Registration Dossier

Administrative data

Description of key information

The value of NOAEL is 50 mg/kg bw/day for males and 150 mg/kg bw/day for females. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12.08.2015-21.03.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
short term deviations from microclimate for details see Remarks on results
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: SPF breeding, VELAZ s.r.o., Únětice, Czech Republic, RČH CZ 11760500- Age at study initiation: 6-7 weeks-on arival, sexually adult- Fasting period before study: no- Housing: All the study proceeded in SPF (Specified Pathogen Free) animal house of CETA in SPF conditions according to internal SOP No. 12. Animals were housed in animal room, 2 rats of the same sex in one plastic cage (polycarbonate cage TECHNIPLAST). - Bedding: Sterilized Lignocel (raw material: spruce; producer: J. Rettenmaier&söhne, Germany).- Diet: ad libitum- Water: ad libitum, Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Ministry of Health. - Acclimation period: 5 days- Identification: It was made by 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 substance and mark of group (according to internal SOP No. 26) ENVIRONMENTAL CONDITIONS- Temperature (°C): 22±3°C- Humidity (%): 30-70%- Air changes (per hr): 15 air changes per hour- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle Study Time ScheduleTest substance delivery: 08. 04. 2015Date of animal arrival: 12. 08. 2015 Acclimatisation: 12. 08. – 16. 08. 2015Start of administration: 19. 08. 2015End of administration: 19. 11. 2015Clinical examinations: main groups 19. 08. – 19. 11. 2015 satell.groups 19. 08. – 17. 11. 2015 Urinalysis: main groups 16. – 19. 11. 2015 satell.groups 14. – 15. 12. 2015Blood taking and necropsies: main groups 17. – 20. 11. 2015 satell.groups 15. – 16. 12. 2015Histopathological examination: 08.01. – 24. 03. 2016 Evaluation of results and final report elaboration: 18.01. – 21. 04. 2016
Route of administration:
oral: gavage
Vehicle:
other: in 0.5% methylcelulose in aqua pro injectione.
Details on oral exposure:
The Preparation of Application form The application form was prepared by mixing with 0.5% methylcelulose in aqua pro injectione. Two concentrations of application form were prepared (50 mg/10 mL and 600 mg/10 mL). Concentration level 50 mg/10mLCa. 250 mg of the test substance was weighted into 100 mL glass beaker calibrated to 50 mL. 50 mL of the vehicle were measured out by volumetric cylinder and added to the beaker. Dissolving in ultrasonic bath for a 10 minutes follows. Then the application form (white suspension) was stirred by using of laboratory stirrer for 5 minutes (1000 rpm). Concentration level 600 mg/10 mLCa. 3 g of the test substance was weighted into 100 mL glass beaker calibrated to 50 mL. 50 mL of the vehicle were measured out by volumetric cylinder and added to the beaker. Dissolving in ultrasonic bath for a 10 minutes follows. Then the application form (white pasty suspension) was stirred by using of laboratory stirrer for 5 minutes (1000 rpm).DIET PREPARATION- Type of food: Diet was sterilised before using. Complete pelleted diet for rats in SPF breeding – Altromin (Standard maintenance diet for mice and rats), Manufacturer: Spezialfutter GmbH & Co., KG, Im Seelenkamp 20, D-32791, Lage, Německo- Composition of diet: Wheat, Oats, Fish meal powder, Dried snail-clover, Soya extracted groats, Wheat sprouts, Dehydrated yeast, Calcium carbonate, Vitamin and Mineral complex.- Nutrient content of the diet: Dry matter – 89%, Crude protein – min. 19%, Fat – min. 4%, Fiber – max. 6%, Ash – max. 7.5%.- Storage temperature of food: at labotarory conditions- Additional information: The standard pelleted laboratory animal diet is analysed for nutrients (once a year) and bacteriological contaminants every 2 months on a regular basis. Results are retained in the CETA archives. Certificates of drinking water analysis (performed twice a year) are retained in the CETA archives. Analysis of diet and water did not reveal any findings that could affect study integrity.VEHICLEMethylcellulose Lot: 1E02012N12 Expiration: 04/2017Producer: Dr. Kulich Pharma Hradec Králové, Czech RepublicAqua pro iniectione Lot: 1503130162 (exp.:03/2017), 1505050287 (exp.:05/2017) Producer: Ardeapharma Ševětín a.s, Czech Republic
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and the homogeneity of application form were determined in CETA analytical laboratories. Stability and homogeneity were determined by means of measuring of a peak area of the test substance by a high-performance liquid chromatography based on a method developed at the test facility.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:50 mg/kg/dayBasis:actual ingested
