6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-15 to 2015-10-20

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

21st September 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

EC Number:

403-830-5

EC Name:

6'-(dibutylamino)-3'-methyl-2'-(phenylamino)spiro[isobenzofuran-1(3H),9-(9H)-xanthen]-3-one

Cas Number:

89331-94-2

Molecular formula:

C35H36N2O3

IUPAC Name:

6'-(dibutylamino)-3'-methyl-2'-(phenylamino)-3H-spiro[2-benzofuran-1,9'-xanthen]-3-one

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The strain has been chosen, because CRO has long experience of testing with this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, VELAZ s.r.o.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 wks
- Weight at study initiation: males: 216-217 g; females: 173-174 g
- Housing: 2/sex in one cage
- Diet: ad libitum, pelleted diet (Atromin 1324, Altromin Spezialfutter GmbH & Co. KG; Germany)
- Water: ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: Diet was sterilised before use. Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Ministry of Health

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Concentration level 50 mg/10 mL
Approximately 500mg of the test substance was weighed into a 150 mL glass beaker. One hundred mL of the vehicle (methylcellulose, MC) was added to the beaker. The suspension was stirred using a glass stirrer and dissolved. The suspension was placed on a magnetic stirrer and stirred for approximatley 60 min at ca. 1400 rpm. Approximately 1 mL of the suspension was pippeted into a 50 mL volumetric flask (suspension was weighed) dissolved and filled to volume by mobile phase. This solution was analysed.

Concentration level 1000 mg/10 mL
Approximately 1000 mg of the test substance was weighed into a 150 mL glass beaker. One hundred mL of vehicle (MC) was added to the beaker. The suspension was stirred using a glass stirrer and dissolved. The suspension was placed on a magnetic stirrer and stirred for approximatley 60 min at ca. 1400 rpm. One mL of suspension was pippeted into a 100 mL volumetric flask (suspension was weighed) dissolved and filled to volume by mobile phase (SS-A). Two mL of SS-A was pippeted into a 25 mL volumetric flask, dissolved and replenished to volume by mobile phase. This solution was analysed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance stability and homogeneity were determined by means of measuring of peak area of test substance by high-performance liquid chromatography (HPLC) based on a method developed for the purpose of this study at the test facility.

Stability of the test item formulation
For the determination of stability, 1 mL of suspension was pipetted into a 100 mL volumetric flask from each sampling, diluted to the appropriate concentration by mobile phase and analysed by HPLC. The samples were taken from the middle of the beaker content at required time intervals (0, 30, 60, 90 and 120 minutes) for the determination of stability. The time interval 0 min represents, for both concentrations, the time after 60 minutes of mixing by the magnetic stirrer (ca 1400 rpm).

The homogeneity of the application form
The determination of homogeneity was performed in the same way as the stability. The samples were taken after 3 minutes of mixing by the magnetic stirrer (ca 1400 rpm) for both concentration levels from 3 given places - the bottom, the middle and the surface of the beaker content.
From the results of the analyses (homogeneity and stability), the suspension of the test substance in 1% water solution of methylcellulose at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) is homogenous and stable at least for 120 minutes.

