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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
short deviations from microclimate limits: Increased temperature 30.07 – 2h(28.2°C) and 25. 08 –2h(28.5°C) Decreased temperature 03-04.08 – 10h(17.7°C), 13.08 –1h(18.9°C), 23.08 -8h(17.9°C), 24.08 – 5h(18.5°C) Increased rel. humidity 24.08 - 10h(73.1%)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 4,4'-bis[[4-anilino-6-[(2-carbamoylethyl)(2-hydroxyethyl)amino]-1,3,5,-triazin-2-yl]amino]stilbene-2,2'-disulphonate
EC Number:
248-420-5
EC Name:
Disodium 4,4'-bis[[4-anilino-6-[(2-carbamoylethyl)(2-hydroxyethyl)amino]-1,3,5,-triazin-2-yl]amino]stilbene-2,2'-disulphonate
Cas Number:
27344-06-5
Molecular formula:
C42H46N14O10S2.2Na
IUPAC Name:
disodium 4,4'-bis[[4-anilino-6-[(2-carbamoylethyl)(2-hydroxyethyl)amino]-1,3,5,-triazin-2-yl]amino]stilbene-2,2'-disulphonate
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: females – sexually adult (6 - 7 weeks on arrival)
- Housing: 2 rats of the same sex in one plastic cage (polycarbonate cages for rats TECNIPLAST, type 1291H, Italy)
- Housing conditions: Animals were housed in a controlled environment, in which optimal conditions were considered to be: approximately 15 air changes per hour, a temperature of 22+3°C, a relative humidity of 30-70% and 12-hour light/12-hour dark cycle
- Identification: made using colour marks (colour for veterinary usage) on their fur (system 1 – 10 of main groups, 1 - 6 of survival group), each cage was marked with the number of study, number of animals, sex, number of cage, name and dose of the test item and mark of group
- Feeding: free access to food (ad libitum).
- Diet: complete pelleted diet for rats – Altromin 1321
Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany
- Water: free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Ministry of Health.

Preparation of Experimental Animals: during the acclimatisation period, the health condition of all animals was monitored daily. Then the animals were randomly divided into the control and test groups and they were marked individually. The first examinations were then performed - weighing of animals, detailed clinical observations and ophthalmological examination.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqua pro iniectione
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighted into glass beaker and the beaker was replenished by aqua pro iniectione and this was insered in ultrasonic bath for 25 minutes . The test solution was stirred by magnetic stirrer (350 rpm) for 35 minutes. For each dose level concentration, the solution was prepared separately. The application forms were prepared daily just before administration. The administration of the test item to animals was performed during two hour after preparation of application form. The stirring of solutions continued during administration.

Animals were fasted 2 hours before to test item administration. After the test item was administered, the animals were starved next 2 hours.

The concentrations of solutions at all dose levels (12 mg/mL, 35 mg/mL and 75 mg/mL corresponding to 120, 350 and 750 mg/kg/day respectively) were adjusted to ensure the administration of 1 mL per 100 g of body weight.

VEHICLE:
Supplier: Dr. Kulich Pharma, Czech Republic
Batch No.: 1902210147, Exp.: 02/2021
Batch No.: 1903050175, Exp.: 03/2021
Batch No.: 1902220150, Exp.: 02/2021
Batch No.: 1903260235, Exp.: 03/2021
Batch No.: 1905170334, Exp.: 05/2021
Batch No.: 1906130400, Exp.: 06/2021
Batch No.: 1907110470, Exp.: 07/2021
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical method
The test item stability and homogeneity were determined by means of measuring of peak area of test item by high-performance liquid chromatography (HPLC) based on a method developed for the purpose of this study at the test facility.

Preparation of test item formulation
The test item formulation was prepared by mixing with Aqua pro iniectione. Two concentrations of test item formulation were prepared (1000 mg/10 mL and 10 mg/10 mL).

Preparation of application forms
- Concentration level 1000 mg/10 mL
The test item was weighed into glass beaker calibrated to 100 mL and dissolved in vehicle (ca 80% of total volume) in ultrasonic bath together with occasional mixing by glass rod for 15 minutes. After this the glass beaker was diluted to mark by vehicle and the application form was stirred by magnetic stirrer (350 rpm) for 5 min.
- Concentration level 10 mg/10 mL
The test item was weighed into glass beaker calibrated to 100 mL and dissolved in vehicle in ultrasonic bath for 5 min. After this the solution was stirred by magnetic stirrer (350 rpm) for 5 minutes.