Remarks:
Doses / Concentrations:150 mg/kg/dayBasis:actual ingested
Remarks:
Doses / Concentrations:450 mg/kg/dayBasis:actual ingested
Remarks:
Doses / Concentrations:600 mg/kg/dayBasis:actual ingested
No. of animals per sex per dose:
Main groupvehicle 10F+10M50 mg/kg/day 10F+10M150 mg/kg/day 10F+10M450mg/kg/day 10F600 mg/kg/day 10MSatellite groupvehicle 6F+6M450mg/kg/day 6F600 mg/kg/day 6M
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for study (50, 150 and 600 mg/kg/day for males and 50, 150 and 450 mg/kg/day for females) were chosen on the basis of the results of Study No.194/11/7 Centralit - Repeated Dose (28 days) Toxicity (Oral), VUOS-CETA Report No. 12-131, 2012. - Rationale for animal assignment: random- Post-exposure recovery period in satellite groups: 28 days
Observations and examinations performed and frequency:
MORTALITY CONTROL: Yes- Time schedule: dailyHEALTH CONDITIONS CONTROL- Time schedule for examinations: daily before, during and immediately after applicationCLINICAL OBSERVATION- Time schedule: daily im natural conditions in their cagesDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: before the first application and then weekly- Observation in the cages: piloerection, posture, eyes (pupils), breathing, tonic or clonic movements, stereotypes or bizarre behaviour- Removal from cages: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina. FUNCTIONAL OBSERVATION: - Time schedule for examinations: at the end of administration period and after recovery period- the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex and motor activity assessment. - observations of grip strength (using dynamometer) Measurements were made on: 1) pectoral legs, 2) pelvis legs. BODY WEIGHT: Yes- Time schedule for examinations: weeklyFOOD CONSUMPTION AND COMPOUND INTAKE: weekly- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: YesFOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: YesWATER CONSUMPTION AND COMPOUND INTAKE: Yes- Time schedule for examinations: twice a week/three times a weekOPHTHALMOSCOPIC EXAMINATION: Yes- Time schedule for examinations: at the 1st week of study and at the last week of administration and recovery period- Dose groups that were examined: at the control and highest groups, both eyes- Made examination: visual acuity, eyelids, palpebral fissure, eyelash, bulb, lacrimal apparatus, conjunction, sclera, cornea, iris, pupil, lens, eye groundHAEMATOLOGY: Yes, from orbital plexus- Time schedule for collection of blood: 91st (main group); 119th (satellite group) day of study - Anaesthetic used for blood collection: Yes (light ether narcosis)- Animals fasted: Yes - How many animals: all animals- Parameters checked in table [No.2] were examined.BIOCHEMICAL CHEMISTRY: Yes, from orbital plexus- Time schedule for collection of blood: 91st (main group); 119th (satellite group) day of study - Anaesthetic used for blood collection: Yes (light ether narcosis)- Animals fasted: Yes - How many animals: all animals- Parameters checked in table [No.3] were examined.URINALYSIS: Yes- Time schedule for collection of urine: 90st (main group); 118th (satellite group) day of study - Metabolism cages used for collection of urine: Yes, for two hours-Details: Immediately before entering metabolic cages the animals were administered 2 mL of drinking water for 100g of body weight by gavage to the stomach.- Parameters checked in table [No.4] were examined.PATHOLOGICAL EXAMINATION- Time schedule for examinations: 91st (main group); 119th (satellite group) day of study- Details: narcotised and sacrificed by cutting the neck spine and medulla, gross necropsy of the cranial, thoracic and abdominal cavitiesBIMETRY ORGANS- Time schedule for examinations: 91st (main group); 119th (satellite group) day of study- Absolute weights of: liver, kidneys, adrenals, testes or ovaries, epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary gland and heart
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, gross necropsy of the cranial, thoracic and abdominal cavitiesHISTOPATHOLOGY: Yes (see table No.:5)
