Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
OECD 422
Type of information:
other: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From January 13th to December 10th, 2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Justification for Read Across is reported in the endpoint summary and in the Category Justification Report attached to the Section 13 of this dossier.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

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)
Version / remarks:
2016
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 4,4'-bis[[6-anilino-4-[(2-hydroxyethyl)methylamino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate
EC Number:
237-600-9
EC Name:
Disodium 4,4'-bis[[6-anilino-4-[(2-hydroxyethyl)methylamino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate
Cas Number:
13863-31-5
Molecular formula:
C38H40N12Na2O8S2
IUPAC Name:
disodium 2,2'-ethene-1,2-diylbis[5-({4-anilino-6-[(2-hydroxyethyl)(methyl)amino]-1,3,5-triazin-2-yl}amino)benzenesulfonate]

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
CRL (SPF quality - guaranteed)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River SPF breeding.
- Age at study initiation: DRFE: 11 weeks on arrival; main study: males, females: sexually adult; males – 9 weeks on arrival, females – 9 weeks on arrival.
- Fasting period: animals were without feed two hours before application and two hours after application of the test item.
- Housing: DRFE: monitored conditions, micro-biologically defined background; main study: SPF conditions.
- 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: maintenance pelleted diet for rats and mice - Altromin for rats/mice.
- Water: ad libitum
- Acclimation period: DRFE: 5 days; main study: 13 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
aqua pro iniectione
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into glass beaker calibrated to 100 ml and dissolved in vehicle (aqua pro iniectione, ca 80 % of total volume) in ultrasonic bath together with occasional mixing by glass rod for 10 minutes. After this the glass beaker was diluted to mark by vehicle and the application form was stirred by magnetic stirrer (400 rpm) for 10 min.
The concentrations of solution at all dose levels were adjusted to ensure the administration of 1 ml per 100 g of body weight. For each dose level concentration, the solution was prepared separately. The application forms were prepared daily just before administration and covered with aluminium foil to avoid potential degradation due to light. The administration of the test item to animals was performed during two hours after preparation of application form. The stirring of solutions continued during administration.
The test item was administered to the stomach by gavage. The animals were treated 7 days per week at the same time (7.00 – 10.00 am). The vehicle control group was administered by vehicle - aqua pro iniectione in the same volume.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity were determined by means of measuring of a peak area of the test item by a high-performance liquid chromatography based on a method developed at the test facility.

The application form for analysis was prepared in the same manner as for application to animals – i.e. solution in aqua pro iniectione.

Concentration level 10 mg/10 ml
Ca 0.10 g of the test item was weighed with wider end of glass Pasteur pipette into a 150 ml glass beaker calibrated to 100 ml and the beaker was slowly replenished by the vehicle. The test item was dissolved in ultrasonic bath for 8 min. The solution was stirred by magnetic stirrer at 350 rpm for 10 minutes.
The beaker with test item was during dissolving and homogenisation covered by aluminium foil due to possible light unstability of test item.

Concentration level 1000 mg/10 ml
Ca 10.0 g of the test item was weighed with laboratory spoon into a 150 ml glass beaker calibrated to 100 ml. The beaker was slowly replenished by the vehicle together with mixing by glass rod. The test item was dissolved in ultrasonic bath together with occasional mixing by glass rod for 10 minutes. After this the application form (yellow fine slurry) was stirred by magnetic stirrer at 400 rpm for 10 min.
The beaker with test item was during dissolving and homogenisation covered by aluminium foil due to possible light unstability of test item.

Stability of the application form
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 of both application forms. Two samples were taken at all time intervals.

Concentration level 10 mg/ 10 ml: time interval 0 min represents for this concentration the time after 8 minutes of ultrasonication and 10 minutes of mixing by magnetic stirrer at 350 rpm.
Concentration level 1000 mg/ 10 ml: time interval 0 min represents for this concentration the time after 10 minutes of ultrasonication together with occasional mixing with glass rod and 10 minutes of mixing by magnetic stirrer at 400 rpm.

Homogeneity of the application form
Concentration level 10 mg/ 10 ml: the samples were taken after 8 minutes in ultrasonic bath and 10 minutes of mixing by magnetic stirrer (350 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content. Two samples were taken from each place.
Concentration level 1000 mg/ 10 ml: the samples were taken after 10 minutes in ultrasonic bath together with occasional mixing with glass rod and 10 minutes of mixing by magnetic stirrer (400 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content. Two samples were taken from each place.

Results of analysis
Both the application forms of the test item (10 mg and 1000 mg /10 ml), at defined laboratory conditions are homogenous and stable at least for 120 minutes from the finalization of application form preparation.
Duration of treatment / exposure:
DRFE
Administration: 19 days as total time of application in DRFE study; females became pregnant gradually, each female was applied from next day after confirmed mating to 19th day of pregnancy)

Main study
Administration
Parental males (totally 49 days of administration):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 43rd day – 63rd day (administration period) → 64th day (necropsy)
Satellite males (totally 49 days of administration + 14 days of observation):
1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 65th day - 77th day (observation period) → 78th day (necropsy)

Parental females:
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration)→ gestation → lactation → day 12 post partum
Satellite females (totally 49 days of administration + 14 days of observation):
1st day – 14th day (pre-exposure period) → 15th day - 63rd day (administration period) → 65th day - 77th day (observation period) → 78th day (necropsy)

Non-pregnant females (without evidence of copulation):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 25 days after the end of mating period

Non-pregnant females (with evidence of copulation):
1st day – 14th day (pre-exposure period) → 15th day - 28th day (pre-mating period, administration) → 29th day – 42nd day (mating, administration) → 25th day after confirmed mating
Frequency of treatment:
Once daily.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control - main study
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Remarks:
main study
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
main study
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
main study
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control - DRFE
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
DRFE
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
DRFE
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
DRFE
No. of animals per sex per dose:
DRFE
1. Control 0 6 males + 6 females
2. Low dose 100 mg/kg/day 6 males + 6 females
3. Intermediate dose 300 mg/kg/day 6 males + 6 females
4. High dose 1000 mg/kg/day 6 males + 6 females

Main study
The first six males and six females with delivered pups per group and satellite groups of animals (control and treated) are a part of the repeated dose toxicity part of study.
Basic groups:
1. Control 0 12 males + 12 females
2. Low dose 80 mg/kg/day 12 males + 12 females
3. Intermediate dose 250 mg/kg/day 12 males + 12 females
4. High dose 750 mg/kg/day 12 males + 12 females