Analysis
Homogeneity of the application forms was checked by determination of a peak area of the test item in three places of application form (at the bottom, in the middle and on the surface).

Stability of the application forms was checked by analyses of the application forms within 120 min (at the time 0, 30, 60, 90 and 120 min). Time interval 0 min represents for concentration 10 mg/ 10mL the time after 5 minutes dissolving in ultrasonic bath and 5 minutes of mixing by magnetic stirrer (350 rpm) and for concentration 1000 mg/10mL the time after 15 minutes dissolving in ultrasonic bath and 5 minutes of mixing by magnetic stirrer (350 rpm).

The beaker with test item was during dissolving, homogenisation and stability measuring covered by alluminium foil due to possible light unstability of test item.
This measure is recommended for further test item application form preparation.

Results:
Both application forms (10 mg and 1000 mg /10 mL) of the test item at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) are homogenous and stable at least for 120 minutes from the finalization of application form preparation.

Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
120 mg/kg bw/day (actual dose received)
Dose / conc.:
350 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
one as main group and one as satellite group
No. of animals per sex per dose:
10
6 for satellite groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
- Other:

Examinations

Observations and examinations performed and frequency:
Body weight: recorded weekly on automatic balances with group mean computing module. All animals were weighed immediately before euthanasia too. Weight increment was calculated as a mean per group per day (in grams).

Food consumption: weekly. The mean values in groups were calculated for each week of the study. Food consumption for animal/day was calculated from mean values of each group: total initial food weight – total remainder of food = group consumption → group consumption/number of animals/number of days = mean food consumption per animal and per day. Calculation of food conversion in %: weight increment/food consumption x 100.

Water consumption: twice a week. The mean values in groups (water consumption per animal and per day) were calculated for each week of the study: total initial volume – total remainder of water = group consumption → group consumption/number of animals/number of days = mean water consumption per animal and per day.

Mortality observations: twice daily

Health condition control: daily.

Clinical observations: daily. This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (11.00 – 13.00 pm.) what was the time of expected maximal effect of the test item. Animals were observed in natural conditions in their cages.

Functional observation:at the last week of administration and recovery period
During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli (reaction to approximation, to contact point, to noise and to pain) and pupillary reflex, were evaluated and motor activity assessment (number of upstanding, emiction and defecation within 3 minutes) was conducted. Moreover the individual observations of grip strength (grip power in Newton) were performed using dynamometer. Measurements were made on pectoral legs and pelvis legs.

Ophthalmological examination: at the 1st week of study and at the last week of administration and recovery period.
Examination of eyes was made in a dark room without treatment (dilatation). Both eyes were examined by means of an ophthalmoscope. The following examinations were made: visual acuity, eyelids, palpebral fissure, eyelash, bulb, lacrimal apparatus, conjunction, sclera, cornea, iris, pupil, lens, eye ground.

Urinalysis: 90th (main groups); 118th (satellite groups) day of study.
The rats were kept in the metabolic cages for the collection of urine for one hours. Immediately before entering metabolic cages the animals were administered 2 mL of drinking water for 100g of body weight by gavage to the stomach.

Haematology examination: 91st (main groups); 119th (satellite groups) day of study.
The blood samples were collected from the orbital plexus by glass micropipette under the light ether narcosis into the PVC test tubes containing anticoagulation systems.

Clinical biochemistry: 91st (main group); 119th (satellite group) day of study .
The animals were starved approximately for 18 hours before blood collection but they were supplied with drinking water ad libitum. The blood samples were collected from the orbital plexus under the light ether narcosis. Biochemical parameters were measured in serum.


Sacrifice and pathology:
At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary gland and heart in control, all treated and satellite groups of animals were recorded. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out. Organs for consequent histopathological examination were taken out and stored in containers with fixative (buffered 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.

