3-methyl-1,1-diphenylurea

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.01.-02.09.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

The decrease of relative humidity (to 22.18 %) in SPF animal room was observed during study. The deviation from the limits was short-term and did not influence the wellness of animals and the study results.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1,1-diphenylurea

EC Number:

236-039-7

EC Name:

3-methyl-1,1-diphenylurea

Cas Number:

13114-72-2

Molecular formula:

C14H14N2O

IUPAC Name:

3-methyl-1,1-diphenylurea

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Name of test material: 3-Methyl-1,1-diphenylurea- Physical state: white solid powder- Composition of test material, percentage of components: main component: 3-Methyl-1,1-diphenylurea CAS:13114-72-2 >98% (w/w) impurities: unlisted additives: unlisted-Molecular formula: C14H14N2O-Molecular weight: 226,27-Batch No.: 14015- Expiration date of the lot/batch: 11/2019- Stability under test conditions: stable- Storage condition of test material: in closed container, in dry room at room temperature (at laboratory conditions)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: SPF breeding, VELAZ s.r.o., Únětice, Czech Republic, RČH CZ 21760118- Age at study initiation: sexually adult (9 weeks on arrival)- 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). - Animal per cage: 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage- Diet: ad libitum, Complete pelleted diet for rats in SPF breeding - ST 1, manufacturer: VELAS a.s., Hrabanov 535, 289 22 Lysá nad Labem CZ 801080-01, Diet was sterilised before using.- 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: Crude protein – min. 21%, Drip – max. 14%, Fat – min. 3%, Fiber – max. 4.1%, Ash – max. 7%, Calcium – min. 1%, Phosphorus – min. 0.8%, Magnesium – min. 0.2%, Sodium – max. 0.25%.- Water: ad libitum- Acclimation period: 5 days, During the acclimatisation period the health condition of all animals was controlled daily.Then the animals were randomly divided into the control and test groups and they were marked individually. ENVIRONMENTAL CONDITIONS- Temperature (°C): 22±3°C- Humidity (%): 30-70%- Air changes (per hr): 15 air - Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle STUDY TIME SCHEDULETest substance delivery: 16. 12. 2014 Determination of stability and homogeneity:19.01. – 29. 01. 2015Dose-range finding experiment: 11. 02. – 10. 03. 2015Main Study:Animal arrival:11. 03. 2015Acclimatisation: 5 daysAdministration started: 17. 03. 2015 Administration and observation: Parental males:1st day – 14th day (pre-mating) → 28th day (mating) → 42nd day of study Satellite males:1st day → 42nd day (administration) → 56th day (recovery period)Parental females:1st day – 14th day (pre-mating) → 28th day (mating) → gestation → lactation → day 3 post partum Satellite females:1st day → 42nd day (administration) → 56th day (recovery period)Non-pregnant females (without evidence of copulation):1st day – 14th day (pre-mating) → 28th day (mating) → 54th day of study Non-pregnant females (with evidence of copulation):1st day – 14th day (pre-mating) → 28th day (mating) → 25th day after confirmed mating (max. 54th day of study) Urinalysis: only males – 42nd and 56th day of studyHaematology and necropsies: parental males – 43th day of study satellite males – 57th day of study parental females – 4th day of lactation satellite females – 57th day of studynon-pregnant females – 55th day of study or 26th day after confirmed mating Examination of blood and necropsies:26. 04. – 02. 05. 201512. – 13. 05. 2015 (satellite groups) End of histopathological examination:02. 09. 2015Final report elaboration:to 16.10.2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:The application form was prepared by mixing with olive oil. Two concentrations of application form were prepared (10 mg/10 ml and 1000 mg/10 ml). Concentration 10 mg/10 mlThe 50 mg of the test substance was mixed and dissolved in 50 mL of the vehicle in ultrasonic bath for 15 minutes. Then the application form (yellow solution without visible solid particles) was shaked for 45 minutes (400 Mot).Concentration 1000 mg/10 mlThe 5 g of the test substance was mixed and dissolved in 50 mL of the vehicle in ultrasonic bath for 15 minutes. Then the application form (milky suspension) was shaked for 45 minutes (400 Mot).VEHICLEOlive oilBatch No.: 5523502 Producer: Dr. Kulich Pharma, Hradec Králové, Czech RepublicThe test solution/suspension was dissolved in ultrasonic bath for a 15 minutes and then the suspension was stirred by magnetic stirrer (400 rpm) for 45 minutes. The concentrations of solution/suspension at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The vehicle control group was administered by olive oil in the same volume. The application form (test substance solution/suspension in olive oil) was prepared daily just before administration.