Satellite groups:
5. Control – vehicle – satellite 0 6 males + 6 females
6. High dose – satellite 750 mg/kg /day 6 males + 6 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on the result of the DRF study on the substance, the selected highest tested dose for the main study should have been 1000 mg/kg bw/day, since no significant toxicological effects have been observed at this dose and it could have been considered as limit dose. However, it should be noticed that this study has been performed not only to investigate the toxicological properties of the substance in itself, but also and mainly to compare the effects within the members of the category; therefore the highest tested dose has been selected as below reported, taking into account the existing following studies:

• 1-DSA – Subchronic toxicity study – Relevant toxicological effects on kidney at 750 mg/kg bw/day
• 1-DSA – Subchronic toxicity study – Relevant toxicological effects on testes at 750 mg/kg bw/day
• 3a-MSA – Subacute toxicity study – Effects to be confirmed on haematological parameters, liver and kidney starting from 200 mg/Kg bw/day
• 3a-MSA – Two generation toxicity study - Relevant toxicological effects mainly in terms of maternal toxicity at 1000 mg/kg bw/day and minor effects at 300 mg/kg bw/day

Indeed, in this respect all systemic studies concerning repeated dose, reproductive and developmental toxicity (OECD 422, 408 and 414) have been set on similar dose level in order to be able to compare all members of the category on the specific organ effects.
The lowest and the medium doses have been selected in order to have a coherent spacing of doses respect to the highest selected one, also taking into account all the available data on the repeated dose toxicity ( 3a-MSA, 1-DSA).

- Fasting period before blood sampling for clinical biochemistry: 18 hours
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
Body weight:
males and satellite animals - the first day of administration and then weekly,
females - the first day of administration and then weekly,
during pregnancy: 0., 7th, 14th, 20th day,
during lactation: 1st, 4th day, 12th day and 13th day
pups (litters) - 1st, 4th day, 12th day and 13th day
pups – individually – 4th day of lactation
The body weight of animals was recorded on automatic balances with group mean computing module on specified days. All animals were weighed immediately before euthanasia too.
Weight increment was computed as a mean per group (in grams). Non-pregnant females (females without parturition) were not included in calculation of means in pregnancy and lactation period.

Food consumption: weekly and on the same days as body weight (except the mating period); satellite males and females – weekly
In 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 the mating period). 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 pre-mating period. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Nonpregnant females (females without parturition) were not included in calculation of mean food consumption in pregnancy and lactation period.
Food conversion in % (weight increment/food consumption × 100) was calculated for animals of the repeated dose toxicity part of study. In the pre-mating period, the food consumption and conversion of females was calculated from values of all females.

Water consumption: satellite males and females – twice a week
The mean values in groups (water consumption per animal and per day) were calculated for each week of the study.

Mortality control
All rats during the treatment periods were examined for vitality or mortality twice daily.

Health condition control
All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before application, during application and immediately after application.

Clinical observations of parental males and females
All rats were observed daily during the administration period. This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (12.00 – 14.00 p.m.). Animals were observed in natural conditions in their cages.

Detailed clinical parental observation
This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, 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.

Functional observation of parents
This observation was done at the end of administration period (only in 6 males and 6 females of each group) and recovery period. During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli and pupillary reflex were evaluated and motor activity assessment was conducted. The individual observations of grip strength were performed using a grip strength meter. Measurements were made on: 1) pectoral legs, 2) pelvis legs. Grip power was expressed in Newtons.

Laboratory examinations
Examination of vaginal smears
Vaginal smears were made from the 1st till the 14th day of study (pre-exposure period) for monitoring of oestrous cycle of females. Only females with regular cyclicity were put into the study. Each morning in the mating period vaginal smears were prepared from all the mated females. These smears were examined for presence of spermatozoa. Vaginal smears have been made also on necropsy day to determine the stage of oestrous cycle.

Urinalysis
This examination was performed only in 6 males of each group and in satellite males. In females this examination was not performed (dams should not be removed from the pups for prolonged periods). The rats were kept in the metabolic cages for the collection of urine for two hours. Immediately before entering metabolic cages the animals were administered 2 ml of drinking water per 100 g of body weight by gavage to the stomach.
Urinalysis parameters:
Volume
Colour
Cloud
Odour
Glucose - GLU
Protein - PRO
Bilirubin - BIL
Urobilinogen - URO
pH
Specific gravity
Blood - BLD
Ketones - KET
Nitrite - NIT
Leucocytes - LEU

Haematological examination
This examination was performed only in 6 males and 6 females of each group and in satellite males and females. The blood samples were collected from the orbital plexus by glass micropipette under the light ether narcosis into the PVC test tubes containing anticoagulation system.
Haematological parameters:
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
Lymphocytes - LYM
Monocytes - MON
Neutrophiles - NEU
Eosinophiles - EOS
Basophiles - BASO
Reticulocyte count was examined by the help of light microscope.

Biochemical examination
This examination was performed only in 6 males and 6 females of each group and in satellite males and females. The animals were fasted approximately for 18 hours before blood collection but they were supplied drinking water ad libitum.
The blood samples were collected from the orbital plexus under the light ether narcosis. Biochemical parameters were measured in serum.
Biochemical parameters:
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
Bilirubin total - T-Bil
Glucose - GLU
Calcium - Ca
Phosphorus - IP
Cholinesterase - CHE
Bile acids - BA
Sodium - Na
Potassium - K
Chloride - Cl

Blood samples from the day 13 pups (pooled sample, one male + one female pup) and the parental males were assessed for serum levels of thyroid hormone thyroxine (T4 total) and rat thyroid stimulating hormone (TSH).
Sacrifice and pathology:
Pathological examination
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.

Biometry of organs
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 + seminal vesicles, thymus, spleen, brain, pituitary gland and heart were recorded (repeated dose toxicity part of study – 6 males and females from each group + satellite groups); testes or ovaries, epididymis or uterus, prostate gland + seminal vesicles, pituitary gland (reproduction part of study – all animals).
Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ × 100/ body weight. From all adult males and females and one male and female day 13 pup from each litter thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.

Histopathological examination
Organs for histopathological examination
In all males and females: pituitary gland, ovaries, uterus incl. cervix of uterus, vagina, epididymis/epididymides, prostate gland + seminal vesicles, testes, thyroid gland, all gross lesions.
In males and females of repeated dose toxicity part of study additionally: adrenal glands, aorta, brain (incl. cerebellum and med. oblongata), caecum, colon, duodenum, pancreas, rectum, salivary glands, sciatic nerve, skeletal muscle, skin, spinal cord – thoracic, spleen, stomach, thymus, thyroid gland, trachea, urinary bladder, female mammary gland area, femur, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes – mesenteric, paraaortal, oesophagus, eye

The mentioned tissues and organs were collected from all killed males and females at necropsy and fixed in buffered 4 % formaldehyde solution (v/v) for further histopathological evaluation. For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.