The mentioned tissues and organs were collected from all males and females at necropsy and fixed in buffered 4% formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.
In the study the full histopathology of the preserved organs and tissues was performed for all high dose, control animals, recovery animals a animals with macroscopical changes
Other examinations:
Blood samples from males and females were assessed for serum levels of thyroid hormones (thyroxine - T4, triiodothyronine - T3 and rat Thyroid Stimulating Hormone -TSH) by ELISA kit. The examination of thyroid hormones was conducted in animals of control groups, all treated groups and recovery groups were analysed.

Results and discussion

Results of examinations

Mortality:
mortality observed, non-treatment-related
Description (incidence):
Males & Satellite Males
No mortality of males was detected.

Females & Satellite Females
Female No. 106 (at the control group) died at the 6th week of study. The exact cause of death was not determined.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males
The statistical analysis was performed for necropsy body weights and body weights of the 13th week. Statistically significant differences were not found.
The body weights of males at the dose level 750 mg/kg/day were slightly decresed compared with the body weight of control males from 7th week to end of study. The body weights of males at the dose levels 120 and 350 mg/kg/day were similar compared to control group. Decreased necropsy body weights at the dose level 750 mg/kg/day were detected. Body weight increments were similar in males of treated groups compared to control for the whole application period.

Satellite males
The statistical analysis was performed for necropsy body weights and body weights of the 17th week. Statistically significant differences were not found.
The body weights and necropsy body weights were similar in males of treated group compared to control for the whole application period and during the recovery period. Body weight increments were similar in males of treated group compared to control for the whole application period and during the recovery period.

Females
The statistical analysis was performed for necropsy body weights and body weights of the 13th week. Statistically significant differences were not found.
The body weights and necropsy body weights were similar in females of treated groups compared to control for the whole application period.
The body weight increments were variable in females of treated and control groups during the whole application period. Negative effect on the body weigh increments were recorded in females of control group at the 12th and 13th week of application period, in females at the dose level 120 mg/kg/day at the 12th week of application period and in females at the dose level 750 mg/kg/day at the 12th week of application period.
Slightly increased total body weight increment of females at the dose levels 120 and 750 mg/kg/day was recorded.

Satellite females
The statistical analysis was performed for necropsy body weights and body weights of the 17th week. Statistically significant differences were not found.
Slightly increased body weights of satellite treated females were recorded during the whole application period and recovery period. The necropsy body weights of treated females were higher compared to control females.
The body weight increments were variable in females of treated and control groups during the whole application and recovery period. Negative effect on the body weigh increments were recorded in control females at the 9th, 12th, 15th and 17th week of study and in treated females at the 12th, 15th and 16th week of study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males & Satellite males
Food consumption of treated males was similar compared to the control males during the whole application period.

Females & Satellite females
Food consumption of treated males was similar compared to the control males during the whole application period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Males
Water consumption of treated males was variable compared to water consumption of control males during the whole application period but adequate to species, sex and age of animals used in experiment.

Satellite males
Water consumption of satellite treated males was similar compared to satellite controls during the application period and recovery period.

Females & Satellite females
Water consumption of treated females was similar compared to water consumption of control females during the whole application period.

Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmologic examination was performed in all animals at the beginning of the study. At the end of study only in animals at the control group, highest group and in both satellite groups (control and 750 mg/kg/day) the ophthalmologic examination was performed. Because in animals at the dose level 750 mg/kg/day (highest dose level) any changes related with application of the test item was not find out, animals of lower dose levels were not examined.

Males & Satellite males
During an examination performed before the start of the study and at the end of the study, no changes were noted.

Females & Satellite females
During an examination performed before the start of the study and at the end of the study, no changes were noted.



Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Males
In haemocoagulation parameters – statistically significantly prolonged APTT in males at the dose level 750 mg/kg/day was recorded. This value of APTT is out of historical control limits.
Percentual representation of reticulocytes in blood in males at the dose level 750 mg/kg/day was statistically significantly decreased (but in range of historical control).
For the white and red blood components, the values were not affected by the administration of the test item.

Satellite males
Statistically significantly decreased percentual count of basophiles in satellite treated males was detected (in a range of historical control).
Other haematological parameters of satellite treated males were similar with the satellite control males.

