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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:
2015-05-12 and 2016-04-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP conform study according to guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation: 43 – 50 days old (male/female)
- Weight at study initiation: males: 200-235 g; females: 124-177 g
- Housing: 2 or 3 animals of the same sex/ cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sunflower oil (Helianthii annui oleum raffinatum)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in sunflower oil in a frequency based on the stability features of the test item in the vehicle (stable not longer than for three days and stored at 5 +/- 3°C).

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 20, 75 and 225 mg/mL
- Amount of vehicle: 2 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of formulations (concentration and homogeneity) in the vehicle was performed in the Analytical Laboratory of Test Facility three times during the study. Five samples (5 mL, each) were taken from different places from each concentration (Groups 2, 3 and 4) for analysis of concentration and homogeneity on 3 occasions. Similarly, five samples were taken from different places from the control substance (Group 1) at each occasion and measured. The samples were stored in a refrigerator until the analysis (at 5 +/- 3°C). Formulation samples were shaken and diluted with tetrahydrofuran and acetonitrile.
Measured concentrations varied between 102 and 109 % of the nominal concentrations and all formulations were considered to be homogeneous.

HPLC conditions:
Detector: 210 nm
Column: HyperPrep HS C18, 250 x 4.6 mm, 8 μm, No.: 10190790
Mobil Phase: Acetonitrile : water = 90 : 10 (v/v)
Flow Rate: 1.5 mL/min
Injection volume: 50 μL
Retention time of CH-80-AL: 10 min
Run time: 13 minutes
Duration of treatment / exposure:
90 days
Frequency of treatment:
7 days/week basis, every day at a similar time (+/- 2 hours)
Doses / concentrations
Remarks:
Doses / Concentrations:
40, 150, 450 mg/kg bw/d
Basis:
other: nominal concentration
No. of animals per sex per dose:
10 animals/sex in the control and dose groups; 5 animals/ sex in the control and high dose groups for recovery observations
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting with 450, 150 and 40 mg/kg bw/day is based on findings obtained in previous repeated dose toxicity studies with the test item in the rat (Reproduction/Developmental Toxicity Screening Test with CH-80-AL in the Rat, study no. 552.439.3249).
- Rationale for selecting satellite groups: five animals per sex in the control and in the high dose group (according to guideline)
- 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: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day). General clinical cage side observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first exposure and once weekly thereafter
- Parameters checked in table [No.1] were examined.

BODY WEIGHT: Yes
- Time schedule for examinations: Weighing was performed on day 0, then weekly. Fasted body weight was measured on day of necropsy (Days 90 and 91 for the main groups and Day 118 for the recovery groups).

FOOD CONSUMPTION AND COMPOUND INTAKE:
- The food consumption was determined on Day 7, then weekly by reweighing the non-consumed diet in the treatment phase and recovery period.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the acclimation period
- Dose groups that were examined: all control and high dose test animals prior to test termination (day 89)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On main group animals one day after the treatment (day 90 and day 91) and on recovery animals at the end of recovery period (day 118)
- Anaesthetic used for blood collection: Yes (Isofluran CP®)
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On main group animals one day after the treatment (day 90 and day 91) and on recovery animals at the end of recovery period (day 118)
- Animals fasted: Yes
- How many animals: all animals at each dose group
- Parameters checked in table [No.3] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: 16 hours before the blood collection
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.6] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week (day 84)
- Dose groups that were examined: all animals
- Battery of functions tested: sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity

ESTROUS CYCLE: Yes
- Time schedule for examinations: During the last two weeks of the treatment period (from Day 77 up to and including Day 90 or 91, depending on the day of the necropsy)
- Dose groups that were examined: all female animals
- Examinations: A vaginal smear was prepared from each female. The type of cycle (regular or irregular), number of days in pro-estrous, estrous and diestrous, number of cycle during the two weeks, number of animals with prolonged diestrous, number of animals with prolonged estrous were evaluated.