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 (main)
6 (satellite)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were determined according to the results of previous studies - Repeated Dose (28 days) Toxicity (Oral) study and One-Generation Reproduction Toxicity Study with this test substance (freely accessible on ECHA website).
- Fasting period before blood sampling for clinical biochemistry: yes, for 18 hours
- Post-exposure recovery period in satellite groups: 28 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included possible clinical effects right after treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- Detailed clinical observations included in the cage observations: piloerection, posture, eyes (pupils), breathing, tonic or clonic movements, stereotypes or bizarre behaviour. After removal from cage the following parameters were checked: reaction to handling, elasticity of skin, colour of visible mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the first week of application and at the end of administration period and recovery period
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 91st day (main group); 119th day (satellite group)
- Anaesthetic used for blood collection: Yes (light ether narcosis)
- Animals fasted: Yes, 16 hours
- How many animals: all animals
- Parameters checked: Total erythrocyte count (RBC), Mean corpuscular volume (MCV), Haematocrit (HCT), Haemoglobin concentration (HGB), Total leucocyte count (WBC), Total platelets count (PLT), Partial thromboplastin time (APTT), Prothrombin time (PT), Fibrinogen (FIB), Granulocytes (Granulo), Lymphocytes (Lympho), Monocytes (Mono)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 91st day (main group); 119th day (satellite group)
- Animals fasted: Yes, 16 hours
- How many animals: all animals
- Parameters checked: Protein total (T-Pro), Alkaline phosphatase (ALP), Cholesterol total (T-Chol), Triglycerides (TG), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Creatinine (Crea), Urea (BUN), Albumin (ALB), Glucose (GLU), Calcium (Ca), Phosphorus (IP), Cholinesterase (CHE), Bile acids (BA), Sodium* (Na), Potassium* (K), Chloride* (CI)

URINALYSIS: Yes
- Time schedule for collection of urine: 90th day (main group); 118th day (satellite group)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked: Volume, Colour, Cloud, Odour, Glucose (GLU), Protein (PRO), Bilirubin (BIL), Urobilinogen (URO), pH, Specific gravity, Blood (BLD), Ketones (KET), Nitrite (NIT), Leucocytes (LEU)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the last week of administration and recovery period
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, 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.

HISTOPATHOLOGY: Yes, performed for all high dose and control animals (see table 1 under "Any other information on materilas and methods incl. tables")

ORGAN WEIGHTS: Yes, 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.

Statistics:

For statistical evaluation the software Statgraphic ® Centurion (version XV, USA) was used.
The parametric tests were used for statistical evaluation of the following parameters:
body weight; selected haematology parameters (haematocrit, haemoglobin concentration, mean volume of erythrocyte, differential leucocyte count, monocytes, granulocytes, lymphocytes, values of blood coagulation: fibrinogen, APPT (Partial thromboplastin time), PT (Prothrombin time)); blood biochemistry (glucose, cholesterol total, anorganic phosphorus, urea, protein total, AST (aspartate aminotransferase), ALT (alanine aminotransferase), albumin, ALP (alkaline phosphatase), calcium, creatinine, sodium, potassium, chloride), bile acids, triglycerides, cholinesterase); urinalysis (volume, pH); biometry of organs (absolute and relative weight).
As the first step the test for normality {Shapiro- Wilk test) was performed. If data were not normally distributed the transformation of data was done (Box-Cox transformation). If data were normally distributed the variance check was performed (Levene's test) to verify if standard deviations within each group were equal. One-Way ANOVA (probability level 0.05) was used to detect whether there are any significant differences amongst the means. If significant differences were find, than the post hoc statistical testing was performed (Fisher's least significant difference - LSD test). If the presence of outliers was noted, the appropriate non-parametric test (Kruskal-Wallis Test, Mann-Whitney test) was chosen.
Non-parametric tests were used for statistical evaluation of following parameters:
selected haematology parameters with non-continuous distribution (total erythrocyte count, total leucocyte count, total platelets count)At first the Kruskal-Wallis test for the comparison of the measured effect in all treatment groups with the vehicle control group was used as global test, and then the two-groups Mann-Whitney test (probability level 0.05) was applied.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Males: In control males and treated males of all dose levels no signs of disease were recorded during the check-in, acclimatisation and application period. During the observation period no changes of health status were noted in treated males. No clinical changes were recorded in control and treated males during the application period. The activity (poise, gait, reaction to handling) of all males in all treated groups was similar during the study and there was no difference in the activity of males in the control group.

Satellite males: In control males and treated satellite males no signs of disease were recorded during the check-in, acclimatisation and application period. During the observation period no changes of health status were noted in treated satellite males. No clinical changes were recorded in control and treated satellite males. The activity (poise, gait, reaction to handling) of all males in the treated group was similar during the study and there was no difference in the activity of males in the control group.