Females
The following statistically significant differences were registered in haemocoagulation parameters – prolonged APTT in all treated groups, shortened PT at the dose level 350 mg/kg/day and decreased value of fibrinogen at the dose levels 120 and 350 mg/kg/day.
In red blood components – statistically significantly increased value of platelet count at the dose level 350 mg/kg/day was recorded.
In white blood component – change of differential leucocyte counts (percentage portion of monocytes – statistically insignificantly increased and percentage portion of neutrophils and eosinopiles - insignificantly decreased in females at the dose level 120 mg/kg/day) was recorded.
All values of heamatological parameters were in historical control limits.

Satellite females
In red blood components – statistically significantly decreased values of haemoglobin and platelet count in satellite treated females were recorded.
In white blood component – change of differential leucocyte counts (percentage portion of neutrophiles – statistically significantly increased in satellite treated females) was recorded.
Other haematological parameters of satellite treated females were similar with the satellite control females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males
Statistically significantly decreased values of protein total and albumin in males at the dose levels 350 and 750 mg/kg/day were recorded (dose dependent). The activity of ALP in males at the doses 350 and 750 mg/kg/day was statistically significantly decreased (dose dependent) compared to the control group. Statistically significantly decreased value of creatinine at the dose level 350 mg/kg/day was recorded. Value of bilirubin total in males at the dose level 750 mg/kg/day was increased. Statistically significantly decreased value of calcium ions in males at the dose levels 350 and 750 mg/kg/day was detected. The value of cholinesterase in all treated groups was significantly decreased compared to the control group. Statistically significantly increased value of chloride ions in males in all treated groups was detected (dose dependent).
Differences in values of other biochemical parameters did not show any significant changes.
All values of biochemical parameters were in historical control limits.

Satellite males
Statistically significantly decreased activities of ALT, AST and ALP were recorded in treated males.
Other biochemical parameters of satellite treated males were similar with the satellite control males.

Females
Statistically significantly increased values of protein total and albumin at the dose level 120 mg/kg/day were recorded on the contrary the values of protein total and albumin at the dose level 750 mg/kg/day were statistically significantly decreased. Statistically significantly decreased value of creatinine at the dose levels 350 and 750 mg/kg/day was detected (with dose-dependence). The value of calcium ions in females at the dose level 120 mg/kg/day was increased with statistical significance. Increase value of cholinesterase in females at the dose levels 120 and 750 mg/kg/day was recorded (at the dose level 120 mg/kg/day with statistical significance). Statistically significantly decreased value of LDL cholesterol in females at the dose level 750 mg/kg/day was detected. Significantly increase of values of sodium and potassium ions in females at the dose levels 350 and 750 mg/kg/day were detected. In all treated groups statistically significantly increased value of chloride ions was recorded (at the dose levels 350 and 750 mg/kg/day the value of chloride ions was in out of historical control limits).
Differences in values of other biochemical parameters did not show any significant changes.

Satellite females
No statistically significant changes of biochemical parameters in satellite females were registered.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Males
Statistically significant differences in volume of urine were not detected but insignificantly decreased volume of urine in males at the dose level 750 mg/kg/day was recorded. Statistically significantly increased pH of urine was detected in males at the dose level 750 mg/kg/day.
Change of colour of urine (yellow) was observed in one male at the dose level 750 mg/kg/day.
Presence of protein in urine was recorded in males of control, low and medium doses. Presence of blood and leukocytes in urine was observed in all doses, including the control group. Presence of protein, blood and leucocytes in urine has relation to the sex and age of animals used in experiments.

Satellite males
Statistically significant differences in volume of urine and pH of urine were not detected. Presence of proteins, of leukocytes and of blood in urine was recorded in both groups. Presence of protein, blood and leucocytes in urine has relation to the sex and age of animals used in experiments.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Males
Reactions to touch, noise, pain and pupillary reflex of treated males were the same as in the control group. The number of upstanding of treated males compared to control males was increased at the dose level 750 mg/kg/day. The variability was adequate to species, sex and age of animals used in experiments. Emiction and defecation of treated animals was without importatnt changes. The time of grip of pectoral legs was slightly increased at the dose level 120 mg/kg/day but without toxicological importance.

Satellite males
No significant differences were detected in examined parameters.