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 (Analytical group I). 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:

21

Frequency of treatment:

7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

140 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

280 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

560 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Basic groupsvehicle 12F+12M140 mg/kg/bw 12F+12M280 mg/kg/bw 12F+12M560 mg/kg/bw 12F+12MSatellite groupsvehicle 6F+6M560 mg/kg/bw 6F+6M

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-range finding experiment with 21-day application period was performed with 4 groups of treated animals without control group. On the basis of the results given above the following dose levels – 140, 280 and 560 mg/kg/day were chosen for the main Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. - Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

MORTALITY CONTROL- Time schedule: dailyHEALTH CONDITION CONTROL- Time schedule: daily - during the acclimatization and the experimental part CAGE SIDE OBSERVATIONS: Yes- Time schedule: males and females - daily during the administration periodThis 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 (12.00 – 14.00 p.m.) – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cagesDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: males and females before the first application and then weekly - At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, position of eyelids, breathing, tonic or clonic movements, stereotypes or bizarre behaviour.- The second part was the observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina. BODY WEIGHT: Yes- Time schedule for examinations: males - weekly females - weekly in premating and mating period during pregnancy: 0., 7th, 14th, 20th day, during lactation: 1st, 3rd and 4th day; satellite males and females - weeklyFOOD CONSUMPTION- Time schedule: parental males - weekly (except the mating period) parental females - weekly during premating period during pregnancy and lactation – on the same days as body weight satellite males and females – weeklyIn a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed. In males mean values were calculated for each week of the study (except of mating period in parental males). Food consumption for animal/day was calculated from mean values of each group. The same way of calculation of mean food consumption was used for females in premating period and for satellite females. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption of parental females for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption of pregnant females.FOOD 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: Yes- Time schedule for examinations: : satellite males and females – twice or three times a week (the 1st and 2nd week – twice a week and from 3rd week to the end of study a week)- The mean values in groups (water consumption per animal and per day) were calculated for each week of the study. OPHTHALMOSCOPIC EXAMINATION: NoHAEMATOLOGY: Yes- Time schedule for collection of blood: at the end of administration/recovery period- Anaesthetic used for blood collection: Yes, light ether narcosis- Animals fasted: Yes- How many animals: 6 males and 6 females of each group and in satellite males and females. - Parameters checked in table [No.3] were examined.BIOCHEMICAL EXAMINATION: Yes- Time schedule for collection of blood: at the end of administration/recovery period- Anaesthetic used for blood collection: Yes, light ether narcosis- Animals fasted: Yes- How many animals: 6 males and 6 females of each group and in satellite males and females. The animals starved approximately for 18 hours before blood collection but they were supplied by drinking water ad libitum.- Parameters checked in table [No.4] were examined.URINALYSIS: Yes- Time schedule for collection of urine: last day of administration/recovery period – only males- Metabolism cages used for collection of urine: Yes, for 2 hours- Immediately before entering metabolic cages the animals were administered 2 mL of drinking water for 100 g of body weight by gavage to the stomach.- Animals fasted: No- How many animals: 6 males of each group and in satellite males- Parameters checked in table [No.2] were examined.