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITYMalesOne male (No. 25) at the dose level 50 mg/kg/day died during application period (on the 49th day of study) – intubation error. This death did not relate to the test substance administration. FemalesTwo females (No. 143 and 150) at the dose level 450 mg/kg and one female in the satellite treated group (No. 154) died after the first application. FOOD CONSUMPTION AND COMPOUND INTAKEMale:Food consumption of treated males was slightly higher than in control males for the whole time of application period. This difference was dependent on dose level.Satellite males: Food consumption of satellite treated males was slightly higher than in satellite control males for the whole time of application period and recovery period.Female: Food consumption of treated females was similar or slightly higher than control group during the whole application period. Satellite female: Food consumption of satellite treated females was similar or slightly higher than in satellite control females for the whole time of application period. In recovery period food consumption of satellite treated females was similar to the satellite control females. FOOD CONVERSIONMalesFood conversion of treated males was similar or slightly lower than control group during the whole application period (except marked fall of food conversion in the 12th week of application period at the dose levels 150 and 600 mg/kg/day). Satellite malesDose independent dis-balance of food conversion was recorded between the satellite treated group and the satellite control group during the whole study. FemalesDose independent disbalance of food conversion was recorded between the treated groups and the control group during the whole application period. Satellite femalesDose independent disbalance of food conversion was recorded between the satellite treated group and the satellite control group during the whole study (the application and recovery period). WATER CONSUMPTION AND COMPOUND INTAKEMalesWater consumption of treated males at the dose level 50 and 150 mg/kg/day was similar compared to water consumption of control males for the whole time of application. At the dose level 600 mg/kg/day the water consumption was similar or increased compared to control males during the application period (except the 2nd week of application when this was decreased). Satellite malesWater consumption of satellite treated males was higher compared to satellite control males in both period - application and recoveryFemalesWater consumption of treated females at the dose level 50 and 150 mg/kg/day was similar compared to water consumption of control females for the whole time of application. At the dose level 450 mg/kg/day the water consumption was similar or slightly higher than control females during the application period.Satellite femalesWater consumption of satellite treated females was similar to satellite control group during the both period – application and recovery. NEUROBEHAVIOURFemales: Straub phenomenon was observed. Straub reaction consisted of the tail becoming rigid and erected across the back of the animals in a S-shaped curve. This reaction is typical for animals after application of opiate substance, e.g. morphine. This reaction was accompanied by stupor and tachypnoe. During next weeks of study, these changes were not observed any more. HAEMATOLOGYMales: in haemocoagulation parameters – significantly decreased value of fibrinogen at the dose level 150 mg/kg/day, significantly prolonged APTT at the dose level 600 mg/kg/day and significantly increased value of platelet count at the dose level 600 mg/kg/day,in white blood component – insignificantly increased value of total leucocyte count at the dose level 600 mg/kg/day, decreased portion of granulocytes in all treated groups (at the dose levels 50 and 150 mg/kg/day statistically significant), insignificantly increased portions of monocytes and lymphocytes in all treated groups and in red blood component - parameters of red components were not altered in all treated groups. The value of APTT was above histocal control limits at the dose level 600 mg/kg/day. Satellite males: in haemocoagulation parameter - significantly shortened APTT and in white blood component – insignificantly increased portion of granulocytes. Parameters of red blood component were not altered.Females: in red blood component - significantly decreased value of total erythrocyte count and haematocrite at the dose level 450 mg/kg/day,in haemocoagulation parameters – significantly decreased value of fibrinogen at the dose level 150 mg/kg/day. and in white blood component – significantly increased value of total leucocyte count at the dose level 450 mg/kg/day, significantly decreased value of granulocytes in all treated groups (dose dependent) and significantly