular cysts was observed.
- In recovery animals, increased incidences and severities of tubular basophilia, granular casts, mononuclear cell foci and tubular dilation along with increased incidence of hyaline casts were still recorded in males treated with 800 mg/kg/day.
- All these degenerative/regenerative and inflammatory changes correlated with increased absolute and relative organ weights of main study males treated with 80, 240 and 800 mg/kg/day as well as increased kidney/body weight ratio in recovery males treated with 800 mg/kg/day. The histopathologic changes in the kidney were consistent with possible irritant effect of the test item and/or its metabolites excreted in the urine, and were considered to be adverse.
- A minor incidence of centrilobular hepatocellular hypertrophy at minimal severity without any further indicator for liver injury in males treated with 240 and 800 mg/kg/day correlated with accentuated lobular pattern and increased absolute and relative liver weights. Minimal hepatocellular hypertrophy was also recorded in few females treated with 800 mg/kg/day. Due to the absence of any further lesion, this finding is deemed to be by metabolic adaption.
- The stomach may also be regarded as a primary target organ. The lesions observed in the stomach represented a localized stomach reaction to a repeatedly gavaged slightly irritant test item. There were minor incidences and/or severities of epithelial hyperplasia, hyperkeratosis, focal glandular erosion and forestomach ulceration in animals treated with 240 and 800 mg/kg/day. In males treated with 800 mg/kg/day, these findings sometimes occurred along with minimal parakeratosis. These lesions may be related to regurgitation (see below) and regressed after the recovery period.
- A number of findings recorded in the nasal cavity, nasopharyngeal duct and pharynx were deemed to be due to accidental uptake of the vehicle/test item by regurgitation.
- In the prostate, minimal focal or multifocal glandular inflammation, characterized by degenerating granulocytic cell infiltrates and inspissated colloid, was recorded in three males treated with 800 mg/kg/day. Minimal concretions were observed also in one male treated with 240 mg/kg/day, but without inflammatory cell reaction. This lesion disappeared completely in recovery animals. The cause remains unclear.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: estrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
240 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: This conclusion is supported by the observations in males because besides the signs of α2u-globulin nephropathy, that was considered not relevant for humans, no additional adverse effects occurred at 240 mg/kg/day.
Key result
Dose descriptor:
NOAEL
Effect level:
240 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: The cause of hyaline droplets, tubular basophilia, and casts observed in males treated with 80 mg/kg/day is most likely caused by α2u-globulin nephropathy and is therefore not relevant for humans.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
80 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Key result
Remarks on result:
not measured/tested
Key result
Critical effects observed:
not specified
Key result
Reproductive effects observed:
no

Analysis of dose formulation:

- The application formulations investigated during the study were found to comprise the substance in the range of 97.9% to 109.9% and thus, the required content limit of ±20% with reference to the nominal concentration was met.

- The homogeneous distribution of the substance in the preparations was approved because single results did not deviate more than 3.1% (<20%) from the corresponding mean.

- The test substance was found to be stable in application formulations when kept eight days at room temperature due to recoveries which met the variation limit of 20% from the time-zero (homogeneity) mean.

Conclusions:
Oral administration of the test substance to Wistar RccHanTM: WIST(SPF) rats at dose levels of 0, 80, 240 and 800 mg/kg bw/day for 90 days with a 28 days recovery period revealed a no observed adverse effect level (NOAEL) of 240 mg/kg bw/day for fertility in male rates based on minimal focal or multifocal glandular inflammation in the prostate of males treated with 800 mg/kg bw/day.
Executive summary:

In a sub-chronic study according to OECDTG408 and GLP, Wistar rats were orally (gavage) exposed to 0, 80, 240 and 800 mg/kg bw/day for 90 days with a 28 days recovery period. The groups comprised 10 females and 10 males which were sacrificed after 91 and 92 days of treatment, respectively. Additional 5 rats per sex and group were used in the control group (treated with corn oil only), the vehicle group (treated with 10% ethanol in corn oil) and the high-dose group (treated with 800 mg/kg/day). Recovery animals were treated for 91 days, and then allowed a 28-day recovery period after which they were sacrificed. Viability/mortality, clinical signs, food consumption and body weights were recorded periodically during acclimatization, treatment and recovery. Weekly behavioral observations were performed during treatment. Functional observational battery, locomotor activity and grip strength tests were performed during week 13. At the end of the treatment period and the 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 animals of the control group (treated with corn oil only), the vehicle group (treated with 10% ethanol in corn oil) and the high-dose group. From the animals of the low and mid dose groups (treated with 80 and 240 mg/kg/day, respectively) liver, kidneys, stomach, prostate, nasal cavities and pharynx were examined in an attempt to establish a no-effect level. 

No substance related mortality, (neuro)behavioral orophthalmoscopic effects were observed. Males and females in the high dose group showed slight to moderate dyspnea and slight to moderate salivation, which was considered treatment related. Body weight and bodyweight changes were significantly affected in the high dose group, which was not evident during the recovery period. Hematological effects were within the range of historical control data. The observed changes in clinical biochemistry parameters except for triglyceride levels in females treated with 800 mg/kg/day and phospholipids and globulin levels in males treated with 800 mg/kg/day, were within the range of historical control data. The alterations were not evident after the 28 day recovery period. Observed effects on urinalysis parameters were within the range of historical control data. The organ/ body weight of the liver of females in the high dose group was increased in the high dose group. In males of the mid and high dose group, absolute organ weight, organ/body weight ratio and organ/brain weight ratio of liver and kidneys was increased. In addition, these parameters were also affected for the kidney in low dose males. Organ/body weight ratio of kidneys of males treated with 800 mg/kg/day was still statistically significantly increased after the recovery period. The primary target organ for the test item is considered to be the kidney. Lesions were recorded predominantly in animals treated with 800 mg/kg/day and affected more males than females. They consisted of increased incidence or incidence and severity of hyaline droplets, tubular basophilia and mononuclear cell foci along with granular casts and tubular dilation in males treated with 80, 240 and 800 mg/kg/day. In addition, hyaline casts were increased in incidence animals treated with 240 and 800 mg/kg/day, and tubular cysts were recorded in animals treated with 800 mg/kg/day. In high dose group females, an increase in incidence or incidence and severity of tubular basophilia along with minimal granular casts, tubular dilation and tubular cysts was observed. In recovery animals, increased incidences and severities of tubular basophilia, granular casts, mononuclear cell foci and tubular dilation along with increased incidence of hyaline casts were still recorded in males treated with 800 mg/kg/day. In addition, accentuated lobular pattern in livers of 2 and 3 males from the mid and high dose group were observed. The stomach may also be regarded as a primary target organ. The lesions observed in the stomach represented a localized stomach reaction to a repeatedly gavaged slightly irritant test item. There were minor incidences and/or severities of epithelial hyperplasia, hyperkeratosis, focal glandular erosion and forestomach ulceration in animals treated with 240 and 800 mg/kg/day. A number of findings recorded in the nasal cavity, nasopharyngeal duct and pharynx were deemed to be due to accidental uptake of the vehicle/test item by regurgitation. Concerning the reproductive system, in the prostate, minimal focal or multifocal glandular inflammation, characterized by degenerating granulocytic cell infiltrates and inspissated colloid, was recorded in three males treated with 800 mg/kg/day. No effects on organ weight or observations in examination of reproductive organs were reported. For systemic effects, a LOEL of 80 mg/kg bw/day is derived for male rats based on α2u-globulin nephropathy which is not relevant for humans. A NOAEL (No-Observed-Adverse-Effect-Level) for effects relevant to human risk assessment may be established at 240 mg/kg/day for females This conclusion is supported by the observations in males because besides non-relevant human relevant nephrotoxic effects no additional adverse effects occurred at 240 mg/kg/day. For fertility, a NOAEL is derived bases on minimal focal or multifocal glandular inflammation in the prostate of males in the high dose group.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
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)
Remarks:
Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen, Switzerland
Limit test:
no