Sacrifice and pathology:

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.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, epididymis/epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary gland and heart were recorded (repeated dose toxicity part of study – 6 males and females from each group + satellite groupsHISTOPATHOLOGY: Yes (see table No. 5)

Statistics:

For statistical evaluation the software Statgraphic ® Centurion (version XV, USA) was used. Males/females from control group were compared with males/females from three treated groups. Satellite males/females from control group were compared with satellite males/females from treated group.The parametric tests were used for statistical evaluation of•body weight •mean pups weight •litter weight •selected haematology parameters •blood biochemistry parameters•data from urinalysis •data from biometry of organs

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males In control males and treated males of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period. In treated males at the dose level 140 mg/kg/day no clinical changes of health condition before, during and after application of the test substance were observed. At the dose level 280 mg/kg/day immediately after application the following symptoms were observed: poking the nose to the bedding and disquiet/irritation. No clinical changes in males at the dose level 280 mg/kg/day were detected during afternoon clinical observation.At the dose level 560 mg/kg/day immediately after application the following symptoms were observed: poking the nose to the bedding, disquiet/irritation, salivation, piloerection. Bizarre behaviour - automutilation (injured tail or ears, sore in skin on the neck) and symptoms of stress (mainly - focal loss of fur, salivation and disquiet/irritation) at the dose level 560 mg/kg/day was observed irregularly during the study. Satellite malesIn satellite control males and satellite treated males of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period. In satellite treated males the following changes were observed immediately after application: poking the nose to the bedding, salivation and piloerection. During clinical observation the bizarre behaviour - automutilation (injured tail) and symptoms of stress (salivation, disquiet/irritation) were observed haphazardly. Swelling of mandibular in one treated male was observed during the study – this finding did not relate with the test substance treatment. Females In control females and treated females of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period. In treated females at the dose level 140 mg/kg/day no changes of health condition before, during and after application of the test substance were observed. At the dose level 280 mg/kg/day immediately after application the following symptoms were observed: poking the nose to the bedding, salivation, hunched posture and disquiet/irritation.Clinical findings (piloerection and hunched posture) in females at the dose level 280 mg/kg/day were detected during afternoon clinical observationAt the dose level 560 mg/kg/day immediately after application the following symptoms were observed: poking the nose to the bedding, hunched posture, salivation, piloerection. Bizarre behaviour - automutilation (injured tail, sore in skin on the neck), symptoms of stress (mainly - focal loss of fur, salivation, disquiet/irritation) and hunched posture at the dose level 560 mg/kg/day was observed irregularly during the study. Satellite femalesIn satellite control females and satellite treated females of all dose levels no signs of diseases were recorded during the check-in and acclimatisation period. In satellite treated females the following changes were observed immediately after application: poking the nose to the bedding, piloerection, secretion around nostrils and disquiet/irritated. Males The activity (poise, gait, reaction to handling) of all males of all treated groups was similar during the study and not different from the activity of males of the control groups.No significant changes were found at all dose levels during the examinations of skin, eyes, lacrimation, visible mucous membrane and secretion and respiration. Focal loss of fur and salivation in males at the dose level 560 mg/kg/day was observed.Satellite males The activity (poise, gait, reaction to handling) of all males of all treated groups was similar during the study and it was not different from the activity of males of the control groups.No significant changes were found at all dose levels during the examinations of skin, eyes, lacrimation, visible mucous membrane and secretion and respiration. Piloerection, salivation and injured tail in the treated animals was observed. Swelling of mandibular in one treated male was observed in the 5th and 6th week of application period – this finding did not relate with the test substance treatment. Females The activity (poise, gait, reaction to handling) of all females of all treated groups was similar during the study and not different from the activity of females of the control groups.No significant changes were found at all dose levels during the examinations of skin, eyes, lacrimation, visible mucous membrane and secretion and respiration. Focal loss of fur, piloerection, salivation, injured tail and hunched posture in females at the dose level 560 mg/kg/day was observed.Satellite females The activity (poise, gait, reaction to handling) of all females of all treated groups was similar during the study and it was not different from the activity of females of the control groups.No significant changes were found at all dose levels during the examinations of skin, eyes, lacrimation, visible mucous membrane and secretion and respiration. Piloerection, salivation, injured tail, sore in skin on the neck and hunched posture in the treated animals was observed.