SPERM EXAMINATION
- Time for examinations: at Necropsy
- Dose groups that were examined: 10 animals of the control and 10 animals at high dose group
- Quantitative examinations: Testes and epididymides were frozen at the necropsy and enumeration was performed later. The total number of homogenization of one side testis was enumerated.
- Qualitative examinations: Sperm motility was determined from sample of ductus deferens at the necropsy. For the evaluation of the sperm motility the mean percentage of motile and immotile sperms was determined. The total sperm count and number of immotile sperms were recorded. A morphological evaluation of ductus deferens sperms sample was performed from the same animals. Sperms were examined and classified as either normal or abnormal (isolated heads, misshapen heads and/or tails).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, including organ weights (see table No. 4); All animals were necropsied one day after the last treatment (main groups) or after four weeks recovery period (recovery groups).
HISTOPATHOLOGY: Yes (see table No. 5); Full histopathology was performed on the preserved organs or tissues of the animals of the control (Group 1) and high dose (Group 4) groups including recovery groups. Further α2μ-globulin in the kidney of male animals of control and high dose group was investigated.
Statistics:
Statistical analysis was done for the following data: body weight, food consumption, hematology, blood coagulation, clinical chemistry, urinalysis (numerical values), sperm parameters, estrous cycle and organ weight data.
The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences.
Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
For the evaluation of data in the recovery group, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.
The rate of mortality, frequency of clinical signs, ophthalmological data, pathology and histopathology findings was calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”. The use of the word “significant” or “significantly” indicates a statistically significant difference between the control and the experimental groups. Significance was judged at a probability value of p < 0.05 and < 0.01. Male and female rats were evaluated separately.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No animals died during the course of the study. No signs of toxicity related to the test item were detected at any dose level (450, 150 or 40 mg/kg bw/day) at the daily and detailed weekly clinical observations.

BODY WEIGHT AND WEIGHT GAIN
The body weight development of male and female animals was not affected in the course of the entire study (450, 150 or 40 mg/kg bw/day). No test item-related body weight or body weight gain changes were observed with respect to controls at any dose level during the treatment or recovery periods.

FOOD CONSUMPTION AND COMPOUND INTAKE
The mean daily food consumption was comparable in animals of the control and test item treated groups (450, 150 and 40 mg/kg bw/day).

OPHTHALMOSCOPIC EXAMINATION
There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 450 mg/kg bw/day).

HAEMATOLOGY
Hematology examinations did not reveal test item related adverse changes in the evaluated parameters (450, 150 or 40 mg/kg bw/day).

CLINICAL CHEMISTRY
Adverse changes were not detected at the evaluation of clinical chemistry parameters in male or female animals at 450, 150 or 40 mg/kg bw/day.

URINALYSIS
Dose related elevation of the protein, ketone and leucocytes content of the urine was observed in male animals at 450 and 150 mg/kg bw/day at termination of the treatment. The change in protein content of the urine was not fully reversible because slightly elevated levels were also detected at the end of the recovery period in male animals at 450 mg/kg bw/day.

NEUROBEHAVIOUR
Functional observation battery did not demonstrate any treatment-related difference with respect to the controls in the behavior or in reactions to different type of stimuli at the end of the treatment period (male and female, 450, 150 or 40 mg/kg bw/day).
The behavior and reactions to different type of stimuli or manipulations of animals were considered to be normal in the control and all test item treated groups.

ORGAN WEIGHTS
A test item related elevation was detected on the kidney weights in male animals at 450 mg/kg bw/day in accordance with histopathological observations. A test item influence on the hepatic and renal functions, due to changes in weights of liver in male and female animals and of kidneys in female animals, could not be excluded but the effect was regarded as not adverse as the test item did not induce histopathological changes in these organs.

GROSS PATHOLOGY
Specific macroscopic alterations related to treatment with the test item were not observed at the terminal necropsy or at the end of the recovery period.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathology examinations revealed hyaline-like droplets in the epithel cells of some proximal convoluted tubules in the kidneys of male animals at 450 mg/kg bw/day. These findings were not seen in the kidneys of male animals in the middle dose group as well as in high dose female animals. Hyaline droplet nephropathy was not associated with interference to α2μ-globulin according to the results of the immunohistochemistry investigation.

SPERM ANALYSIS
Sperm analysis did not reveal any test item related influence on the sperm parameters (count, motility and morphology) at 450 mg/kg bw/day.

ESTROUS CYCLE
A test item influence on the estrous cycle was not detected (450, 150 or 40 mg/kg bw/day). Slight differences between the control and high dose treated female animals in the percentage of animals with irregular cycle, mean number of days in estrous or diestrus were considered to be indicative of biological variation and not related to the test item. In accordance with this finding, histopathological examinations did not reveal changes in the morphology of uterus or ovaries in this study. Moreover, the same compound did not adversely influence the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in female animals in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD no.422; Study no. 552.439.3249).