Females
Reactions to touch, noise, pain and pupillary reflex of treated females were the same as in the control group. The number of upstanding of treated females compared to control males was increased at the dose level 750 mg/kg/day. The variability was adequate to species, sex and age of animals used in experiments. Emiction and defecation of treated animals was without importatnt changes. The time of grip of pectoral legs was slightly increased at the dose level 350 mg/kg/day but without toxicological importance.

Satellite females
No significant differences were detected in examined parameters.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males
Absolute weights of liver and spleen were significantly decreased at all dose levels compared to the control group (with dose-dependence). Statistically significantly decreased absolute weight of testes was recorded in males at the dose level 750 mg/kg/day.
Absolute weights of other organs of treated males were balanced with control males.
Relative weight of liver and spleen were significantly decreased at all dose levels compared to the control group (in spleen with dose-dependence). Statistically significantly increased relative weights of kidneys was recorded in males at the dose level 750 mg/kg/day. At the dose level 350 mg/kg/day relative weights of kidneys was increased insignificantly.
Relative weights of other organs of treated males were balanced with control males.

Satellite males
Statistically significant increased absolute weight of kidneys in satellite treated males was detected.
Absolute weights of other organs of treated males were balanced with control males
The relative weight of organs of satellite treated and control males were similar.

Females
Absolute weights of liver and spleen were significantly decreased at all dose levels compared to the control group (in liver with dose-dependence).
Insignificantly decreased absolute weight of uterus in females at the dose level 120 mg/kg/day was detected. This finding probably related to the oestrus cycle of females.
Absolute weights of other organs of treated females were balanced with control females.
Relative weights of liver and spleen were significantly decreased at all dose levels compared to the control group (without dose-dependence).
Relative weights of other organs of treated females were balanced with control females.

Satellite females
Statistically significant increased absolute weights of brain, heart and uterus in satellite treated females were detected. Increased absolute weight of uterus probably related to the oestrus cycle of females.
Statistically significant changes in relative organ weights of treated females were not detected. Insignificantly increased relative weight of uterus in treated females was detected. This finding probably related to the oestrus cycle of females.
Relative weights of other organs of treated females were balanced with control females.

Gross pathological findings:
no effects observed
Description (incidence and severity):
Males
In all males of control and of treated groups, no macroscopical findings were recorded during necropsy.

Satellite males
No macroscopical findings were recorded during necropsy; only as for thymus, dark red colour in one satellite treated male was recorded.

Females
In all females of control and of treated groups, no macroscopical findings were recorded during necropsy. Non-pathological finding in terms of uterus dilatation in some females of control and of treated groups was detected.

Satellite females
No macroscopical findings were recorded during necropsy.
Non-pathological finding in terms of uterus dilatation was detected in two animals of the treated group.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The full histopathology of the preserved organs and tissues was performed for all high dose and control animals and satellite animals. Organs with macroscopical changes in females (uterus: dilatation) were examined at the lowest and middle dose level groups.

Males
The histopathological examination organs in males showed only sporadical microscopical changes and these changes did not related with the test item treatment.

Satellite males
The histopathological examination organs in satellite males showed only sporadical microscopical changes and these changes did not related with the test item treatment.

Females
The histopathological examination organs in females showed only sporadical microscopical changes and these changes did not related with the test item treatment.

Satellite females
The histopathological examination organs in satellite females showed only sporadical microscopical changes and these changes did not related with the test item treatment.

Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Biochemical examination – concentration of T4, T3 and TSH hormone

Blood samples from the all groups of males and females were assessed for serum levels of thyroid hormones (T4, T3 and TSH).

Males
Statistically significant differences in concentrations of T4, T3 and TSH hormone were not detected.
Mean of concentrations of T4, T3 and TSH hormone were similar in treated males in comparison with the control males.

Satellite males
Statistically significant differences in concentrations of T4, T3 and TSH hormone were not detected.
Mean of concentrations of T4, T3 and TSH hormone were similar in satellite treated males in comparison with the satellite control males.

Females
Statistically significant differences in concentrations of T3 and TSH hormone were not detected.
Mean of concentrations of T3 and TSH hormone were similar in all treated females in comparison with the control females.
Statistically significantly decreased concentration of T4 hormone at the dose levels 120 and 350 mg/kg/day was detected (values of T4 concentration of hormone were in historical control limits).