In repeated dose toxicity part of study, the full histopathology of the preserved organs and tissues was performed for all high dose and control animals and satellite animals. Because of treatment related changes were recorded at the highest dose level in the liver, the histopathological examination of liver was performed also in males and females at the middle and at the lowest dose level.
In reproductive toxicity part of study, the full histopathology of the preserved organs and tissues was performed for all high dose and control animals. Detailed histological examination of testes was performed with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure. Histopathological examination of macroscopically changed organs was performed in one female (uterus dilatation) at the dose level 80 mg/kg/day and in one female (ovarian cyst) at the dose level 250 mg/kg/day.
Statistics:
For statistical evaluation the software Statgraphic® Centurion (version XVII, 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 results statistically significant on probability level (p ≤ 0.05) were indicated in the summary tables.

In general:
The parametric tests were used for statistical evaluation of
• body weight of males and females
• selected haematology parameters
• blood biochemistry parameters
• data from urinalysis (pH, volume)
• data from biometry of organs

As the first step the test for normality (Shapiro-Wilk test) was used. If the data were not normally distributed than the transformation of data was performed (Box-Cox transformation). If still the normal distributed distribution was not achieved than non-parametric tests (Kruskal-Wallis Test, Mann-Whitney test) were used.
For normally distributed data have at first the variance check has been performed (Levene´s test)to verify if standard deviations within each group are equal. One-Way ANOVA (probability level p ≤ 0.05) was used to detect whether there are any significant differences amongst the means. In case of significant differences, the post hoc statistical testing was performed (Fisher's least significant difference - LSD test).

The non-parametric tests were used for statistical evaluation of
• selected haematology parameters with non-continuous distribution (total erythrocyte count, total leucocyte count, total platelet count)

The Kruskal-Wallis test was used for the comparison of the measured effect in all treatment groups with the vehicle control group (as global test) and the two-groups Mann-Whitney test (probability level p ≤ 0.05).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No signs of disease and clinical signs of intoxication were recorded during the application period in all treated animals. No clinical changes were recorded in control or treated males during the application period.
The behaviour, clinical status and activity of treated animals were similar during the study and not different from males of the control group.

Satellite animals
No signs of disease and clinical signs of intoxication were recorded during the application period in all treated males. During the observation period (recovery; weeks 8 and 9) no changes of health status were noted in satellite treated animals.
No clinical changes were recorded in satellite control or satellite treated animals during the application period.
No clinical signs of toxicity were observed during the detailed clinical observation in satellite treated animals in comparison with the satellite control group.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males
The body weight of males was slightly decreased at the dose level 750 mg/kg/day compared to the control group before start of administration and during the whole study and at the dose level 250 mg/kg/day during the 3rd week. Statistically significant differences in necropsy body weight were not found in treated males.

Satellite males
Body weight of satellite treated males was slightly decreased in comparison with satellite control males for the whole time of application and during recovery period.
Statistically significant differences in necropsy body weight were not found in satellite treated males.

Females
Before the mating period, body weights of females in all treated groups was similar to the control group. The body weights of females at the dose level 250 mg/kg/day was slightly decreased compared to the control females during the pregnancy and lactation periods.
Statistically significant differences in necropsy body weight were not found in treated females.

Satellite females
Body weight of satellite treated females was comparable with satellite control animals for the entire application and recovery periods. Statistically significant differences in necropsy body weight were not found in satellite treated females.

Mean body weight increment
Males
Weight increments of treated males were variable as well as in control males and not adversely influenced by the test item administration. The negative body weight increments were recorded in males during the 3rd week at the dose level 250 mg/kg/day and during the 6th week at the dose levels 80 and 250 mg/kg/day.

Satellite males
Weight increments of satellite treated males in application and recovery period were comparable with control satellite males, except the 3rd and 6th week. The body weights increments of the treated satellite males were decreased compared to the control satellite males during the 3rd and 6th week.

Females
The negative body weight increment was recorded in control as well as in treated females during the 1st week of application. During the 2nd week of application were the body weight increments in treated females comparable with control.

Satellite females
Weight increments in treated females were variable and not affected by the test item treatment. The negative body weight increments were recorded in control as well as in treated satellite females.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males
The food consumption of treated males was similar in comparison with the control males during the whole application period of study.

Satellite males
The food consumption of satellite treated males was comparable to the control group during the entire application and recovery periods.

Females
During the pre-mating period, pregnancy and lactation period the food consumption of treated females was similar with the control females.

Satellite females
The food consumption of satellite treated females was similar with the control group during the entire application and recovery periods.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
Males
The food conversion of treated males compared to control animals was variable and without treatment relation.

Satellite males
The food conversion of satellite treated males was quite similar or higher in comparison with satellite control (exc. the 3rd and the 6th week of study).

Females
During the 1st week was the food conversion decreased in females at the dose level 250 mg/kg/day compared to the control group but during the 2nd week it was the opposite.
During the pregnancy and lactation period, the food conversion of treated females was similar with the control group, except lactation period 1 – 4 at the dose level 750 mg/kg/day.

Satellite females
The food conversion of satellite treated females was variable compared to satellite control females, but not adversely influenced by the test item treatment.
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Satellite males
The water consumption of satellite treated males was comparable to the satellite control group during the entire application and recovery period, except the 9th week. The water consumption of satellite treated males was increased in comparison with control satellite males during the 9th week.

Satellite females
The water consumption of satellite treated females was slightly increased compared to the satellite control group during the entire application and recovery period (except the 5th week).
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males
The parameters of red blood components (RBC, MCV, Hct, Hgb) of treated males were not adversely influenced by the administration of the test item. The value of reticulocytes was well-balanced and not statistically significantly changed in treated groups of males.
The white blood component were quite well-balanced in treated males compared to control males. Only differential percentage count of monocytes was slightly increased and the count of neutrophils was very slightly decreased in comparison with control group at the dose level 80 mg/kg/day (without statistical significance and without dose dependence). The value of monocytes count was over the historical control range at the dose level 80 mg/kg/day.
The values of haemocoagulation parameters were not significantly affected by the test item treatment, except significantly decreased value of APTT (p ≤ 0.05) at the dose level 80 mg/kg/day, but still in range of historical control. The platelet count was slightly decreased at the dose level 750 mg/kg/day in comparison with control, but without statistical significance and in range of historical control range.