DETERMINATION of α2µ-globulin
No increased of α2µ-globulin levels in male rats were oberserved at 450 mg/kg bw/day. The values obtained by image analysis using previously a monoclonal antibody were generally lower as compared with control animals.

Effect levels

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) was determined to be 150 mg/kg bw/day for male and female Wistar rats.
Executive summary:

The objective of this study was to obtain information on the possible health hazards after repeated exposure with the test item at three dose levels over a prolonged period of time (90 days) followed by a 28-day recovery period in the high dose and control dose animals in order to assess reversibility, persistence or delayed occurrence of potential toxicological effects.

The test item was administered orally (by gavage) to male and female Hsd.Han: Wistar rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 450, 150 and 40 mg/kg bw/day doses corresponding to concentrations of 0, 225, 75 and 20 mg/mL, applied in a dose volume of 2 mL/kg bw for 90 or 91 days. Each five animals/sex in the control and high dose groups assigned to the recovery groups were treated identically up to Day 89 then they were observed without administration for four weeks (recovery observations).

The suitability of the chosen vehicle (sunflower oil) for the test item and its sufficient stability in the vehicle was analytically verified up front. Recovery of the test item was between 97 and 104 % of nominal concentrations at 1 and 500 mg/mL in sunflower oil, respectively. Thus, it was proved to be stable at room temperature for one day (112 and 105 % of the starting vales at 1 and 500 mg/mL, respectively) and at 5 +/- 3°C for 3 days (94 and 92 % of the starting vales at 1 and 500 mg/mL, respectively). Concentrations of the test item in the dosing formulations varied between ranges of 92 % and 98 % of nominal concentrations at each analytical occasion confirming proper dosing.

Animals were observed for mortality twice a day in the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted in the last week of treatment. The body weight and food consumption were measured and evaluated weekly. The estrous cycle of all female animals was examined during the last two weeks of the treatment period. Clinical pathology examinations (including hematology, blood coagulation, clinical chemistry and urinalysis) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Sperm examinations were conducted in animals of the control and high dose groups at termination of the treatment. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups. Further α2μ-globulin in the kidney of male animals of control and high dose group was investigated.

The results of study were interpreted comparing test item treated groups with controls, which were administered concurrently with vehicle only.

No animals died during the course of the study. No signs of toxicity related to the test item were detected at any dose level (450, 150 or 40 mg/kg bw/day) at the daily and detailed weekly clinical observations or in the course of the functional observation battery. The behavior and physical condition of animals were normal during the entire observation period. The body weight development of male and female animals was not affected in the course of the entire study. No test item-related body weight or body weight gain changes were observed with respect to controls at any dose level during the treatment or recovery periods. The mean daily food consumption was comparable in animals of the control and test item treated groups (450, 150 and 40 mg/kg bw/day). There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 1000 mg/kg bw/day). A test item related influence on the estrous cycle was not detected (450, 150 and 40 mg/kg bw/day). Hematology examinations did not reveal test item related adverse changes in the evaluated parameters (450, 150 and 40 mg/kg bw/day). Adverse findings were not detected in any of the clinical chemistry parameters in male or female animals at 450, 150 and 40 mg/kg bw/day. Dose related elevation of the protein, ketone and leucocytes content of the urine was observed in male animals at 450 and 150 mg/kg bw/day at termination of the treatment. The change in protein content of the urine was not fully reversible because slightly elevated levels were also detected at the end of the recovery period in male animals at 450 mg/kg bw/day. A test item related elevation was detected on the kidney weights in male animals at 450 mg/kg bw/day in accordance with histopathological observations. A test item influence on the hepatic and renal functions, due to changes in weights of liver in male and female animals and of kidneys in female animals, could not be excluded but the effect was regarded as not adverse as the test item did not induce histopathological changes in these organs. Histopathology examinations revealed hyaline-like droplets in the epithel cells of some proximal convoluted tubules in the kidneys of male animals at 450 mg/kg bw/day. These findings were not seen in the kidneys of male animals in the middle dose group as well as in high dose female animals. Hyaline droplet nephropathy was not associated with interference to α2μ-globulin according to the results of the immunohistochemistry investigation as no increased α2µ-globulin levels in male rats were observed. Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined to be 150 mg/kg bw/day for male and female Hsd.Han:Wistar rats.