Mortality:

mortality observed, treatment-related

Description (incidence):

MalesOne male at the dose level 140 mg/kg/day died during mating period (in the 3rd week of study) – intubation error. This decease did not relate to the test substance administration. FemalesOne female (No. 152) at the dose level 280 mg/kg died during study (in the 6th week of study - at the twenty-second day of pregnancy) – delivery. This death did not relate to the test substance administration. Two females (No. 171 and 162) at the dose level 560 mg/kg died during study (in the 5th and 7th week of study).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MalesThe statistical analysis was performed for body weight. The statistically significant differences were not found. The different mean body weight of groups before application was caused by gradual start-up of application. Loss of body weight in males at the dose level 560 mg/kg/day was detected in the 1st week of application period. Insignificantly decreased body weight was recorded at the dose level 560 mg/kg/day during the whole application period. The body weight increment was decreased at the dose level 280 mg/kg/day in the 1st week of application period. Satellite malesStatistically significantly decreased body weight was recorded during the whole study. Loss of body weight of treated males was recorded in the 1st and 5th week of application periodFemalesThe evaluation of weight increments during pregnancy and lactation period is included in reproduction part of study.Before mating period – the body weight of all treated groups and control group was similar. Body weight increment of treated groups and control group was similar before mating period.Satellite femalesDecreased body weight was recorded in satellite treated females during the whole study. From the 2nd week of application to end of study the difference was with statistical significance. Loss of body weight in treated females was detected in the 1st, 3rd, 4th and 5th week of application period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

MalesThe food consumption of treated males and control males was relatively well-balanced during the application period (except the 1st week of application period at the dose level 560 mg/kg/day). Satellite males Decreased mean food consumption at the treated group was recorded in the 1st, 4th, 5th, 6th, 7th and 8th week of study. FemalesThe mean food consumption before mating period was decreased at the dose level 560 mg/kg/day. During pregnancy period decreased mean food consumption was recorded at the dose levels 280 and 560 mg/kg/day. On the 3rd day of lactation period decreased food consumption was detected in all treated groups (at the dose level 560 mg/kg/day the difference was marked). Satellite femalesDecreased mean food consumption at the treated group was recorded during the whole application period.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

MalesDis-balance of mean food conversion between control group and the middle and the highest treated groups was recorded during the whole study. Satellite males Dis-balance of mean food conversion between treated group and control group was recorded during the whole study. FemalesDecreased food conversion in all treated groups was recorded before mating period (at the dose level 560 mg/kg/day the difference was marked). During pregnancy and lactation period food conversion was decreased at the dose level 560 mg/kg/day. Satellite females Decreased food conversion in treated group was recorded during the whole application period and on the contrary during recovery period it was increased

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Satellite males The mean water consumption at the treated group and control group was similar during the whole application period. During the recovery period the mean water consumption of treated group was decreased.Satellite females Increased mean water consumption at the treated group was recorded during the application period. During recovery period it was relatively well-balanced compared with control group.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males The mean corpuscular volume was increased at the dose levels 280 and 560 mg/kg/day (at the dose level 560 mg/kg/day statistically significant). Value of total erythrocyte count at the dose levels 280 and 560 mg/kg/day was only insignificantly decreased. Significantly decreased value of prothrombin time and significantly dose-dependent increased value of active partial thromboplastin time (APTT) was recorded in all treated groups. Other measured parameters were similar to the control group.Satellite males In treated group the value of total leucocyte count and percentual portion of granulocytes was increased (value of granulocytes with statistical significance). Significantly decreased percentual portion of lymphocytes was found at the treated group. Other measured parameters were similar to the control group.Females The value of prothrombin time at the dose level 560 mg/kg/day was decreased with statistical significance. The value of platelet count was decreased at the dose level 560 mg/kg/day significantly and at the dose level 280 mg/kg/day it was decreased insignificantly. Increased value of total leucocyte count was recorded in all treated groups, but at the dose level 280 mg/kg/day the difference was significant. Percentual portion of granulocytes at the dose levels 280 and 560 mg/kg/day was decreased. Percentual portion of lymphocytes was insignificantly increased at the dose level 560 mg/kg/day. Other measured parameters were similar to the control group. Satellite females Statistically significantly increased value of mean corpuscular volume and concentration of haemoglobin were recorded in treated females. Statistically significantly increased percentual portion of monocytes in treated females was detected. Insignificantly increased percentual portion of granulocytes and decreased percentual portion of lymphocytes in treated females were detected. Other measured parameters were similar to the control group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