Females: In control females and treated females of all dose levels no signs of disease were recorded during the check-in, acclimatisation and application period. During the observation period no changes of health status were noted in treated females. No clinical changes were recorded in control and treated females. The activity (poise, gait, reaction to handling) of all females in all treated groups was similar during the study and there was no difference in the activity of females in the control group.

Satellite females: In control females and treated satellite females no signs of diseases were recorded during the check-in, acclimatisation and application period. During the observation period no changes of health status were noted in treated satellite females. No clinical changes were recorded in control and treated satellite females during the application period. The activity (poise, gait, reaction to handling) of all females in the treated group was similar during the study and there was no difference in the activity of females in the control group.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males: The body weight of males at all dose levels was comparable to the body weight of control males. Statistical analysis was performed only for necropsy body weight. Statistically significant differences were not found. Body weight increment was similar in males of treated groups compared to control for the entire application period.

Satellite Males: The body weight of treated males in comparison with the control group of males was higher within the whole study. Body weight increment was slightly higher for almost the entire study. Various differences in body weight increments within the treated groups and the control group was recorded during the recovery period.

Females: The body weight of females at all dose levels was well-balanced with the body weight of control group of females. Statistical analysis was performed only for necropsy body weight. Statistically significant differences were not found. The body weight increments were similar in females of treated and control groups for seven weeks of the study, thereafter body weight increments in treated groups were variable without negative effect on the body weight of the animals.

Satellite Females: Slightly decreased body weight of satellite treated females was recorded within the whole study. Various differences in body weight increments within the treated groups and the control group was recorded during application and recovery period. No regular increase or decrease in body weight increment of treated satellite females compared to control satellite females were recorded, therefore this imbalance is considered to be unrelated with application of the test substance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of treated males was similar to control males during the entire application period. Food consumption of satellite treated males was slightly higher than in satellite control males during the entire application period.
Food consumption of treated and satellite treated females was similar to control females during the entire application period.

Food efficiency:

no effects observed

Description (incidence and severity):

Males: Similar food conversion was recorded in the treated groups and the control group during the entire study.

Satellite males: Food conversion of satellite treated males was relatively well-balanced in the satellite control group, imbalance was recorded during the recovery period.

Females: An imbalance in food conversion was recorded in the treated groups and the control group from the 8th week of the study. This difference was not dependent on dose level.

Satellite females: During the recovery period, an imbalance in food conversion of satellite treated females and satellite control females were recorded from the 8th week of the study. No regular increase or decrease in food conversion of treated satellite females compared to control satellite females were recorded, therefore the imbalance is considered to be unrelated with application of the test substance.

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

no effects observed

Description (incidence and severity):

Males: Water consumption of treated males was similar or slightly increased compared to water consumption of control males during the entire application period.

Satellite males: Water consumption of satellite treated males was similar or higher compared to satellite controls in both periods - application and recovery.

Females: A slight imbalance of water consumption was recorded in all treated groups and the control group during the entire study. This difference was not dependent on dose level. The variability was adequate for species, sex and age of animals used in the experiment.

Satellite females: Water consumption of satellite treated females was relatively well-balanced in comparison with the satellite control group in both periods - application and recovery.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

During an examination performed before the start of the study and at the end of the study, no changes were noted in male and female animals of the main and satellite groups.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males: Red blood components (RBC, Hct, Hgb) were influenced by administration of the test substance. Decreased total erythrocyte count in comparison with the control group was recorded at all treated groups with the dose-dependency. Statistically significant decreases in total erythrocyte count and related decreased haemoglobin concentration and haemocrite were registered in the dose levels 250 and 1000 mg/kg/day. These changes persisted to the end of the recovery period and were irreversible.
Total leucocyte count was not influenced by administration of the test substance. Only a percentage value of monocytes was decreased at dose levels 250 and 1000 mg/kg/day and the percentage value of granulocytes was increased at the dose level 1000 mg/kg/day (without dose-dependency). The concentration of fibrinogen was statistically significantly decreased at the dose levels 62.5 and 1000 mg/kg/day.