Satellite females
Statistically significant differences in concentrations of T3 and TSH hormone were not detected.
Mean of concentrations of T3 and TSH hormone were similar in satellite treated females in comparison with the satellite control females.
Statistically significantly decreased concentration of T4 hormone in satellite treated females was detected (the value of T4 concentration of hormone was in historical control limits).

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

120 mg/kg/day

No toxicologically adverse effects of the test item on growth of animals, food and water consumption and health condition of animals were detected in both sexes. The mild variability observed during the study was adequate to species, sex and age of animals used in experiments.

Changes of haematological parameters were detected in females: statistically significantly prolonged APTT and statistically significant decrease of value of fibrinogen. These changed haematological parameters were in historical control limits.

Changes of biochemical parameters were detected in both sexes: in males statistically significant decrease of value of cholinesterase and statistically significantly increased concentration of chloride ions, in females statistically significantly increased values of protein total albumin, cholinesterase, concentrations of calcium and chloride ions. Statistically significantly decreased concentration of T4 hormone was recorded in females. These changed biochemical parameters were in historical control limits.

Examination of urine showed statistically significantly decreased volume of urine in females.

During the biometry of organs statistically significantly decreased absolute and relative weight of liver and spleen in both sexes were found out. 

During pathological inspection of organs, no changes were detected only dilatation of uterus in two females. This finding of uterus was not considered as related with test item treatment.   

Histopathological examination detected hydrometra in uterus in three females. This finding was not considered as related with test item treatment.

 

350 mg/kg/day

No toxicologically adverse effects of the test item on growth of animals, food and water consumption and health condition of animals were detected in both sexes. The mild variability observed during the study was adequate to species, sex and age of animals used in experiments.

Changes of haematological parameters were detected in females: statistically significantly prolonged APTT, statistically significantly shortened PT, statistically significantly increased value of platelet count and statistically significant decrease of value of fibrinogen. These changed haematological parameters were in historical control limits.

Changes of biochemical parameters were detected in both sexes: in males statistically significant decrease of values of protein total albumin, cholinesterase, creatinine, ALP, concentration of calcium ions and statistically significantly increased concentration of chloride ions; in females statistically significantly decreased value of creatinine and statistically significantly increased concentrations of sodium, potasium and chloride ions. Statistically significantly decreased concentration of T4 hormone was recorded in females. These changed biochemical parameters were in historical control limits (except for value of chloride ions in females, this value was over historical limits).

Examination of urine showed statistically significantly decreased volume of urine and statistically significantly decreased pH value in females.

During the biometry of organs, statistically significantly decreased absolute and relative weight of liver and spleenin both sexeswere found out. 

Duringpathological inspection of organsno changes were detected except for dilatation of uterus in one female. This change finding of uterus was not related with test item treatment.   

Histopathological examination showed hydrometra in uterus in three females. This finding was not related with test item treatment.

 

750 mg/kg/day

No toxicologically adverse effects of the test item on growth of animals, food and water consumption and health condition of animals were detected in both sexes. The mild variability observed during the study was adequate to species, sex and age of animals used in experiments.

Changes of haematological parameters were detected in both sexes: in males statistically significantly prolonged APTT, statistically significant decrease of value of reticulocytes and delayed statistically significant decrease of percentage portion of basophiles; in females statistically significantly prolonged APTT and delayed statistically significantly decreased value of platelet count and haemoglobin and delayed statistically significantly increased percentage portion of neutrophiles. These changed haematological parameters were in historical control limits (except the value of APTT in males, this value was over historical limits). In satellite treated males the value of APTT was conformable with satellite control males. 

Changes of biochemical parameters were detected in both sexes: in males statistically significant decrease of values of protein total, albumin, cholinesterase, statistically significantly increased value of bilirubin total, statistically significantly decreased activity of ALP, concentration of calcium ions, statistically significantly increased concentration of chloride ions and delayed statistically significantly decreased activity of ALP, ALT and AST; in females statistically significantly decreased value of protein total, albumin, creatinine, value of LDL cholesterol and statistically significantly increased concentrations of sodium, potasium and chloride ions and delayed statistically significantly increased concentration of T4 hormone. These changed biochemical parameters were in historical control limits (except for value of chloride ions in females, this value was over historical limits). In satellite treated females the concentration of chloride ions was conformable with satellite control females.