Satellite males
The percentage count of lymphocytes (p ≤ 0.05) was statistically significantly increased and the count of monocytes (p ≤ 0.05) was statistically significantly decreased at the satellite treated males in comparison with control satellite group. Statistically significantly decreased value of reticulocytes was recorded in satellite treated males. Other parameters of red and white component and haemocoagulation were comparable with parameters in the control satellite group. All values were in a range of historical control.

Females
The parameters of red blood components in treated females were not changed in comparison with the control females. The value of reticulocytes was comparable in treated groups of females with control group.
The value of the total leucocyte count and the five-population differential of white blood cells were not affected by the test item treatment.
Haemocoagulation parameters (APTT, PT, FIB) were not influenced by administration of the test item – statistically significant changes were not recorded.
All values of haematological parameters were in a range of historical control.

Satellite females
All the values of haematological parameters in treated satellite females were comparable with parameters in the control satellite group, except reticulocytes. Statistically significantly decreased value of reticulocytes was recorded in satellite treated females. All values of haematological parameters were in a range of historical control.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males
Significantly changed values (p ≤ 0.05) of creatinine (decreased) and Ca (increased) were detected in males at all treated groups compared to the control animals. The values of creatinine recorded at the dose levels 80 and 750 mg/kg/day were out of the historical control range. The values of alanine aminotranferase (ALT) were dose-dependently increased in treated males, at the dose levels 250 and 750 mg/kg/day with statistical significance and out of the historical control range at the dose level 750 mg/kg/day.
The value statistically significantly altered at all dose levels was cholinesterase (CHE) - decreased at the dose level 80 mg/kg/day and dose-dependently increased at the dose levels 250 and 750 mg/kg/day compared to the control animals (but in historical control range).
Statistically significantly increased values of T-Bil in males at the dose levels 80 and 750 mg/kg/day were recorded, but still in range of the historical control.
In males at the dose levels 250 and 750 mg/kg/day statistically significantly decreased values (p ≤ 0.05) of chlorides (Cl) were detected (in historical control range).
The values of bile acids (BA) were increased at all treated groups in comparison with control males, but without statistical significance (not dose-dependently and in historical control range).
Values of other biochemical parameters of treated males were in a historical control range and not significantly altered in comparison with the controls.

Satellite males
The statistically significantly decreased values of T-Pro, TG and ALB (but in range of historical control) were recorded in satellite treated males in comparison with the satellite control males. Values of other biochemical parameters of satellite treated males were similar to the satellite control group.

Females
Statistically significantly increased value of T-Chol was reported in females at the dose level 250 mg/kg/day (in the historical control range). Statistically significant difference was found out in treated females in value of Ca at the dose levels 250 and 750 mg/kg/day, but still in the historical control range. The value of TG was statistically insignificantly increased at the dose level 250 mg/kg/day in comparison with control (over the historical control range). The values of AST and ALT were increased at the dose level 750 mg/kg/day, without statistical significance but out of the range of historical control. The value of ALP was insignificantly increased at the dose level 750 mg/kg/day.
Values of other biochemical parameters of treated females were comparable to the control group.

Satellite females
Statistically significantly decreased value of CHE and increased value of T-Chol were noted in satellite treated females in comparison with the satellite control females. The value of ALT and AST were statistically insignificantly decreased in treated satellite females (in range of the historical control).Values of other biochemical parameters of treated satellite females were comparable to the control satellite group.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males
A statistically significantly decreased volume of urine was detected in males at the dose level 80 mg/kg/day. Presence of proteins, blood and leukocytes were recorded in treated males as well as in control males. These findings were not associated with the application of the test item.

Satellite males
The volume of urine was statistically significantly increased in satellite treated males. The presence of leucocytes and blood was recorded only in satellite control males. Other recorded findings were comparable at the treated satellite males with the control satellite males.
Behaviour (functional findings):
effects observed, non-treatment-related
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 in treated males was slightly higher compared to the control. Emiction and defecation in treated males was similar with the control males. 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 in females at the dose level 250 mg/kg/day was slightly decreased in comparison with the control females. The values of grip strength of pectoral and pelvic legs were without 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, non-treatment-related
Description (incidence and severity):
Absolute organ weight
Males
Statistically significant differences were not recorded. The insignificantly decreased absolute weight of adrenal glands was recorded in males at the dose level 80 mg/kg/day in comparison with the control males. Absolute weights of other organs were in dosed males comparable with the control males.

Satellite males
A statistically significantly changed absolute weight of organs were not recorded in treated satellite males in comparison with the satellite control males. Absolute weights of organs at dosed group of satellite males was similar with the control satellite males.

Females
No statistically significant changes were found out. Absolute weights of organs were in dosed females comparable with the control females.

Satellite females
Statistically significant differences were not recorded. Absolute weights of organs were similar in satellite treated and control females.

Relative organ weight
Males
The relative weight of the kidneys was statistically significantly increased in males at the dose level 750 mg/kg/day in comparison with the control males. Relative weight of testes was slightly increased at the dose level 750 mg/kg/day in comparison with control. Relative weights of other organs were in dosed males comparable with the control males.

Satellite males
Statistically significant differences were not recorded. Relative weight of testes was slightly increased in comparison with control satellite males. Relative weights of other organs were similar in satellite treated and control males.

Females
The statistically significantly changed relative weights of organs were not recorded. Relative weights of organs were similar in treated and control females.

Satellite females
Relative weight of brain of treated females was statistically significantly decreased in comparison with satellite control females. Relative weights of other organs were similar in satellite treated and control females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males
Control: no macroscopic findings were recorded in all 6 males
80 mg/kg/day: no macroscopic findings were recorded in all 6 males
250 mg/kg/day: no macroscopic findings were recorded in 6 males
750 mg/kg/day: no macroscopic findings were recorded in all 5 males, in 1 male were found three hard objects (3 mm diameter) in urinary bladder and its mucosa was irritated and swollen.

Satellite males
Control satellite: no macroscopic findings were recorded in all 6 males.
750 mg/kg/day satellite: no macroscopic findings were recorded in 5 males, in one satellite male was recorded yellow formation (5 × 5 mm) in left epididymis.