Satellite males: Statistically significant decreases in total erythrocyte count and related decreased haemoglobin concentration and haematocrite were registered in treated satellite males at the end of the recovery period. Also decreased platelet count, concentration of fibrinogen and a percentage value of monocytes (statistically significant) were recorded in treated males.

Females: The following statistically significant differences were registered in red blood components -statistically significant decreases in total erythrocyte count (dose-dependent), haemoglobin concentration, haematocrit and platelet count at the dose levels 250 and 1000 mg/kg/day. These changes persisted to the end of the recovery period and were irreversible. Extended prothrombin time at the dose levels 62.5 and 250 mg/kg/day and decreased fibrinogen concentration at the dose levels 62.5 and 1000 mg/kg/day were recorded. The white blood components were not significantly influenced by test substance treatment (except a decrease in the percentage value of monocytes at the dose level 1000 mg/kg/day).

Satellite females: Statistically significant decreases in the total erythrocyte count haemoglobin concentration haematocrite and platelet count were registered in treated satellite females at the end of the recovery period. Also, shorter activated partial thromboplastin times (statistically significant) were recorded in treated females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males: Biochemical paramaters of treated males were well-balanced with the control males except for decreased concentration of albumin at the dose level 250 mg/kg/day and decreased bile acids at the dose level 1000 mg/kg/day (statistically significant). Values of other parameters were well-balanced in control and treated males.

Satellite males: Decreased concentration of cholinesterase (statistically significant) and bile acids were recorded in treated males at the end of recovery period. Values of other parameters were relatively balanced in satellite control and treated males.

Females: The value of ALT was statistically significantly increased in females at the dose level 250 mg/kg/day, glucose was increased in females of the dose level 1000 mg/kg/day (statistically significant). The following statistically significant differences were registered in ion concentration: phosphorus ions were increased in all treated groups (without dose-dependency), sodium and potassium ions were decreased (without dose-dependency) and chloride ions were increased in the dose levels 250 and 1000 mg/kg/day. Values of other parameters of biochemical examination were similar to the control group.

Satellite females: Only the cholinesterase concentration was statistically significantly decreased in treated females. Values of other parameters of satellite treated females were similar to satellite control females.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

Statistical evaluation was performed for pH and volume of urine.
Males: A statistically significant decrease of urine pH was detected only in males at the dose level 250 mg/kg/day. This finding can be considered to be incidental - no dose-dependency was recorded. Many factors can influenced pH of urine - e.g. age and sex, so this minor finding has no toxicologjcal importance. Volume of urine was slightly increased in all treated groups (compared to the control group). Change of colour of urine was not observed. Presence of protein was recorded in control and treated groups (except high dosed males). Presence of leucocytes in urine was observed in control and all treated groups. Presence of protein and leucocytes in urine has relation to the sex and age of animals used in experiments.

Satellite males: A change of colour in urine was observed in satellite treated males. Presence of proteins and leucocytes was detected in both satellite groups.

Females: The volume of urine was decreased in all treated groups (dose-dependently) compared to the control group. Statistically significant differences in pH and volume of urine were not detected. Presence of proteins and leucocytes in urine was observed only in females at the highest dose level, which is a finding appropriate to the age and sex of animals.

Satellite females: Statistically significant differences in pH and volume of urine were not detected. Examination of urine revealed only insignificantly increased volume of urine in satellite treated females. Presence of leucocytes was detected only in the control group (finding appropriate to age of animals.

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 slightly decreased at the dose level 1000 mg/kg/day. The variability was adequate to species, sex and age of animals used in experiments. There were no dose dependent decrease or increase in upstanding recorded. Emiction of treated animals was similar to control animals, defecation was slightly higher in treated animals compared to control animals but without dose-dependency. The values of grip strength of pectoral legs and pelvic legs did not show any significant differences between control and treated males.