MalesStatistically significantly increased value of cholesterol total (dose dependent) was recorded at the dose levels 280 and 560 mg/kg/day. Activity of ALT and value of bilirubin total (dose dependent) was increased at the dose levels 280 and 560 mg/kg/day (at the dose level 560 mg/kg/day statistically significant). Value of creatinine (dose dependent) was increased in all treated groups (at the dose level 560 mg/kg/day statistically significant). Decreased value of glucose in all treated groups was detected with statistical significance. Increased value of cholinesterase in all treated groups was detected with statistical significance. Statistically significantly increased value of bile acids (dose dependent) was recorded at the dose levels 280 and 560 mg/kg/day. Increased concentration of chloride ions at the dose levels 280 and 560 mg/kg/day was detected with statistical significance.All other measured parameters were similar to the control group. Satellite males The value of cholesterol total, concentrations of calcium ions, inorganic phosphorus and potassium ions were increased in treated males with statistical significance. Increased activity of ALP in treated males was detected with statistical significance. Significantly decreased value of cholinesterase in treated males was found out. All other measured parameters were similar to the control group. FemalesStatistically significantly increased value of cholesterol total (dose dependent) at the dose levels 280 and 560 mg/kg/day was recorded. Activity of AST at the dose level 560 mg/kg/day was decreased with statistical significance. Increased value of cholinesterase (dose dependent) at the dose levels 280 and 560 mg/kg/day was detected with statistical significance. Decreased value of triglycerides was detected in all treated groups (statistically significant). Statistically significantly increased value of bile acids at the dose level 560 mg/kg/day was recorded. Statistically insignificantly increased value of bilirubin total at the dose level 560 mg/kg/day was recorded. Value of sodium ions was increased in all treated groups (at the dose levels 140 and 560 mg/kg/day statistically significant). Significantly increased value of chloride ions at the dose level 560 mg/kg/day was detected.All other measured parameters were similar to the control group. Satellite females The value of cholesterol total, concentrations of calcium ions and inorganic phosphorus were increased in treated females with statistical significance. Significantly increased values of urea, bile acids and significantly decreased activity of AST and concentration of chloride ions in treated females were found out. All other measured parameters were similar to the control group.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

The statistical analysis was performed for urine volume and pH of urine. Statistically significantly increased pH of urine was recorded at the dose level 280 mg/kg/day. Change of colour of urine (yellow brown or brown) was recorded at the dose levels 140, 280 and 560 mg/kg/day. Higher specific weight of urine was detected at the dose levels 280 and 560 mg/kg/day. Presence of bilirubin was recorded in one male at the dose level 280 mg/kg/day and in five males at the dose level 560 mg/kg/day. Presence of protein was recorded in one male in control group and one male at the dose level 280 mg/kg/day. Presence of ketones was recorded in one male at the dose level 280 mg/kg/day and in two males at the dose level 560 mg/kg/day and presence of leucocytes was recorded in all groups (one male in the control group, two males at the dose levels 140 and 280 mg/kg/day and three males at the dose level 560 mg/kg/day). Satellite males Statistically significantly increased pH of urine was recorded in the treated group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