Test material

Reference
Name:
Unnamed

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHanTM: WIST(SPF)
Details on species / strain selection:
Recognized by international guidelines as a recommended test system.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
- Age: 7 weeks at delivery
- Weight at acclimatization: Males: 143.9 to 203.8 g, Females: 102.8 to 166.7 g
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK).
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 20/10) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: 7 days

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

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Administration by gavage is a common and accepted route of exposure for studies of this type.
Vehicle:
other: 10% ethanol in corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- The dose formulations were prepared weekly. Based upon the results of dose formulation analyses performed during a non-GLP dose range finding study, the stability of the test item formulations was considered to be sufficient to justify weekly preparation.
- Prior to the preparation of the dose formulations, the test item was molten in the flask at 80 °C for 24 h to ensure homogeneity of the test item. Thereafter, the test item was allowed to cool down and solidify. Small flakes of test item were scraped from the block and weighed into a tared glass container on a suitable precision balance. Then ethanol was added (The volume of ethanol equalled 10% of the final volume of the dosing solution) and the mixture was stirred until the test item was completely dissolved. To ensure complete solution of the test item in ethanol, the dose formulation of group 5 with a concentration of 160 mg/mL was slightly heated. Then, corn oil was added to give the appropriate final concentration of the test item in the solution. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on a previous formulation trial conducted for the dose range finding study where 10% ethanol in corn oil was found to be the only vehicle suitable to achieve sufficient test item concentrations. Effects of ethanol administration to rats are well characterized, and 8 mL/kg of a 10% solution for 90 days are “well tolerated”.
- Concentration in vehicle: 16, 48 and 160 mg/mL dose solutions are corresponding to the doses of 80, 240 and 800 mg/kg bw/day
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- After treatment start and during weeks 6 and 13, the following samples of approximately 2 g were drawn in duplicate prior to dosing for analysis of homogeneity and concentration: one sample of the control group (group 1) as well as three samples (top, middle and bottom) of the vehicle group (group 2) and of each test item-treated group (groups 3, 4 and 5).
- Duplicate samples of about 2 g of each dose formulation of groups 2 to 5 were taken after treatment start to confirm stability (4 hours and 8 days at room temperature, 20 ± 5 °C).
- The test item was used as analytical standard.
- Acceptance criteria for dose formulation concentration, homogeneity and stability determinations were ±20% of nominal content.
Duration of treatment / exposure:
91/ 92 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Dose / conc.:
240 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15 females and 15 males in control, vehicle and top-dose group
10 females and 10 males in low and mid-dose group
Control animals:
yes, concurrent vehicle
other: corn oil only
Details on study design:
- Dose selection rationale:
The dose levels were selected based on a previous 14-day dose range-finding toxicity study in Wistar rats, using dose levels of 100, 300 and 1000 mg/kg/day. Body weight gain of males treated with 1000 mg/kg was reduced. In both sexes, liver and kidney weight was increased in animals treated with 1000 mg/kg. In females, increased liver weight was also observed in animals treated with 300 mg/kg, and increased adrenal weight was observed in females treated with 1000 mg/kg.

- Post-exposure recovery period:
28 days, for 5 additional animals in control, vehicle control and high dose group. After 91 days of treatment with ethanol-containing vehicle, rats may become addicted. To prevent ethanol-withdrawal symptoms, treatment with vehicle was continued in the vehicle group and the high-dose group throughout recovery. The control group was treated with corn oil during recovery.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
Observations for viability / mortality were recorded twice daily.

DETAILED CLINICAL OBSERVATIONS:
The animals were observed for clinical signs once before commencement of administration as well as twice daily during days 1 - 3 and daily on days 1 - 91/92 of the treatment period and once daily during days 1 - 28 of the recovery period.