Females
Control: no macroscopic findings were recorded in all 6 females.
80 mg/kg/day: no macroscopic findings were recorded in all 6 females.
250 mg/kg/day: no macroscopic findings were recorded in all 6 females.
750 mg/kg/day: no macroscopic findings were recorded in all 6 females.

Satellite females
Control satellite: no macroscopic pathological findings were recorded in all 6 females (dilatation of uterus – non-pathological finding was recorded in two of six females).
750 mg/kg/day satellite: no macroscopic pathological findings were recorded in all 6 females (dilatation of uterus – non-pathological finding was recorded in two of six females).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males
For examination of the repeated toxicity of the test item, the full histopathology was performed for the first six males of control (No. 1-6) and high dose (No. 61-66). Because of probably treatment related changes at the highest dose level in liver were recorded, the histopathological examination of liver was performed also at the middle (No.41-46) and at the lowest dose level (No.21-26).
In liver histological changes related to the test item treatment were recorded. Focal polymorphonuclear inflammation of the liver parenchyma was detected in 0 – 0 – 0 – 5 males. The finding in the lungs was evaluated as the agonal change associated with the euthanasia of the animals. The non-specific change as the focal lymphocytic infiltration of the interstitium was detected in prostate of 3 – / – / – 3 males. The incidence of other microscopical findings was very sporadic.

Satellite males
The satellite animals (control No. 81 – 86 and high dosed No. 91 – 96) as a part of the repeated dose toxicity study were histopathologically examined. The full histopathology was performed.
In liver the focal polymorphonuclear necrotic inflammation of the liver parenchyma was detected in 1 – 2 satellite males. The non-specific change as the focal lymhocytic infiltration of the interstitium of epididymis was recorded in 0 – 2 satellite males. The findings in the lungs, trachea and kidneys were evaluated as the agonal change associated with the euthanasia of the animals. The non-specific finding dilatation of gastric glands in stomach was recorded in two treated satellite males. The incidence of other microscopical findings was very sporadic.

Females
For examination of the repeated toxicity of the test item, the full histopathology was performed for the first six females delivered pups from control (Nos. 101,103,106,109,110,112) and high dose group (Nos.161,162,165,166,170,172). Because of probably treatment related changes in animals at the highest dose level in liver were recorded, the histopathological examination of liver was performed also in females at the middle (Nos.142,143,144,146,148,150) and at the lowest dose level (Nos.121,123,124,125,128,131).
The incidence of affected females is expressed in numeric form and ranged in sequence of dose levels 0 – 80 – 250 – 750 mg/kg/day and 0 S – 750 S in satellite groups.
The histological changes related to the test item treatment were not recorded. Only sporadic findings were recorded in the control group of females as well as in dosed group of females. Focal polymorphonuclear inflammation of the liver parenchyma was detected only in 0 – 0 – 0 – 1 female. The changes related to pregnancy were found in both control and high treated females: accumulation of siderophages in the mucosa of the uterus in 2 – / – / – 1 females. The incidence of other microscopical findings was very sporadic.

Satellite females
The satellite animals (control No. 181 – 186 and high dosed No.191 – 196) as a part of the repeated dose toxicity study were also histopathologically examined. The full histopathology was performed.
The hemangiectasia was recorded in pituitary gland of 0 – 2 satellite females (the non-specific change). The finding in the kidneys was evaluated as the agonal change associated with the euthanasia of the animals. The incidence of other microscopical findings was very sporadic.
Histopathological findings: neoplastic:
no effects observed
Details on results:
Repeated oral administration of the test item to rats by gavage did not cause the death of animals. Two females died at the dose level 80 mg/kg/day, due to accidental and not treatment-related reasons.

The effect of the test item treatment on growth of animals was not adverse. The food consumption was similar between control and treated animals. Individual variability of body weight increments was adequate to species, sex and age of animals used in the study and to period of gravidity or lactation in females.

The water consumption in satellite treated males was comparable to the satellite control group during the whole study (except the 9th week). The water consumption in satellite treated females was slightly increased compared to the satellite control females (except the 5th week).

Test item treatment did not produce clinical changes in health status of animals.

Repeated dose toxicity part of study:
Six males per group and first six mothers per group that delivered pups were examined from the main groups. Also, satellite animals were part of the examination of the repeated dose toxicity of the test item.

Haematological examination of treated animals showed only sporadic significant differences in haematological parameters in treated animals compared to control animals.
In males statistically significantly decreased value of APTT at the dose level 80 mg/kg/day was recorded, but still in range of historical control. The differential percentage count of monocytes was slightly increased and the count of neutrophils was very slightly decreased in comparison with control group in males at the dose level 80 mg/kg/day (without statistical significance), but the value of monocytes count was over the historical control range. This exceed of the historical control range was due to a marked increase in the differential percentage count of monocytes and the concurent decrease in the differential percentage count of neutrophils in one male at this dose level.
In the satellite treated males were recorded statistically significantly increased percentage count of lymphocytes statistically, significantly decreased count of monocytes and statistically significantly decreased value of reticulocytes in comparison with control satellite group. All values were in a range of historical satellite control.
No significant changes in haematological parameters were observed in treated groups of females. Only the percentage value of neutrophils was very slightly decreased at the dose level 250 mg/kg/day in comparison with control females (in range of historical control).
In the satellite treated females was noted the statistically significantly decreased value of reticulocytes, but still in range of the historical satellite control.
All findings in the haematological parameters observed in both sexes were considered to be of no toxicological significance. These isolated findings in haematological examination did not reveal toxic effect of the test item on administered animals.