increased value of lymphocytes in all trated groups (dose depened). The values of haematological parameters of control and treated females were not out of historic control.Satellite females: in haemocoagulation parameter - significantly decreased value of fibrinogen and in white blood component – insignificantly increased value of total leucocyte count. Parameters of red blood component were not altered in satellite treated females.The values of haematological parameters of satellite control and satellite treated females were not out of historic control.CLINICAL CHEMISTRYMalesStatistically significantly increased value of protein total at the dose level 150 and 600 mg/kg/day was recorded (dose dependent). Significantly increased value of albumin at the dose level 600 mg/kg/day was recorded. Concentration of calcium ions and inorganic phosphorus in males at the dose level 600 mg/kg/day was increased with statistical significance. Dose dependent decreasing of concentration of chloride ions in all treated groups was detected at the dose levels 150 and 600 mg/kg/day statistically significant. Dose dependent decreasing of activity of ALP in all treated groups was detected (at the dose levels 150 and 600 mg/kg/day statistically significant). Decreased activity of ALT and AST at the dose level 600 mg/kg/day and decreased activity of ALT at the dose level 150 mg/kg/day was detected. Value of cholesterol total in all treated groups was increased (at the dose levels 50 and 150 mg/kg/day statistically significant). Significantly decreased value of urea in males at the dose level 150 mg/kg/day was detected. Significantly decreased value of bile acids and significantly dose-dependent increased value of cholinesterase was recorded in all treated groups. Insignificantly decreased value of creatinine in males at the dose level 150 mg/kg/day was recorded.Other parameters values were balanced in control and treated males.The value of protein total and albumin was above histocal control limits at the dose level 600 mg/kg/day. The value of cholinesterase was above historical control limits at the dose levels 150 and 600 mg/kg/day. The value of chloride ions was under historical control limits in all treated groups. Satellite malesSignificantly decreased value of bile acids and significantly increased value of cholinesterase was recorded in treated group. Statistically significantly increased activity of ALP and insignificantly decreased activity of AST in treated males was recorded. Values of other parameters were relatively balanced in satellite control and treated males.Values of all parameters were within the limits of historic control. FemalesSignificantly dose-dependent increased value of protein total and value of albumin in all treated groups was recorded (except value of protein total at the dose level 50 mg/kg/day where it was without statistical significance). Value of creatinine in all treated groups was significantly dose-dependently decreased. Significantly dose-dependent increased value of cholesterol total in all treated groups was recorded. Activity of AST in all treated groups was decreased with statistical significance. Significantly increased concentration of calcium ions (dose dependent) in all treated groups was recorded. Value of inorganic phosphorus in females at the dose levels 150 and 450 mg/kg/day was significantly dose dependently increased. Significantly increased value of triglycerides at the dose levels 150 and 450 mg/kg/day was detected. At the dose level 50 mg/kg/day increased concentration of sodium ions, potassium ions and chloride ions was recorded with statistical significance. The concentration of potassium ions was also significantly increased in females at the dose level 450 mg/kg/day. Insignificantly decreased concentrations of chloride ions in females at the dose levels 150 and 450 mg/kg/day was detected. Values of other parameters of biochemical examination were similar to the control group. The value of protein total was above histocal control limits at the dose level 450 mg/kg/day. The value of cholesterol total was above historical control limits at the dose levels 150 and 450 mg/kg/day. The value of chloride ions was under historical control limits at the dose levels 150 and 450 mg/kg/day. Satellite femalesStatistically significantly decreased concentration of chloride ions was detected in satellite treated females. Insignificantly decreased values of creatinine, albumin and protein total in satellite treated females were recorded. Values of other parameters of