BODY WEIGHT:
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

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.

OPHTHALMOSCOPIC EXAMINATION:
Ophthalmoscopic examinations were performed once on all animals during acclimatization and on groups 1, 2 and 5 during week 13. The ophthalmoscopic examinations of both eyes of the animals were performed after the application of a mydriatic solution (Ciba Vision AG, 3172 Niederwangen / Switzerland) using a direct ophthalmoscope.

HAEMATOLOGY:
- Time schedule for collection of blood: After 13 and 17 (recovery) weeks
- Anaesthetic used for blood collection: Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia.
- Animals fasted: The 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. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
- 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 (slides were prepared but not evaluated)
- Coagulation: Prothrombin time (= Thromboplastin time), Activated partial Thromboplastin time

CLINICAL CHEMISTRY:
The following clinical biochemistry parameters were determined: Glucose, Urea, Creatinine, Bilirubin total, Cholesterol total, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Alkaline, phosphatase, Bile acids, Gamma-glutamyl-transferase, Creatine kinase, Sodium, Potassium, Chloride, Calcium, Phosphorus, Protein total, Albumin, Globulin, Albumin/Globulin ratio

URINALYSIS:
- Time schedule for collection of urine: After 13 and 17 (recovery) weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Urine was collected during the 18 hours fasting period into a specimen vial.
- Physical Examinations: Urine volume (18 hour), Specific gravity (relative density), Color, Appearance
- Chemical Examination: pH value, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes

NEUROBEHAVIOURAL EXAMINATION:
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 13) thereafter. Summary of parameters: appearance, motor, behavior, respiration, reflexes, miscellaneous (Lacrimation, Limbs cyanotic, Mydriasis, Miosis, Exophthalmos, Reduced muscle tone), grip Strength, locomotor Activity.
Sacrifice and pathology:
GROSS PATHOLOGY:
- Sacrifice: after 13 or 17 (recovery) weeks
- Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.
- Organs and tissues collected and examined: Adrenal glands, Aorta, Bone (stemum, femur including joint), Bone marrow (femur), Brain - including section of medulla/pons, cerebral and cerebellar cortex, Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes w/optic nerve, Harderioan gland, 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, Pharynx, 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, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland), Tongue, Trachea, Ureter, Urinary bladder filled w/formalin at necropsy, Uterus (w/ cervix, vagina and oviducts), All gross lesions.
- Weights were determined of; Adrenal glands, brain including section of medulla/pons, cerebral and cerebellar cortex, epididymides, heart including auricles, kidneys, liver, ovaries, spleen, testes, thymus, Uterus (w/ cervix, vagina and oviducts). Organs were weighed before fixation and the weights were recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight and brain weight.