MalesReactions to contact, to noise, to pain and pupillary reflex of treated males and satellite treated group were the same as in the control group. The value of upstanding was decreased at the dose level 560 mg/kg/day and in satellite treated males. Results of emiction and defecation in treated males were not the same as in control males but the variation was within the range of the physiological reaction of animals. The values of grip strength of pectoral legs and pelvic legs in males did not show any difference between control and the treated dose levels. FemalesReactions to contact, to noise, to pain and pupillary reflex of treated females and satellite treated group were the same as in the control group. The value of upstanding was decreased at the dose levels 280 and 560 mg/kg/day. Results of emiction and defecation in treated females were not the same as in control females but the variation was within the range of the physiological reaction of animals. The values of grip strength of pectoral legs and pelvic legs in females did not show any difference between control and the treated dose levels.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute organ weightMales Dose-dependent and statistically significantly increased absolute weight of liver was recorded in all treated groups. Increased absolute weight of prostate gland was detected at the dose level 560 mg/kg/day (with statistical significance). Statistically significantly decreased absolute weight of brain was recorded at the dose levels 280 and 560 mg/kg/day (with statistical significance). Insignificantly decreased absolute weight of spleen was detected at the dose levels 280 and 560 mg/kg/day.The absolute weight of other organs was well-balanced with the control group. Satellite males Decreased absolute weight of brain and heart of treated males was recorded (with statistical significance). The absolute weight of other organs was well-balanced with the control group.Females Decreased absolute weight of brain and heart was recorded at the dose level 560 mg/kg/day (with statistical significance). Absolute weight of uterus and pituitary gland at the dose level 560 mg/kg/day was significantly decreased. This decreased absolute weight of uterus and pituitary gland was caused by heterogeneity of the highest group – 3 mothers, one female with stillborn pups and two non-pregnant females. The absolute weight of other organs was relatively well-balanced with the control group.Satellite females Absolute weight of thymus was insignificantly decreased in treated females. The absolute weight of other organs was relatively well-balanced with the control group.Relative organ weightMales Dose-dependent increased relative weight of liver was recorded in all treated groups (with statistical significance). Increased relative weight of heart was detected in all treated groups (at the dose levels 140 and 560 mg/kg/day statistically significant). Increased relative weight of prostate gland was detected in all treated groups (at the dose level 560 mg/kg/day statistically significant). Relative weight of adrenal glands was significantly increased at the dose level 560 mg/kg/day. The relative weight of other organs was well-balanced with the control group. Satellite males The statistical analysis of the data revealed significantly increased weight of testes in the treated group of males. The relative weight of other organs was well-balanced with the control group. Females Dose-dependent increased relative weight of liver was recorded in all treated groups (with statistical significance). Significant increase of relative weight of ovaries at the dose level 280 mg/kg/day was recorded. The relative weight of other organs was well-balanced with the control group.Satellite femalesStatistically significantly increased relative weight of brain, kidneys, liver and ovaries was recorded in treated group. Insignificantly increased relative weight of pituitary gland in treated females was detected.The relative weight of other organs was well-balanced with the control group.