Examination of urine showed statistically significantly increased pH of urine in males and delayed statistically significantly decreased pH value in females. At the dose level 750 mg/kg/day the urine volume was similar compared to control group.

During the biometry of organs, reversible statistically significantly decreased absolute and relative weight of liver and spleen in both sexes, statistically significantly decreased absolute weight of testes, delayed statistically significantly increased absolute weight of kidneys in males, delayed statistically significantly increased absolute weights of brain, heart and uterus in females were found out. 

During pathological inspection of organs, only dark colour of thymusin one satellite male was found out. Uterus dilatation in four females (two from main group and two from main satellite group) was found out. This change finding of uterus was not related with test item treatment.  

Only sporadic occurrence of other microscopical changes in animals were observed during histopatological examination. These findings was not related with the test item treatment. No histopathological findings of liver and spleen related to changed weight of this organs were found out.

Applicant's summary and conclusion

Conclusions:
The value of NOAEL for REPEATED DOSE TOXICITY (90 days) was established as 750 mg/kg body weight/day both for MALES and FEMALES. The value of NOAEL was determined on the basis results of histopathological examinations. No biological significant changes were observed.
Executive summary:

The oral administration of the test item to rats by gavage for a period of 90 consecutive days at all dose levels (including satellite groups) did not cause mortality.

In the control group one female died during the study. The exact cause of death was not determined. This death was not related with the test item treatment.

No effects of the test item on the body weight, food consumption and water consumption were recorded during the study. Slight changes of these parameters were without toxicological importance.

No influence of the test item on clinical status of treated animals and satellite treated animals was recorded.

No findings were detected during the histopathological examination of liver, spleen and kidneys of animals at the highest dose level and satellite treated animals.

Haematological examination showed significantly prolonged APPT in females in all treated groups and at males at the highest dose level (750 mg/kg/day). In females significantly shortened PT at the middle dose level (350 mg/kg/day) and decreased value of fibrinogen at the lowest (120 mg/kg/day) and middle dose levels were recorded. In satellite treated animals changes of these haematogical parameters were not recorded. All changed values of haematological parameters in satellite treated animals were not of biological or toxicological significance.

Biometry of organs showed reversibly decreased relative and absolute weight of liver and spleen in all treated animals of both sexes (with dose dependence) and increased relative weight of kidneys in males in the highest dose level. These changes of organs weights were not recorded in satellite animals. All changed weights of organs in satellite treated animals have no biological or toxicological significance.

Biochemical examination in treated animals of both sexes showed following significant differences: decreased value of protein total and albumin in males (with dose dependence) and increased value of protein total and albumin in females at the lowest dose level and on the contrary at the highest dose level these values were decreased. Decreased values of cholinesterase in all treated male groups and increased value of cholinesterase in females at the lowest dose level were detected. Activity of hepatic enzyme were changed in males: irreversible decreased activity of ALP and delayed decreased activity of ALT and AST were detected (the values of ALP, ALT and AST was in historical control limits). Decreased value of creatinine in females was detected with dose dependence and in males decreased value of creatinine at the middle dose level was recorded. Disbalance of concentration of calcium, sodium, potassium and chloride ions were recorded in males and females in all treated groups. In females at the middle and highest dose levels concentration of chloride ions was out in historical control limits.  

All changes were reversible (except decreased activity of ALP in males and delayed decreased activity of ALT and AST in males). All these changes observed in animals had probably an adaptive character - the organism's response to the test item treatment.A longer recovery time would probably be required to completely eliminate the altered hepatic enzyme activity in males.

All changed values of biochemical parameters in satellite treated animals have no biological or toxicological significance were not not biological or toxicological significantly.

 

The value of NOAEL for REPEATED DOSE TOXICITY (90 days) was established as 750 mg/kgbody weight/day both for MALES and FEMALES. The value of NOAEL was determined on the basis results of histopathological examinations. No biological significant changes were observed.