During biochemical examination of animals, statistically significantly changed values were detected.
In males, significantly increased values of calcium ions (Ca) and decreased values of creatinine (Crea) were detected at all dose levels, without dose dependence. The decreased values of creatinine were out of the historical control range at the dose levels 80 and 750 mg/kg/day. The values of alanine aminotransferase (ALT) were dose-dependently increased in males at all dose levels, at the dose level 250 and 750 mg/kg/day with statistical significance, but reversible (not observed at the satellite treated males). The value of ALT was out of the historical control range at the highest dose level. This exceed of the historical control range was due to a marked increased value of ALT in male No. 61 at the highest dose level. The value altered at all dose levels (statistically significantly) compared to control animals was cholinesterase (CHE) – decreased at the dose level 80 mg/kg/day and increased at the dose levels 250 and 750 mg/kg/day (in range of the historical control range). Also the values of bile acids (BA) were increased at all treated groups, but without statistical significance (in range of historical control).
Significantly decreased values of chlorides ions were reported in males at the dose levels 250 and 750 mg/kg/day, but still in range of historical control range.
In males at the dose level 250 mg/kg/day was detected statistically significantly increased value of phosphorus (in the historical control range). All these changes in biochemical parameters in males were reversible, not observed in satellite treated males.
A statistically significant decreased values of total protein, albumin and triglycerides were recorded in satellite treated males in comparison with the satellite control males, but in range of the historical satellite control. This could be associated with microscopical changes in liver - hepatitis. The values of other biochemical parameters of satellite treated males were similar to the satellite control group.
During the biochemical examination of females, significantly changed biochemical values were recorded only sporadically in treated females. Significantly increased value of total cholesterol was reported in females at the dose level 250 mg/kg/day (in historical control range). Significantly increased values of calcium ions were recorded in females at the dose levels 250 and 750 mg/kg/day, but also in range of the historical control. Values of ALT and AST were insignificantly increased and out of the historical control range at the dose level 750 mg/kg/day. These exceeds of the historical control ranges were due to a marked increased values of ALT and AST in one female at this dose level (female No. 170). The value of triglycerides was out of the historical control range for the same reason at the dose level 250 mg/kg/day, the value of triglycerides was marked increased in female No.142 at this dose level. Other biochemical parameters of treated females were comparable to the control group.
Statistically significantly decreased value of cholinesterase and increased value of total cholesterol were noted in satellite treated females in comparison with the satellite control females (in range of the historical satellite control). Values of other biochemical parameters of treated satellite females were comparable to the control group.
The biochemical examination showed a toxicologically significant effect of the test item on the treated males. The increased values of ALT were caused by liver damage in males. Decreased creatinine levels may be associated with decreased synthetic liver function. Also increased values of cholinesterase, bile acids may be associated with the findings reported during the histological examination of liver.
Biochemical examination of treated females did not show a toxicologically significant effect of the test item.

Urinalysis did not reveal any statistically significant changes of pH. The volume of urine was statistically significantly decreased in males at the dose level 80 mg/kg/day and statistically significantly increased in the satellite treated males. The presence of proteins, blood and leucocytes were recorded in treated males as well as in control males and were not associated with the application of the test item.

During biometry of organs in males and females, significant changes of absolute and relative weights of organs related to the administration of the test item were not recorded.

During the macroscopic examination, no findings related to the test item treatment were found out.

Histological examination recorded changes related to the test item treatment only in the males. The focal polymorphonuclear inflammation (hepatitis) was detected in liver of five males at the dose levels 750 mg/kg/day. This finding was recorded only in one female at the highest dose level. This finding was not fully reversible, because of occurrence of the focal polymorphonuclear necrotic inflammation in two satellite males. The histopathological examination of the genital tract of organs revealed the tubular degeneration of testicles in one control male and in three males at the dose level 750 mg/kg/day. Because the spermiogenesis of the males was not affected with the test item treatment, this change was considered as non-specific, unrelated to the effect of the test item treatment. The incidence of other microscopical findings were in most cases spontaneous, agonal changes, or sporadic, not associated with the test item treatment.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
food efficiency
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
urinalysis
water consumption and compound intake

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Dose-range finding

DRF 422 dose (mg/kg) notes
0 100 300 1000
For dams (per dose)    
Mortality 0 0 0 0  
Body Weight F [group mean ± standard deviation (g)] 442.12 ± 21.36 447.87 ± 37.48 450.16 ± 31.02 433.70 ± 33.40 at 20th day of pregnancy
Clinical signs: description, severity, time of onset and duration No effects No effects No effects No effects  
Haematological findings No effects No effects No effects No effects  
Number of pregnant dams 6/6 6/6 6/6 6/6  
Implantations [mean ± SD] 15.53 ± 1.86 15.17 ± 3.66 17.17 ± 1.94 16.17 ± 2.79  
Resorptions [mean ± SD] 1.00 ± 1.26 0.33 ± 0.52 0.67 ± 0.82 2.17 ± 4.83  
Corpora lutea [mean ± SD] 16.33 ± 1.03 16.50 ± 1.38 17.67 ± 2.25 17.33 ± 2.66  
Pathological examination  No effects No effects No effects No effects  
           
For foetuses/offspring (per dose)  
Total number of live foetuses  86 89 99 84  
Total number of dead foetuses  0 1 0 0  
External, soft tissue and skeletal malformations and other relevant alterations No effects No effects No effects No effects  