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

During microscopical examination vacuolation of cytoplasm of hepatocytes (different intensity) of liver was detected in males and females. Vacuolation of liver cells in males was also detected during focal fatty examination. These changes in liver were observed in all treated groups including satellite treated group but in satellite treated animals this changes of liver were found out only sporadically. Occurrence of these changes in liver probably related with adaptive reaction of organism to the test substance treatment. In two satellite treated males acute subcapsular necrosis of liver during microscopical examination was detected. The focal subcapsular necrosis of liver is already toxicologically significant serious finding of liver and could probably relate to test substance treatment. The test substance had negative effect on the liver and the test substance treatment could probably cause irreversible damage of liver, but this is controversial. Further changes related with the metabolism of the test substance were observed during the biochemical examination. Dose dependent increased value of cholesterol total and/or bilirubin total and/or creatinine and/or cholinesterase and/or bile acids and decreased value of glucose in both sexes was recorded. Increased activity of ALT and value of chloride ions in males and decreased activity of AST and value of triglycerides was also recorded. The biochemical changes in metabolism and the management of ions were observed in animals at the end of recovery period. In males, increased value of cholesterol total, increased concentration of calcium ions, inorganic phosphorus and potassium ions, increased activity of ALP and decreased value of cholinesterase was recorded. In females, increased value of cholesterol total, urea, bile acids, increased concentration of calcium ions, inorganic phosphorus and decreased activity of AST and concentration of chloride ions was recorded. Presence of bilirubin accompanied by a change of color of urine was recorded in males during examination of urine. Hemosiderosis of spleen was found out during microscopical examination in both sexes, but during biometry of organs no significant changes of spleen were detected. And no significant hematology changes of red blood components were recorded. This microscopical change of spleen related to adaptation of the organism to the test substance treatment, but increased incidence (compared with the control group) of this microscopical finding was also recorded at the end of recovery period. The reversible microscopic changes of the stomach and forestomach mucosa (inflammation, erosion and oedema) which was detected during microscopic examination related to oral application of the test substance (site of application).

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

140 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

140 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

other: urine parametrs

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

140 mg/kg bw/day (actual dose received)

System:

haematopoietic

Organ:

blood

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

3

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

140 mg/kg bw/day (actual dose received)

System:

other: biometry of organs

Organ:

liver

Treatment related:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

The value of NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY was established as 140 mg/kg body weight/day both for MALES and FEMALES.

Executive summary:

Introduction

The test substance,Akardit,was tested for reproduction and subacute toxicity using the OECD Test Guideline No. 422:Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on March 22^nd1996.

 

Methods

Wistar rats of SPF quality were used for testing. The test substance was administered in the form of solution/suspension in olive oil. Oral application by stomach tube was performed daily. The study includes four main groups and two satellite groups of animals. Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 140, 280, 560 mg/kg of body weight /day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (560 mg/kg/day).The dose levels for study were determined on the basis of results of adose-range finding experiment study phase.

 

The treated groups were administered daily for the following periods:

males and females – 2 weeks prior to the mating period and during the mating period,

pregnant females – during pregnancy and till the 3^rdday of lactation,

males – after mating period – totally for 42 days,

nonpregnant females (mated females without parturition) – for 25 days after the confirmed mating, non-mated females – to the 54^thday of study.

After the end of administration period the animals of main groups were sacrificed and satellite animals were observed for the next 14 days without treatment.

   

During the study clinical observation and health status control were performed daily. The body weight and food consumption were measured weekly or in the specified time intervals. Detailed clinical observation was carried out weekly. Functional observation was performed at the end of application and recovery period. Vaginal smears were prepared daily during the mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, weight of litters, sex or vitality) were also recorded.

The study was finished by urinalysis, haematological and biochemical analysis and gross necropsy of animals. In all males of main groups the sperm parameters, sperm motility and sperm morphology were examined. The selected organs from parental animals were removed for weighing and histopathological examination.

 

 

Summary of Results

The Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening with the test substance,Akardit, was performed.

 

Repeated Dose Toxicity Part of Study:

At the dose level 140 mg/kg/day one male died during the study. This death did no relate to the test substance treatment. Post mortem examination of male revealed a perforated oesophagus by the reason of intubation error.

 

The test substance at the dose level 140 mg/kg/day did not influence body weight of animals and health conditions of animals. Changes of haematological blood parameters in males (significantly increased value of APTT and significantly decreased value of prothrombin time), biochemical blood parameters (significantly decreased value of glucose and significantly increased value of cholinesterase in males and significantly decreased value of triglycerides and significantly increased concentration of sodium ions in females), biometry of organs (significantly increased weight of liver in both sexes and significantly increased weight of heart in males) were detected at the dose level 140 mg/kg/day. Occurrence of these changes probably related with adaptive reaction of organism to the test substance treatment.