HISTOPATHOLOGY:
- Slides of all collected organs were 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. Organ and tissue samples taken from animals which died spontaneously or which were killed in extremis were evaluated similarly to those organs taken from animals of the high-dose group. From the animals of the low and mid dose groups, liver, kidneys, stomach, prostate gland, nasal cavities level I (males only), nasal cavities levels II, III and IV and pharynx were examined to establish a no-effect level.
Statistics:
The following statistical methods were used to analyze body weight, grip strength, locomotor activity, 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):
- No clinical signs were recorded in any animal during the acclimatization period and during the recovery period. During the treatment period, no clinical signs were recorded in animals treated with corn oil (group 1) and with vehicle (10% ethanol in corn oil, group 2) as well as in females treated with 80 and 240 mg/kg/day.
- One male treated with 80 mg/kg/day was noted with moderate salivation on a single day during week 13, this was probably caused by accidental regurgitation of the dosing solution and is thus related to the treatment procedure. No test item-related clinical signs were noted in males treated with 80 mg/kg/day.
- One male treated with 240 mg/kg/day was noted with slight dyspnea and slight to moderate salivation on two consecutive days during week 8. No other test item-related clinical signs were noted in males treated with 240 mg/kg/day.
- In males and females treated with 800 mg/kg/day, slight to moderate dyspnea and slight to moderated salivation were observed in single animals throughout the treatment period. These clinical signs were noted in various individuals and were mostly observed for a short period of several days only, never affecting more than 30% of the group. These clinical signs are considered test item-related.
- Any other findings recorded in single animals such as fissures, scabs and wounds are considered incidental.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- No premature deaths occurred during the acclimatization period and during the recovery period.
- Four animals died prematurely during the treatment period. One female treated with 240 mg/kg/day and one female treated with 800 mg/kg/day were found dead during week 6. One male treated with 800 mg/kg/day spontaneously died during week 11 and one male treated with vehicle, 10% ethanol in corn oil, spontaneously died after treatment during week 13. The death of the female treated with 240 mg/kg/day was caused by renal failure; the cause of death of the other decedents could not be established.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Compared to vehicle-treated animals, absolute body weight and body weight gain were statistically significantly reduced in males treated with 800 mg/kg/day from the second week and first week onwards, respectively, until the end of the treatment period.
- In females treated with 800 mg/kg/day, statistically significantly lower absolute body weight and body weight gain was observed during weeks 1-4, 6 and 8 of the treatment period compared to vehicle-treated animals.
- In females treated with 240 mg/kg/day, body weight gain was slightly reduced during the first half of the treatment period compared to vehicle-treated animals, with the difference attaining statistical significance during week 4; however, the animals recovered towards the end of the treatment period.
- During the 4-week recovery period, no statistically significant differences in absolute body weight between animals treated with corn oil, vehicle and 800 mg/kg/day were observed. Body weight gain of females treated with 800 mg/kg/day was increased in week 4 of the recovery period and body weight gain of males treated with 800 mg/kg/day was increased in weeks 2 to 4 of the recovery period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not affected by treatment.
Food efficiency:
not examined
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 observed. Findings such as slight corneal opacity, persistent pupillary membranes or persistent hyaloid vessels observed in all groups were within the normal range of background lesions observed for animals of this strain and age.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of treatment, the following changes in hematology parameters observed in animals treated with the test substance compared to animals treated with vehicle only were considered test item-related:
- Increased prothrombin time (PT) and decreased partial thromboplastin time (PTT) observed in animals treated with 800 mg/kg/day.
- A shift in the reticulocyte count in females treated with 800 mg/kg/day characterized by an increase in low-fluorescence reticulocytes and a decrease in medium and high-fluorescence reticulocytes. This shift was also observed in males treated with 800 mg/kg/day; however, only the decrease in medium-fluorescence reticulocytes attained statistical significance.
All values were within the range of historical control data.

After the 4-week recovery period, no statistically significant differences between animals treated with corn oil, vehicle and 800 mg/kg/day were observed.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of treatment, the following changes in clinical biochemistry parameters observed in animals treated with the test substance compared to animals treated with vehicle only are considered test item-related:
- Decreased glucose levels in males and females treated with 800 mg/kg/day
- Increased urea levels in males treated with 800 mg/kg/day and females treated with 240 and 800 mg/kg/day.
- Increased creatinine levels in males and females treated with 800 mg/kg/day.
- Increased cholesterol levels in males and females treated with 800 mg/kg/day.
- Increased triglyceride levels in males and females treated with 800 mg/kg/day.
- Increased phospholipid levels in males and females treated with 800 mg/kg/day.
- Increased lactate dehydrogenase levels (LDH) in males treated with 800 mg/kg/day.
- Increased alkaline phosphatase levels (ALP) in females treated with 800 mg/kg/day.
- Increased potassium levels in females treated with 240 and 800 mg/kg/day.
- Increased calcium levels in males treated with 800 mg/kg/day and in females treated with 80 and 800 mg/kg/day.
- Increased phosphorus levels in males treated with 800 mg/kg/day and in females treated with 80, 240 and 800 mg/kg/day.
- Increased protein levels in males treated with 800 mg/kg/day.
- Increased globulin concentration and decreased albumin/globulin ratio (A/G ratio) in males and females treated with 800 mg/kg/day.