satellite treated females were similar to satellite control females.Values of all parameters were within the limits of historic control.URINALYSISMale/Satellit male: Statistically significant differences in pH and volume of urine were not detected. Presence of protein and of leucocytes in urine.Female: degrease of pH of urine at dose 150 and 450 mg/kg/day and leucocytes only in one female at dose 150 mg/kg/day.Satellite female: Presence of leucocytes was detected in one female from each satellite group.NEUROBEHAVIOURORGAN WEIGHTS1) ABSOLUTE ORGAN WEIGHTMales Absolute weight of liver was significantly increased in all treated groups with dose dependence. Increased absolute weight of kidneys in all treated groups was recorded (at the dose level 600 mg/kg/day statistically significant increase). Insignificantly increased absolute weight of spleen at the dose level 600 mg/kg/day was recorded. Increased absolute weight of pituitary gland at the dose levels 150 and 600 mg/kg/day was detected. Absolute weights of other organs of treated males were balanced with control males. Satellite malesStatistically significant increase of absolute weight of organs in treated satellite males was recorded in spleen, heart, kidneys and liver. Absolute weights of other organs of satellite treated and control males were similarFemales Absolute weight of spleen was significantly increased at the dose levels 150 and 450 mg/kg/day. Increased absolute weight of liver at the dose levels 150 and 450 mg/kg/day was recorded (at the dose level 450 mg/kg/day statistically significant increase). Absolute weight of kidneys was insignificantly increased at the dose level 450 mg/kg/day. Decreased absolute weight of uterus in females at the dose levels 50 and 450 mg/kg/day was recorded. This change of uterus weight probably related with oestrus cycle. Absolute weights of other organs of treated females were balanced with control females. Satellite femalesStatistically significant increase of absolute weight of ovaries was recorded in satellite treated females. Absolute weight of kidneys, spleen, liver and pituitary gland of satellite treated females were insignificantly increased compared to satellite control females. Absolute weights of other organs of satellite treated and control females were similar.2)RELATIVE ORGAN WEIGHTMales Relative weight of liver was significantly increased in all treated groups with dose dependence. Increased relative weight of kidneys in all treated groups was recorded (at the dose level 600 mg/kg/day statistically significant increase). Slightly increased relative weight of spleen was recorded at the dose level 600 mg/kg/day.Satellite malesStatistically significantly increased relative weight of kidneys and liver in satellite treated males was recorded. Relative weight of other organs of satellite treated and control males were similar.Females Relative weight of spleen was significantly increased in all treated groups with dose dependence. Statistically significant increase of relative weight of liver was detected in females at dose level 450 mg/kg/day. Relative weight of kidneys in females at the dose level 450 mg/kg/day was increased insignificantly. Relative weight of uterus in females at the dose levels 50 and 450 mg/kg/day was decreased against to control. Satellite femalesStatistically significant increase of relative weight of spleen, ovaries and kidneys was recorded in satellite treated females.Insignificant increase of relative weights of liver was recorded in satellite treated females. Relative weight of other organs of satellite treated and control females were similar.GROSS PATHOLOGYSee attached documentHISTOPATHOLOGY: NON-NEOPLASTICSee attached document
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
The value of NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established 50 mg/kg body weight/day for MALES and 150 mg/kg body weight/day for FEMALES. The value of NOAEL was determined on the base of following findigs: microscopical examination (early stage of chronic progressive nepropathy of kidneys in males), haematological examination (changes of value of total erythrocyte count and value of haemoglobin in females), biochemical examination (changes of values of chloride ions, calcium ions, inorganic phosphorus, protein total, albumin, bile acids, cholinesterase and activity of ALP in males, changes of value of triglycerides and aktivity of AST in females) and biometry of organs (changes of weight of kidneys, liver in males and weight of liver in females).
Executive summary:

Introduction

The test substance, Centralit ,was tested for subchronic toxicity using the Method B.26 Sub-Chronic Oral Toxicity Test: Repeated Dose 90-day Oral Toxicity Study in Rodents, Council Regulation (EC) No. 440/2008, Published in O.J. L142, 2008. This sub-chronic oral toxicity test method is a replicate of theOECD Test Guideline No. 408:Repeated Dose 90-day Oral Toxicity Studyin Rodents, Adopted 21stSeptember 1998.

 

Methods

Wistar rats of SPF quality were used for testing. The test substance was administered in 0.5% methylcellulose in aqua pro injectione by stomach tube; oral application of rats was made daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 10 males and 10 females; each satellite group consisted of 6 males and 6 females.Main groups contained 3 treated groups of males (doses 50, 150, 600 mg/kg of body weight /day), 3 treated groups of females (doses 50, 150, 450 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only), one treated group of males (600 mg/kg/day) and one treated group of females (450 mg/kg/day).

The administration period lasted 90 days. After that animals of main groups were sacrificed and satellite animals were observed for the next 28 days without treatment.

   

Dose levels for the main study for males - 50, 150, 600 mg/kg/day and for females 50, 150, 450 mg/kg/day were chosen on the basis of results ofstudyCentralit -Repeated Dose (28 days) Toxicity (Oral), Report No. 12-131, VUOS 2011-2012. The reason for choice of different high dose levels for males and females was that the females were found more sensitive to the test substance treatment.

 

During the 90-day study clinical observation and health status control were performed daily. The body weight, food consumption were measured weekly and the detailed clinical observation was carried out in the same time interval. Water consumption was measured twice and sequentially three times a week. Ophthalmologic examination was done at the beginning and at the end of the study. Before the end of study the functional observation was accomplished. The study was finished by urinalysis, haematological and biochemical analysis, and gross necropsy of animals. The selected organs for weighing and histopathological examination were removed.

 

 

Conclusion

During the study the higher sensitivity to the test substance treatment in females was found out (two females at the highest dose level and one female at the highest satellite dose level died after the first application).In these females distorsion of tail was observed during the pathological examination. Other pathological findings were not found out.

The effect of the test substance on theheath condition in femaleswas observed during the 1stweek of study. Negative effect on neurology system of animals manifested as Straub phenomenon accompanied by stupor and tachyphoe.

In males no death related to the test substance was recorded. No effect of the test substance on theheath condition and no effect on neural system in maleswas observed.

 

Microscopical exanimations of organs in males showedirreversible, serious and toxicologically significantnegative effects on kidneys (early stage of chronic progressive nephropathy) at the highest dose level. Chronic progressive nephropathy was found out in almost all high dose males and also in one control males and in three satellite control males (but only sporadic incidence in control). This finding at kidneys was accompanied by irreversibly increased weight of kidneys and changes of biochemical parameters (unbalance of inorganic ions - mainly decreased value of chloride ions and increased value of sodium ions and value of inorganic phosphorus). The value of chloride ions was under historical control limits. The biochemical examination also showed increased levels of protein and albumin. The value of protein total and albumin was above historical control limits at the highest dose level and value of chloride ions was under historical control limits in all treated groups.

 

Microscopical exanimations of organs in males showed basophile pigment in cytoplasm of hepatocytes in liver at the middle and highest dose levels. The changes in liver were probably adaptive response of organism to application of the test substance and after finishing of application these changes either completely disappear or were observed only sporadically without toxicological significance. This findings in liver (basophile pigment) was accompanied by reversible increased weight of liver and changes in some biochemical parameters related to liver function: decreased activity of ALP, value of bile acids, increased value of cholinesterase. The value of cholinesterase was above historical control limits at the highest dose level.

Similarly in females only reversible microscopical change in liver related to adaptive response organism to the test substance application was recorded: basophile pigment in cytoplasm of hepatocytes in liver at the middle and highest dose levels. This finding in liver was accompanied by others changes: biometry of organs - significantly increased weight ofliver, haematological parameters – decreased value of total erythrocyte count and value of haemoglobin, biochemical parameters – decreased activity of AST and increased value of triglycerides. After finishing of the test substance application these findingseither completely disappear or were observed only sporadically without toxicological significance.

 

In both sexes occurred also other changes recorded during pathological, haematological, biochemical examinations and during biometry of organs. Occurrence of these findings were rather sporadic and without toxicological significance.

 

After consideration of all findings the NOAEL was determined as follows:

 

The value of NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established 50 mg/kgbody weight/day for MALES and 150 mg/kgbody weight/day for FEMALES.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study is a GLP compliant and has Klimish score 1.

Additional information

The value of NOAEL was determined on the base of following findigs in 90 -days repeated dose toxicity study: microscopical examination (early stage of chronic progressive nepropathy of kidneys in males), haematological examination (changes of value of total erythrocyte count and value of haemoglobin in females), biochemical examination (changes of values of chloride ions, calcium ions, inorganic phosphorus, protein total, albumin, bile acids, cholinesterase and activity of ALP in males, changes of value of triglycerides and aktivity of AST in females) and biometry of organs (changes of weight of kidneys, liver in males and weight of liver in females).


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
90-day repeated dose toxicity study caried out base on testing proposal is available in this endpoint.

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: kidneys

Justification for classification or non-classification