 

At the dose level 280 mg/kg/day one female died during the study. Death of females related with complications during delivery (at thetwenty-seconddayof pregnancy). 

The test substance treatment had influence on growth of animals (decreased body weight increment, imbalance of food conversion in males and decreased body weight, food consumption and food conversion during the pregnancy and lactation period in females). Clinical observation showed slight influence on health condition (poking the nose to the bending and disquiet/irritation in males and poking the nose to the bedding, salivation, hunched posture, piloerection and disquiet/irritation in females). The haematological blood parameters (significantly increased value of APTT and significantly decreased value of prothrombin time in males and significantly increased value of WBC in females), biochemical blood parameters (significantly decreased value of glucose and significantly increased value of cholinesterase, value of cholesterol total, value of bile acids, concentration of chloride ions, insignificantly increased value of bilirubin total in males and significantly decreased value of triglycerides and significantly increased value of cholesterol total and value of cholinesterase, insignificantly increased value of bile acids in females), properties of urine in males (significantly increased pH of urine and change of colour) were changed. The changes of absolute and/or relative weights of organs (significantly increased weight of liver and significantly decreased weight of brain in males and significantly increased weight of liver and ovaries in females) were recorded.

The occurrences of microscopical changes of liver, stomach and/or forestomach (increased occurrence of vacuolation of cytoplasm of hepatocytes in liver and inflammation or oedema in stomach) in males and/or females were detected.

 

At the dose level 560 mg/kg/day one female died on the 35^thday of study and one female died on the 47^thday of study.

Growth of animals was influenced by the test substance treatment (loss of body weight, imbalance of food conversion and food consumption in both sexes). Clinical status of animals after application was also influenced by the test substance treatment (poking the nose to the bending, salivation, hunched posture, piloerection, bizarre behaviour – automutilation and symptoms of stress).

The haematological examination (significantly increased value of APTT and value of MCV and significantly decreased value of prothrombin time, delayed significantly changed percentual portion of granulocytes and lymphocytes in males and significantly decreased value of platelet count and value of protrombin time, delayed significantly increased value of MCV, value of haemoglobin and percentual portion of monocytes in females), blood biochemical examination (irreversible significant increase value of cholesterol total, significantly increased activity of ALT, value of creatinine, value of bilirubin total, value of cholinesterase, value of bile acids and value of chloride ions, significantly decreased value of glucose, delayed significantly increased activity of ALP, value of calcium ions, value of inorganic phosphorus and value of potassium ions, delayed significantly decreased value of cholinesterase in males and irreversible significant increase value cholesterol total, value of bile acids, irreversible significant decrease activity of AST, significantly increased value of cholinesterase, value of sodium and chloride ions, significantly decreased value of triglycerides, delayed significantly increased value of urea, value of calcium ions and inorganic phosphorus, significantly decreased value of chloride ions biologically significant increased value of bilirubin total in females), urinalysis in males (change of colour of urine, presence of bilirubin and ketones, delayed significantly decreased pH of urine) were changed.

Duringbiometry of organs the following changes of absolute and/or relative weight of organs were recorded: significantly changes of absolute weight of organs – increased weight of liver, irreversible decreased weight of brain, increased weight of prostate gland, delayed decreased weight of heart in males and decreased weight of brain, heart, uterus and pituitary gland in females and significantly changes of relative weight of organs – increased weight of liver, adrenal glands, heart, prostate gland, delayed increased weight of testes in males and irreversible increased weight of liver, delayed increased weight of brain, kidneys and ovaries in females. The histological examination of organs and tissues (vacuolation of cytoplasm of hepatocytes (different intensity) and acute necrosis of liver, hemosiderin of spleen, inflammation, erosion and oedema in stomach and/or forestomach at the dose level 560mg/kg/day revealed changes attributable to the test substance administration.