All values, except for triglyceride levels in females treated with 800 mg/kg/day and phospholipids and globulin levels in males treated with 800 mg/kg/day, were within the range of historical control data.

After the 4-week recovery period, no test item-related differences between animals treated with corn oil, vehicle and 800 mg/kg/day were observed.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following changes in urinalysis parameters observed in animals treated with test substance compared to animals treated with vehicle only are considered test item-related:
- Increased urinary volume in males treated with 800 mg/kg/day
- Increased protein in males and females treated with 800 mg/kg/day
All values were within the range of historical control data.

After the 4-week recovery period, no statistically significant differences between animals treated with corn oil, vehicle and 800 mg/kg/day were observed.

Considering that all clinical laboratory values, except for triglyceride levels in females treated with 800 mg/kg/day and phospholipids and globulin levels in males treated with 800 mg/kg/day, were within the range of historical control data, and that all changes observed were reversible within the 4 week recovery period, the few changes in clinical laboratory values observed in females treated with 240 mg/kg/day are regarded to be adaptive rather than adverse.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Weekly Behavioral Observations: No test item-related findings were recorded during the acclimatization, treatment and recovery period. Slightly decreased activity was recorded in one male treated with corn oil and one male treated with 240 mg/kg/day during week 13 as well as in two females treated with 240 mg/kg/day during weeks 1 and 5, respectively. These findings are considered incidental.
- Functional Observational Battery: No test item-related findings were recorded. Grip strength was not affected by treatment. Total locomotor activity was not affected by treatment. A statistically significant increase of locomotor activity observed in females treated with 800 mg/kg/day in the second 10-minute recording interval was attributed to the low value of the vehicle-treated group in this interval and is therefore considered incidental. Similarly, statistically significantly decreased locomotor activity observed in test item-treated males of all dose groups observed in the first 10-minute recording interval was attributed to the exceptionally high value of the vehicle-treated group in this interval and is therefore considered incidental.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
After 13 weeks of treatment, no test item-related changes in absolute organ weight, organ/body weight ratio or organ/brain weight ratio was observed in females of any dose group, except for an increase in organ/body weight ratio of the liver of females treated with 800 mg/kg/day. In males treated with 240 and 800 mg/kg/day, absolute organ weight, organ/body weight ratio and organ/brain weight ratio of liver and kidneys was increased. Absolute organ weight, organ/body weight ratio and organ/brain weigh ratio of kidneys was also increased in males treated with 80 mg/kg/day.