Main study

  dose (mg/kg) notes
  0 80 250 750 0S 750S
Repeated-dose part  
Body Weight M [group mean ± standard deviation (g)] 499.59 ± 45.49 490.84 ± 37.65 493.88 ± 46.92 471.59 ± 29.05 515.74 ± 28.58 491.19 ± 23.14 Necropsy body weight
Body Weight F [group mean ± standard deviation (g)] 315.86 ± 17.92 314.75 ± 19.49 308.59 ± 20.79 330.34 ± 13.98 281.52 ± 14.25 291.03 ± 12.51 Necropsy body weight
Food Consumption M [group mean (grams/animal/day)] 28.57 30.97 29.72 28.26 25.79 28.86 at 7th or 9th week
Food Consumption F [group mean (grams/animal/day)] 66.70 64.56 60.74 66.52 17.16 21.25 at 12th lactation day or 9th week
Water Consumption M [group mean (mL/animal/day)] Not examined Not examined Not examined Not examined 63.33 72.62 at 9th week
Water Consumption F [group mean (mL/animal/day)] Not examined Not examined Not examined Not examined 48.81 63.33 at 9th week
Mortality 0 0 0 0 0 0 at 7th or 9th week
Signs of toxicity M+F No signs of disease and clinical signs of intoxication  No signs of disease and clinical signs of intoxication  No signs of disease and clinical signs of intoxication  No signs of disease and clinical signs of intoxication  No signs of disease and clinical signs of intoxication  No signs of disease and clinical signs of intoxication  at 7th or 9th week
Nature, severity and duration of clinical observations M + F No clinical changes  No clinical changes  No clinical changes  No clinical changes  No clinical changes  No clinical changes  at 7th or 9th week
Haematological tests M No effects increased value of monocytes count [10.14 % WBC vs 6.5] No effects No effects No effects No effects The white blood components were quite well-balanced in treated males compared to control males. Only differential percentage count of monocytes was slightly increased and the count of neutrophils was very slightly decreased in comparison with control group at the dose level 80 mg/kg/day (without statistical significance and without dose dependence).
The values of haemocoagulation parameters were not significantly affected by the test item treatment, except significantly decreased value of APTT (p ≤ 0.05) at the dose level 80 mg/kg/day, but still in range of historical control. The platelet count was slightly decreased at the dose level 750 mg/kg/day in comparison with control, but without statistical significance and in range of historical control range.
Haematological tests F No effects No effects No effects No effects No effects Statistically significantly decreased value of reticulocytes [2.18 % vs 2.65 %]  
Clinical biochemistry M No effects Effects Effects Effects No effects No effects The value statistically significantly altered at all dose levels was cholinesterase (CHE) - decreased at the dose level 80 mg/kg/day and dose-dependently increased at the dose levels 250 and 750 mg/kg/day compared to the control animals (but in historical control range).
Statistically significantly increased values of T-Bil in males at the dose levels 80 and 750 mg/kg/day were recorded, but still in range of the historical control.
In males at the dose levels 250 and 750 mg/kg/day statistically significantly decreased values (p ≤ 0.05) of chlorides (Cl) were detected (in historical control range).
The values of bile acids (BA) were increased at all treated groups in comparison with control males, but without statistical significance (not dose-dependently and in historical control range).
The statistically significantly decreased values of T-Pro, TG and ALB (but in range of historical control) were recorded in satellite treated males in comparison with the satellite control males. 
Creatinine [µmol/L] 31.40 25.73 27.15 24.32 24.48 26.92 Decreased values of creatinine were detected in males at all treated groups compared to the control animals. The values of creatinine recorded at the dose levels 80 and 750 mg/kg/day were out of the historical control range.
Calcium [mmol/L] 2.47 2.69 2.70 2.66 2.64 2.63 Increased values of calcium were detected in males at all treated groups compared to the control animals. 
Alanine aminotranferase (ALT) [µkat/L] 0.67 0.74 0.81 1.27 0.89 0.91 The values of alanine aminotranferase (ALT) were dose-dependently increased in treated males, at the dose levels 250 and 750 mg/kg/day with statistical significance and out of the historical control range at the dose level 750 mg/kg/day.
Clinical biochemistry tests with relevant baseline values F No effects No effects No effects No effects No effects No effects Statistically significantly increased value of T-Chol was reported in females at the dose level 250 mg/kg/day (in the historical control range). Statistically significant difference was found out in treated females in value of Ca at the dose levels 250 and 750 mg/kg/day, but still in the historical control range. The value of TG was statistically insignificantly increased at the dose level 250 mg/kg/day in comparison with control (over the historical control range). The values of AST and ALT were increased at the dose level 750 mg/kg/day, without statistical significance but out of the range of historical control. The value of ALP was insignificantly increased at the dose level 750 mg/kg/day.
Statistically significantly decreased value of CHE and increased value of T-Chol were noted in satellite treated females in comparison with the satellite control females. The value of ALT and AST were statistically insignificantly decreased in treated satellite females (in range of the historical control).
Urinanalysis M No effects Statistically significantly decreased volume of urine No effects No effects No effects Statistically significantly increased volume of urine Presence of proteins, blood and leukocytes were recorded in treated males as well as in control males. These findings were not associated with the application of the test item.
Circulating thyroid hormones (T4) M [µg%] 4.605 ± 1.233  5.376 ± 0.819 4.172 ± 1.271 5.807 ± 0.774 Not examined Not examined Mean concentrations of T4 hormone was significantly increased at the dose level 750 mg/kg/day, but in range of historical control. 
Circulating thyroid hormones (TSH) M [pg/ml] 1213.5 ± 422.1  930.3 ± 165.4 1033.6 ± 446.3 916.3 ± 369.4 Not examined Not examined  
Organ weights M No effects No effects No effects No effects No effects No effects  
Organ weights F No effects No effects No effects No effects No effects No effects  
Relative weight of organs M No effects No effects No effects Relative weights of the kidneys and tests statistically significantly increased [0.7143 vs 0.6328 and 0.9723 vs 0.8111, respectively] No effects No effects  
Relative weight of organs F No effects No effects No effects No effects No effects Relative weight of brain statistically significantly decreased [0.6856 vs 0.7258]  
Histopathological findings M No effects No effects No effects 5/6 Focal polymorphonuclear inflammation of the liver 1/6 Focal polymorphonuclear inflammation of the liver 2/6 Focal polymorphonuclear inflammation of the liver The incidence of other microscopical findings was very sporadic 
Histopathological findings F No effects No effects No effects 1/6 Focal polymorphonuclear inflammation of the liver No effects 2/6 hemangiectasia in pituitary gland The incidence of other microscopical findings was very sporadic 

Applicant's summary and conclusion

Conclusions:
Toxicologically significant changes were recorded mainly in treated males. Damage of liver, confirmed by the histological examination and evaluation of biochemical parameters, was observed in males at the highest dose level. The statistically significantly increased value of alanine aminotransferase and statistically significantly decreased value of creatinine (out of the historical control ranges) was recorded in males at the dose level 750 mg/kg/day. Histopathological finding – the focal polymorphonuclear inflammation in the liver (hepatitis) – was reported in males at the dose level 750 mg/kg/day and only in one female at this dose level. This finding was not fully reversible. Two satellite high dose males showed also this lesion (focal polymorphonuclear necrotic inflammation) at the end of recovery period.

NOAEL (No Observed Adverse Effect Level) for repeated dose toxicitity in males was established as 250 mg/kg body weight/day and in females was established as 750 mg/kg body weight/day.
Executive summary:

Study design

Before the start of study with laboratory animals, the stability and homogeneity of application form were determined at the test facility. A dose-range finding experiment (DRFE) was performed to determine the dose levels for the main study.

The doses of 80, 250, 750 mg/kg/day were determined on the basis of results of DRFE and approved by Sponsor.

Methods

Wistar rats (SPF quality) were used for testing. The test item was administered in the form of a solution in aqua pro iniectione. Oral application by stomach tube was performed daily. The animals were without feed two hours before application and two hours after application of the test item. The main study includes 3 treated groups (doses 80, 250, 750 mg/kg/day) and one control group (vehicle only) and two satellite groups of animals - one control group (vehicle only) and one treated group (750 mg/kg/day).

Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females.

The first 6 males and 6 mothers who delivered pups per group (as per internal SOP) and a satellite groups of animals (control and treated) are part of the repeated dose toxicity study and examined with respect to toxicity of the test item. Satellite animals were used for observation of reversibility, persistence or delayed occurrence of systemic toxicity effects up to 14 days post treatment. All 12 males and females per group are a part of the reproduction study and examined with respect to reproduction parameters.