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 females. The number of upstanding was slightly lower in females at the dose level 250 mg/kg/day in comparison with the control femals. The variability was adequate to species, sex and age of animals used in experiments. The values of grip strength of pectoral legs and pelvic legs did not show any significant differences between control and treated females.

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: Statistically significant changes in absolute organ weights of males were not detected. Absolute weights of thymus and spleen were insignificantly increased at all dose levels compared to the control group. The absolute weight of the adrenal glands were increased in males at the dose levels 62.5 and 1000 mg/kg/day. The absolute weight of the pituitary gland was decreased in males at the dose level 62.5 mg/kg/day. The absolute weight of the epididymides in males at the dose level 62.5 mg/kg/day was decreased in comparison with the control group. Absolute weights of other organs of treated males were balanced with control males. Statistically significant changes in relative organ weights of males were not detected. Slight increases in the weights of the thymus, adrenal glands and spleen were recorded in all treated groups. An insignificant decrease in the weight of the testes and prostate gland was recorded in all treated male groups.

Satellite males: Statistically significant changes in absolute organ weights of satellite males were not detected. Absolute weights of the thymus, adrenal glands, spleen and liver were slightly increased compared to the control group. Absolute weights of epididymides and pituitary gland were insignificantly decreased. A statistically significant decrease in the relative weight of the epididymides in satellite treated males was recorded. An insignifiant decrease in the relative weights of the brain, heart, testes and pituitary gland were recorded. The relative weight of other organs of satellite treated and control males were similar.

Females: Statistically significant changes in absolute organ weights of females were not detected. The absolute weight of the thymus was decreased in all treated groups compared to the control group. Absolute weights of adrenal glands, brain, kidneys and liver were increased in all treated groups. The weight of the pituitary gland was decreased in females at the dose level 62.5 mg/kg/day. A decrease in the absolute weight of the uterus in females at the dose level 250 mg/kg/day was recorded. This finding was related to the oestrus cycle (low number of females with uterus dilatation compared to other groups). A statistically significant decrease in the relative weight of the uterus was detected in females at the dose level 250 mg/kg/day. An insignificant decrease in the weight of the thymus in all treated groups of females, and in the pituitary gland in the treated group of females at the dose level 62.5 mg/kg/day compared to the control group was recorded. An increase in the relative weight of the adrenal glands and liver in all treated groups was recorded.

Satellite females: A statistically significant decrease in the absolute weight of the brain was recorded in satellite treated females. The absolute weight of the spleen and uterus of satellite treated females were insignificantly decreased compared to satellite control females. An increase in the absolute weight of the pituitary gland was recorded in treated females. Statistically significant changes in relative organ weights of treated females were not detected. An insignificant increase in the relative weight of the ovaries and pituitary gland was recorded in treated females in comparison with the control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Males: In nearly all males (38) no macroscopical findings were recorded during necropsy. Macroscopic findings were sporadic and without dose-dependency. In the liver, dark red foci in one male of the high dose was observed. Oedematous testis was recorded for one male of the control group.

Satellite males: No macroscopical findings were recorded during necropsy in 11 animals. In the stomach mucosa, dark red foci in one male was observed.

Females: In females, no macroscopic changes were recorded during necropsy. Dilatation of the uterus (non-pathological finding) was observed in 11 females throughout the different treatment groups (without dose dependency). The following macroscopic changes were detected sporadically: cyst around ovary in one female, uterine horn filled with liquid in one female and dark red foci in the stomach mucosa of one female.