After the 4-week recovery period, the organ/body weight ratio of kidneys of males treated with 800 mg/kg/day was still statistically significantly increased compared to males treated with vehicle only. No statistically significant differences between females treated with corn oil, vehicle and 800 mg/kg/day were observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- In the liver of two males treated with 240 mg/kg/day and of three males treated with 800 mg/kg/day, accentuated lobular pattern was noted. This finding correlated with hepatocellular hypertrophy. Discoloration of the kidneys was observed in one male treated with 240 mg/kg/day and in two males treated with 800 mg/kg/day. These findings were considered test item-related.
- All other findings were recorded in one or two animals only, did not show any dose-dependency and were within the range of background lesions expected for animals of this strain and age. These findings were considered incidental.
- After the 4-week recovery period, no test item-related findings were recorded in any animal. All findings observed were considered incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- The primary target organ for the test item is considered to be the kidney. Lesions were recorded predominantly in animals treated with 800 mg/kg/day and affected more males than females. They consisted of increased incidence or incidence and severity of hyaline droplets, tubular basophilia and mononuclear cell foci along with granular casts and tubular dilation in males treated with 80, 240 and 800 mg/kg/day. In addition, hyaline casts were increased in incidence animals treated with 240 and 800 mg/kg/day, and tubular cysts were recorded in animals treated with 800 mg/kg/day. In high dose group females, an increase in incidence or incidence and severity of tubular basophilia along with minimal granular casts, tubular dilation and tubular cysts was observed.
- In recovery animals, increased incidences and severities of tubular basophilia, granular casts, mononuclear cell foci and tubular dilation along with increased incidence of hyaline casts were still recorded in males treated with 800 mg/kg/day.
- All these degenerative/regenerative and inflammatory changes correlated with increased absolute and relative organ weights of main study males treated with 80, 240 and 800 mg/kg/day as well as increased kidney/body weight ratio in recovery males treated with 800 mg/kg/day. The histopathologic changes in the kidney were consistent with possible irritant effect of the test item and/or its metabolites excreted in the urine, and were considered to be adverse.
- A minor incidence of centrilobular hepatocellular hypertrophy at minimal severity without any further indicator for liver injury in males treated with 240 and 800 mg/kg/day correlated with accentuated lobular pattern and increased absolute and relative liver weights. Minimal hepatocellular hypertrophy was also recorded in few females treated with 800 mg/kg/day. Due to the absence of any further lesion, this finding is deemed to be by metabolic adaption.
- The stomach may also be regarded as a primary target organ. The lesions observed in the stomach represented a localized stomach reaction to a repeatedly gavaged slightly irritant test item. There were minor incidences and/or severities of epithelial hyperplasia, hyperkeratosis, focal glandular erosion and forestomach ulceration in animals treated with 240 and 800 mg/kg/day. In males treated with 800 mg/kg/day, these findings sometimes occurred along with minimal parakeratosis. These lesions may be related to regurgitation (see below) and regressed after the recovery period.
- A number of findings recorded in the nasal cavity, nasopharyngeal duct and pharynx were deemed to be due to accidental uptake of the vehicle/test item by regurgitation.
- In the prostate, minimal focal or multifocal glandular inflammation, characterized by degenerating granulocytic cell infiltrates and inspissated colloid, was recorded in three males treated with 800 mg/kg/day. Minimal concretions were observed also in one male treated with 240 mg/kg/day, but without inflammatory cell reaction. This lesion disappeared completely in recovery animals. The cause remains unclear.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
240 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: This conclusion is supported by the observations in males because besides the signs of α2u-globulin nephropathy, that was considered not relevant for humans, no additional adverse effects occurred at 240 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
240 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: The cause of hyaline droplets, tubular basophilia, and casts observed in males treated with 80 mg/kg/day is most likely caused by α2u-globulin nephropathy and is therefore not relevant for humans.

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
80 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

Analysis of dose formulation:

- The application formulations investigated during the study were found to comprise the substance in the range of 97.9% to 109.9% and thus, the required content limit of ±20% with reference to the nominal concentration was met.

- The homogeneous distribution of the substance in the preparations was approved because single results did not deviate more than 3.1% (<20%) from the corresponding mean.

- The test substance was found to be stable in application formulations when kept eight days at room temperature due to recoveries which met the variation limit of 20% from the time-zero (homogeneity) mean.

Applicant's summary and conclusion

Conclusions:
Oral administration of the test substance to Wistar RccHanTM: WIST(SPF) rats at dose levels of 0, 80, 240 and 800 mg/kg bw/day for 90 days with a 28 days recovery period revealed a no observed adverse effect level (NOAEL) of 240 mg/kg bw/day in female rats. The effects observed at 80 mg/kg bw in male rats (α2u-globulin nephropathy) are not relevant to humans.
Executive summary:

In a sub-chronic study according to OECDTG408 and GLP, Wistar rats were orally (gavage) exposed to 0, 80, 240 and 800 mg/kg bw/day for 90 days with a 28 days recovery period. The groups comprised 10 females and 10 males, sacrificed after 91 and 92 days of treatment, respectively. Additional 5 rats per sex and group were used in the control group (treated with corn oil only), the vehicle group (treated with 10% ethanol in corn oil) and the high-dose group (treated with 800 mg/kg/day), treated for 91 days, and then allowed a 28-day recovery period after which they were sacrificed. Viability/mortality, clinical signs, food consumption and body weights were recorded periodically during acclimatization, treatment and recovery. Weekly behavioral observations were performed during treatment. Functional observational battery, locomotor activity and grip strength tests were performed during week 13. At the end of the treatment period and the 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 animals of the control group (treated with corn oil only), the vehicle group (treated with 10% ethanol in corn oil) and the high-dose group. From the animals of the low and mid dose groups (treated with 80 and 240 mg/kg/day, respectively) liver, kidneys, stomach, prostate, nasal cavities and pharynx were examined in an attempt to establish a no-effect level. 

No substance related mortality, (neuro)behavioral or ophthalmoscopic effects were observed. Males and females in the high dose group showed slight to moderate dyspnea and slight to moderate salivation, which was considered treatment related. Body weight and body weight changes were significantly affected in the high dose group, which was not evident during the recovery period. Hematological effects were within the range of historical control data. The observed changes in clinical biochemistry parameters except for triglyceride levels in females treated with 800 mg/kg/day and phospholipids and globulin levels in males treated with 800 mg/kg/day, were within the range of historical control data. The alterations were not evident after the 28 day recovery period. Observed effects on urinalysis parameters were within the range of historical control data. The organ/ body weight of the liver of females in the high dose group was increased in the high dose group. In males of the mid and high dose group, absolute organ weight, organ/body weight ratio and organ/brain weight ratio of liver and kidneys was increased. In addition, these parameters were also affected for the kidney in low dose males. Organ/body weight ratio of kidneys of males treated with 800 mg/kg/day was still statistically significantly increased after the recovery period. The primary target organ for the test item is considered to be the kidney. Lesions were recorded predominantly in animals treated with 800 mg/kg/day and affected more males than females. They consisted of increased incidence or incidence and severity of hyaline droplets, tubular basophilia and mononuclear cell foci along with granular casts and tubular dilation in males treated with 80, 240 and 800 mg/kg/day. In addition, hyaline casts were increased in incidence animals treated with 240 and 800 mg/kg/day, and tubular cysts were recorded in animals treated with 800 mg/kg/day. In high dose group females, an increase in incidence or incidence and severity of tubular basophilia along with minimal granular casts, tubular dilation and tubular cysts was observed. In recovery animals, increased incidences and severities of tubular basophilia, granular casts, mononuclear cell foci and tubular dilation along with increased incidence of hyaline casts were still recorded in males treated with 800 mg/kg/day. In addition, accentuated lobular pattern in livers of 2 and 3 males form the mid and high dose group were observed. The stomach may also be regarded as a primary target organ. The lesions observed in the stomach represented a localized stomach reaction to a repeatedly gavaged slightly irritant test item. There were minor incidences and/or severities of epithelial hyperplasia, hyperkeratosis, focal glandular erosion and forestomach ulceration in animals treated with 240 and 800 mg/kg/day. A number of findings recorded in the nasal cavity, nasopharyngeal duct and pharynx were deemed to be due to accidental uptake of the vehicle/test item by regurgitation. In the prostate, minimal focal or multifocal glandular inflammation, characterized by degenerating granulocytic cell infiltrates and inspissated colloid, was recorded in three males treated with 800 mg/kg/day. Based on these effects, a LOEL of 80 mg/kg bw/day is derived for male rats. However the basis for this effect level, α2u-globulin nephropathy, is not relevant for humans. NOAEL (No-Observed-Adverse-Effect-Level) for effects relevant to human risk assessment may be established at 240 mg/kg/day for females This conclusion is supported by the observations in males because besides nephrotoxic effects not relevant for humans, no additional adverse effects occurred at 240 mg/kg/day.