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 up to the 12th day of lactation; males – after mating period – 49 days in total; non-pregnant females (mated females without parturition) – for 25 days after confirmed mating; non-mated females – for 25 days after the end of the mating period. After the end of the administration period, the animals in the main groups were sacrificed and satellite animals were observed for the next 14 days without treatment.    

During the study, clinical observation and health status controls were performed daily. The body weight and food consumption were measured weekly or at the specified time intervals. Detailed clinical observation was carried out weekly. Functional observations were performed at the end of the application and observation periods. Vaginal smears were prepared daily, 2 weeks before start of the administration period (oestrous cycle monitoring), during the mating period (until the presence of spermatozoa) and at necropsy. Reproduction parameters relevant to pups (number of pups, weight of litters and weight of pups, sex and vitality of pups, measurement of anogenital distance, nipple retention, serum levels of thyroid hormones (T4 and TSH in pups) were also recorded. The study was completed with urinalysis, haematological and biochemical analysis and gross necropsy of animals. In all males of the main groups, the sperm parameters, sperm motility and sperm morphology were examined. Selected organs from adult animals and pups were removed for weighing and histopathological examination.

Results - repeated dose toxicity part of the study

Repeated oral administration of the test item did not cause any mortality (except two females from the dose level 80 mg/kg/day – female No. 126 died due to intubation error and female No. 127 during the complicated delivery). These deaths were accidental and not treatment-related.

The 6 males per group (Nos. 1-6, 21-26, 41-46, 61-66) and first six mothers per group that delivered pups were examined from the main groups (control: Nos. 101,103,106,109,110,112; the lowest dose: Nos. 121,123,124,125,128,131; the middle dose: Nos. 142,143,144,146,148,150 and the highest dose: Nos. 161,162,165,166,170,172). Also, satellite animals (control: males - Nos. 81-86, females – Nos. 181-186 and high dosed: males - Nos. 91-96 and females Nos. 191-196) were part of the examination of the repeated dose toxicity of the test item.

Test item treatment did not produce clinical changes in health status of animals, did not affect the normal growth of treated parental males and females.

The haematological examination did not reveal toxic effect of the test item on administered animals. Haematological examination of treated males and females showed only sporadic significant differences in haematological parameters in treated groups compared to control group. These isolated findings in haematological examination did not reveal toxic effect of the test item on administered animals.

During biochemical examination of animals, several statistically significantly changed values were detected.

In males, significantly increased value of calcium ions (Ca) and decreased value of creatinine (Crea) were detected at all dose levels, without dose dependence. The decreased values of creatinine were out of the historical control range at the dose levels 80 and 750 mg/kg/day. The values of alanine aminotransferase (ALT) were dose-dependently increased in males at all dose levels, at the dose level 250 and 750 mg/kg/day with statistical significance, but reversible (not observed at the satellite treated males). The value of ALT was out of the historical control range at the highest dose level. This exceed of the historical control range was due to a marked increase of ALT value in male No. 61 at the highest dose level. The value altered at all dose levels (statistically significantly) compared to control animals was cholinesterase (CHE) – decreased at the dose level 80 mg/kg/day and increased at the dose levels 250 and 750 mg/kg/day (in range of the historical control range). Significantly decreased values of chlorides ions were reported in males at the dose levels 250 and 750 mg/kg/day, but still in range of historical control range. The statistically significantly increased values of total bilirubin (in historical control range) were recorded in males at the dose levels 80 and 750 mg/kg/day. In males at the dose level 250 mg/kg/day was detected statistically significantly increased value of phosphorus (in the historical control range). All these changes in biochemical parameters in males were reversible, not observed in satellite treated males.

A statistically significantly decreased values of total protein, triglycerides and albumin were recorded in satellite treated males in comparison with the satellite control males, but in range of the historical satellite control. Values of other biochemical parameters of satellite treated males were similar to the satellite control group.

During the biochemical examination of females, significantly changed biochemical values were recorded only sporadically in treated females. Significantly increased value of total cholesterol was reported in females at the dose level 250 mg/kg/day (in historical control range). Significantly increased values of calcium ions were recorded in females at the dose levels 250 and 750 mg/kg/day, but also in range of the historical control. Values of ALT and AST were insignificantly increased and out of the historical control range at the dose level 750 mg/kg/day. These exceeds of the historical control ranges were due to a markedly increased values of ALT and AST in one female at this dose level (female No. 170). The value of triglycerides was out of the historical control range for the same reason at the dose level 250 mg/kg/day, the value of triglycerides was markedly increased in female No. 142 at this dose level. Other biochemical parameters of treated females were comparable to the control group.

Statistically significantly decreased value of cholinesterase and increased value of total cholesterol were noted in satellite treated females in comparison with the satellite control females (in range of the historical satellite control). Values of other biochemical parameters of treated satellite females were comparable to the control group.

The biochemical examination of treated males revealed some dose-dependently changed values and some values out of historical control range associated with the test item administration. Specifically, these parameters are: ALT, creatinine and cholinesterase. But this changes were not observed in satellite treated males.

Biochemical examination of treated females did not show a toxicologically significant effect of the test item.

Urinalysis did not reveal any statistically significant changes of pH. The volume of urine was statistically significantly decreased in males at the dose level 80 mg/kg/day and statistically significantly increased in the satellite treated males. The presence of proteins, blood and leucocytes were recorded in treated males as well as in the control males and were not associated with the application of the test item.

During biometry of organs in males and females, significant changes of absolute and relative weights of organs related to the administration of the test item were not recorded.

During the macroscopic examination, no findings related to the test item treatment were found out.

Histological examination showed changes related to the test item treatment only in the males. The focal polymorphonuclear inflammation (hepatitis) was detected in liver of 5 males at the dose level 750 mg/kg/day. This finding was recorded only in 1 female at the highest dose level. This finding was not fully reversible, because of occurrence of the focal polymorphonuclear necrotic inflammation in 2 satellite males.  

The histopathological examination of the genital tract of organs revealed the tubular degeneration of testicles in 1 control male and in 3 males at the dose level 750 mg/kg/day. Because the relative weight of testicles and spermiogenesis of the males were not affected with the test item treatment, this change was considered as non-specific, unrelated to the effect of the test item treatment. The incidence of other microscopical findings were in most cases spontaneous or agonal changes, or sporadic, not associated with the test item treatment.

Conclusions

Accordingly, the NOAEL for repeated dose toxicity in males was established as 250 mg/kg body weight/day and in females was established as 750 mg/kg body weight/day.