Satellite females: No macroscopic pathological changes were recorded in satellite females during necropsy. Dilatation of the uterus (non-pathological finding) was observed in 3 satellite females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Males: Tubular atrophy (minimal intensity) of testes was recorded in 6 males of the control and 2 males of the high dose. Other findings were observed either in control animals only or in both control and administered animals, or they were of a secondary nature with no relation to the test substance treatment.
Focal fatty changes in the liver (presence of small or large spherical fatty vacuoles in the cytoplasm of hepatocytes) were detected by examination of specially stained cryotome sections: irregular (irregularly situated foci in liver lobuli) numerous in 3 males (2 control; 1 high dose), frequent in 2 males (1 control; 1 high dose).

Satellite males: Tubular atrophy (minimal intensity) of testes was recorded in 5 males. Other findings were observed either in control animals only or in both control and treated animals, or they were of a subsidiary nature with no relation to the test substance treatment. Focal fatty changes (presence of small or large spherical fatty vacuoles in the cytoplasm of hepatocytes) were detected by examination of specially stained cryotome sections of the liver: irregular (irregularly situated foci in liver lobuli) numerous (2 males high dose), frequent (3 control; 1 high dose), diffuse (1 control) and centrolobular (1 control).

Females
A bursal cyst on the ovary was found in 2 females of the high dose. This finding was evaluated as a spontaneous lesion. A cyst on the thymus was recorded in 2 control females. Small cysts of spontaneous origin were found in the mucosa of the uterus in 2 high dose females. Hydrometra of the uterus was recorded in 5 control and 6 high dose females (related to the status of their reproductive cycle). Other findings were observed either in control animals only or in both control and treated animals, or they were of a subsidiary nature with no relation to the test substance treatment.
One female from the dose level 62.5 mg/kg/day was also examined. A folicular cyst was recorded.
Focal fatty changes (presence of small or large spherical fatty vacuoles in cytoplasm of hepatocytes) were detected by examination of specially stained cryotome sections of the liver: irregular (irregularly situated foci in liver lobuli) sporadic (1 high dose), numerous (2 control), frequent (4 control; 2 high dose) and centrolobular (1 high dose).

Satellite females: Hydrometra of the uterus was recorded in 3 control and 1 high dose female (related to the status of their reproductive cycle). Other findings were observed either in control animals only or in both control and administered animals, or they were of a subsidiary nature with no relation to the test substance treatment.
Focal fatty changes (presence of small or large spherical fatty vacuoles in cytoplasm of hepatocytes) were not detected by examination of specially stained cryotome sections of the liver: irregular (irregularly situated foci in liver lobuli) frequent (2 control; 2 high dose) and diffuse (1 high dose).

Histopathological findings: neoplastic:

no effects observed

Details on results:

62.5 mg/kg/day
No adverse effects of the test substance on growth of animals, food and water consumption and health condition of animals were detected in either sex. Changes of haematological parameters were detected in both sexes: statistically significant decreases in concentration of fibrinogen in males, statistically significant increases in prothrombin time and decreases in fibrinogen concentration in females. Biochemical parameters of males were not influenced by test substance treatment. In females, decreased concentration of ions - sodium and potassium (statistically significantly) and increased value of phosphorus were recorded. Biometry of organs did not show statistically significant changes in absolute and relative weight of organs. No macroscopic changes related to test substance treatment in males and females were detected.

250 mg/kg/day
No adverse effects of the test substance on growth of animals, food and water consumption and health condition of animals were detected in either sex. Changes of haematological parameters were detected in both sexes: statistically significantly decreased total erythrocyte count and related decreased haemoglobin concentration and haematocrite in males and females. In addition, decreased platelet count and increased prothrombin time were recorded in females. Biochemical parameters of males were altered only in albumin concentration - it was statistically significantly decreased. In females, statistically significant increases in ALT, concentration of phosphorus and chloride ions were recorded. Statistically significant decreases in the concentration of sodium and potassium ions were detected. Biometry of organs did not show statistically significant changes in absolute and relative weight of organs except decreased relative weight of the uterus. No macroscopic changes related to test substance treatment in males and females were detected.

1000 mg/kg/day
No adverse effects of the test substance on growth of animals, food and water consumption and health condition of animals were detected in either sex. Changes of haematological parameters were detected in both sexes: statistically significant decreases in total erythrocyte count and related decreased haemoglobin concentration and haematocrite in males and females. In addition, decreased fibrinogen and percentage value of monocytes, increased portion of granulocytes in males were recorded. Decreased platelet count, fibrinogen concentration and percentage value of monocytes were found in females. Biochemical parameters of males altered only in bile acids concentration. In females, statistically significant increases in glucose concentration, concentration of phosphorus and chloride ions were recorded. Statistically significant decreases in the concentration of sodium and potassium ions were detected. Biometry of organs did not show statistically significant changes in absolute and relative weight of any organs. No macroscopic changes related to test substance treatment in males and females were detected. Microscopical changes did not reveal histopathological findings related to the test substance treatment.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In a oral repeated dose study according to OECD guideline 409 an NOAEL of 1000 mg/kg bw/d in rats was determined.

Executive summary:

The test substance was tested for subchronic toxicity according to OECD Test Guideline No. 408. Wistar rats of SPF quality were used for testing. The test substance was administered in a vehicle (1% water solution of methylcellulose) by stomach tube; oral application to rats occurred daily. The study included 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 (doses 62.5, 250, 1000 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 (1000 mg/kg/day). The administration period lasted 90 days. Animals in the main groups were then sacrificed while satellite animals were observed for the next 28 days without 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 a week. Ophthalmologic examination was performed at the beginning and at the end of the study. Before the end of study, the functional observations were performed. The study was completed by urinalysis, haematological and biochemical analysis, and gross necropsy of animals. The organs selected for weighing and histopathological examination were removed. These parameters (except clinical observation during the recovery period) were also checked for the satellite groups of animals.

The test substance treatment did not produce changes detected in health condition control, opthalmological observation, daily clinical observation after application and functional observation of animals. Food and water consumptions were not negatively influenced by the test substance administration. The test substance did not interfere with normal growth of treated animals. Body weight and body weight increments of treated animals were not affected by the test substance administration. During haematological examination, changes in red blood components of both sexes were recorded: decreased total erythrocyte count and related decreased concentration of haemoglobin and haematocrit (statistically significant decreases in dose levels 250 and 1000 mg/kg/day). This change was irreversible and dependent on dose level. In females, platelet count was statistically significantly decreased at the dose levels 250 and 1000 mg/kg/day. This decrease was also irreversible. This irreversible effect was not accompanied by any significant clinical changes or pathomorphological findings of related tissues and organs (spleen, bone marrow). Upon histological examination, there were no changes that indicated an increase in erythrocyte damage in the spleen or a failure of hematopoiesis in the bone marrow. Therefore, these findings are considered to be without significant toxicological importance. During biochemical examination no significant changes related to the test substance treatment were recorded in males. An unbalanced concentration of ions was detected. Statistically significant decreases in the concentration of sodium and potassium ions and increased concentration of chloride ions were recorded in treated females. Also concentration of phosphorus was significantly increased in all treated groups of females compared to control group. All changes were reversible. During biometry of organs, significant changes in organ weights related to test substance administration were not detected. Toxicologically significant microscopical changes in all examined organs were not found. Urine parameters were not influenced by test substance administration - only a decrease in the value of urine pH in males of the dose level 250 mg/kg/day was recorded and this finding can be considered to be incidental, because it was not dependent on the dose levels. (Many factors can influenced pH of urine - e.g. age and sex, so this minor finding has no toxicological importance). No macroscopical changes related to test substance administration were recorded. Microscopical evaluation showed that the test substance orally administered at the dose of 1000 mg/kg/day (the highest dose level) did not cause histopathological changes indicative of a toxic effect in any examined organs.

The NOAEL (No Observed Adverse Effect Level) value in male and female rats was set at 1000 mg/kg/day.