Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-morpholino-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

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 08 to November 11, 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Reliability of source study is 1. Justification for Read Across is reported in the endpoint summary and in the Category Justification Report attached to 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:

29th July 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

EC Number:

273-468-9

EC Name:

Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

Cas Number:

68971-49-3

Molecular formula:

C40 H38 N12 O22 S6 .6Na (molecular formular in EC inventory is not correct)

IUPAC Name:

hexasodium 2-[[4-[bis(2-hydroxyethyl)amino]-6-[4-[(E)-2-[4-[[4-[bis(2-hydroxyethyl)amino]-6-(2,5-disulfonatoanilino)-1,3,5-triazin-2-yl]amino]-2-sulfonatophenyl]ethenyl]-3-sulfonatoanilino]-1,3,5-triazin-2-yl]amino]benzene-1,4-disulfonate

Test animals

Species:

rat

Strain:

Wistar

Remarks:

SPF quality

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River SPF breeding, supplied via VELAZ s.r.o., Czech Republic, RČH CZ 11760500
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: DRFE: 11 weeks at arrival, Main study: 9-10 weeks on arrival,
- Fasting period before study: the animals were without feed two hours before application and two
hours after application of the test item.
- Housing: main study: SPF conditions according to internal SOP No.12.
- Bedding: sterilized soft wood fibers Lignocel
- 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 (e.g. ad libitum): maintenance pelleted diet for rats and mice - Altromin for rats/mice, Manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 5 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Aqua pro iniectione

Details on oral exposure:

- Concentration in vehicle: 150 mg, 300 mg and 600 mg in 10 ml

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical Method
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 Preparation of Application Form
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 into a 150mL glass beaker calibrated to 100 mL and the beak
er was slowly replenished by the vehicle. The test item was dissolved in ultrasonic bath for 5 min. The solution was stirred by magnetic stirrer at 350 rpm for 5 minutes.
The beaker with test item was during dissolving and homogenisation covered by aluminium foil due to possible light instability of test item.
Concentration Level 1000 mg/10 mL Ca 10.0 g of the test item was weighed into a 150mL glass beaker calibrated to 100 mL. The beaker was slowly replenished by the vehicle (ca 80% of total volume) together with occasional 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 glass beaker was diluted to mark by vehicle and the application form was stirred by magnetic stirrer at 500 rpm for 10 min.
The beaker with test item was during dissolving and homogenisation covered by aluminium foil due to possible light instability of test item.
The 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.
Conc. level 10 mg/ 10 mL: Time interval 0 min represents for this concentration the time after 5 minutes of ultrasonication and 10 minutes of mixing by magnetic stirrer at 350 rpm.Conc. 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 500 rpm.
Note: The beaker with test item was during application form preparation and homogeneity and stability measuring covered by aluminium foil due to possible light instability of test item (information from sponsor).
The Homogeneity of the Application Form
Conc. level 10 mg/ 10 mL: The samples were taken after 5 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.
Conc. level 1000 mg/ 10 mL: The samples were taken after 10 minutes in ultrasonic bath together with occasional mixing with glass rod and 60 minutes of mixing by magnetic stirrer (500 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
The beaker with test item was during dissolving, homogenisation and stability measuring covered by aluminium foil due to possible light instability of test item.
This measure, laboratory conditions and procedures described above are recommended for further test item application form preparation.
It follows from the results of analyses (homogeneity and stability) that the both application forms of the test item (10 mg and 1000 mg /10 mL), at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner) are homogenous and stable at least for 120 minutes from the finalization of application form preparation.

Duration of treatment / exposure:

Parental males totally 49 days of administration
Satellite males totally 49 days of administration + 14 days of observation
Parental females 42 days + gestation → lactation → day 12 post-partum
Satellite females totally 49 days of administration + 14 days of observation
Non-pregnant females without evidence of copulation 42 days → 25th day after the end of mating period
Non-pregnant females with evidence of copulation 42 days → 25th day after confirmed mating

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control of the basic group and satellite

Doses / concentrations 2

Dose / conc.:

80 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

Control of the basic group and satellite

No. of animals per sex per dose:

Basic group: 12 males and 12 females per group
Satellite groups: 6 males and 6 females per group

Control animals:

yes, concurrent no treatment

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: The animals were fasted approxim
ately for 18 hours before blood collection but they were supplied drinking water ad libitum

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first application and then weekly (except the mating period)

BODY WEIGHT: Yes
- Time schedule for examinations: 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

FOOD CONSUMPTION:
- Food consumption: Yes
weekly and on the same days as body weight (except the mating period); satellite males and females – weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: satellite males and females – three times a week

URINALYSIS: Yes. 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. The following parameters were determined by analyser PocketChem PU-4010 (Arkray, Inc., Japan).

MORTALITY CONTROL: twice daily

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGTHS
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. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.
From all adult males and females thyroid glands were preserved in fixation medium. The thyroid weight was determined after fixation.
Full histopathology of the preserved reproductive organs and tissues was performed for all high dose and control animals.

Other examinations:

Postmortem examinations (parental animals)
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.

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.
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 reproduction parameters with non-continuous distribution (number of live born pups, number of pups, number of implantations)
• selected haematology parameters with non-continuous distribution (total erythrocyte count, total leucocyte count, total platelet count)
The Kruskal-Wallis test was used for the comparis on 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):

Clinical Observation
Males
No clinical changes were recorded in control and treated males during the application period.
Satellite males
No clinical changes were recorded in control and treated satellite males during the application period Females
No clinical changes were recorded in control and treated females during the application period.
Satellite females
No clinical changes were recorded in control and treated satellite females during the application period.

Detailed Clinical Observation
Males
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
The activity (poise, gait, reaction to handling) of all males in the satellite treated group was similar during the study and there was no difference in the activity of males in the satellite control group.
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
The activity (poise, gait, reaction to handling) of all females in the satellite treated group was similar during the study and there was no difference in the activity of females in the satellite control group.

Mortality:

no mortality observed

Description (incidence):

Males
There were no unscheduled deaths during the main study.
Females
There were no unscheduled deaths during the main study

Description (incidence and severity):

Body Weight and Body Weight Increment
Mean Body Weight
Males
The statistical analysis was performed for body weights and necropsy body weights. Statistically significant differences were not found.
Body weight of males at the dose levels 250 and 750 mg/kg/day were decreased during the whole study in comparison with the control group. From the 5th week to the end of the study, the decrease of body weight in males mainly at the dose level 750 mg/kg/day was marked and can be considered as biologically significant as also showed total body weight gain.
The necropsy body weight at the dose levels 250 and 750 mg/kg/day was decreased in comparison with the control group.
Satellite males
The statistical analysis was performed for body weight and necropsy body weights.
The body weight of the satellite treated males was decreased during the whole study compared to the control group. This decrease of body weight was statistically significant from the 5th week to the end of the study.
Females
The statistical analysis was performed for necropsy body weight and body weight for all time period (pre-mating, pregnancy and lactation period).
Body weight of treated females in pre-mating period was lower in comparison with the control group. This was caused by sequential inclusion of animal groups to the study. During the pregnancy period, the body weight of treated females was decreased in comparison with the control group. At the dose levels 250 and 750 mg/kg/day a decrease of body weight was statistically significant (except the day 0 of pregnancy period at the dose level 250 mg/kg/day, when this d
ecrease was insignificant).
Statistically significantly decreased body weights of females at the dose levels 250 and 750 mg/kg/day in lactation period were recorded (except the 13th day of the lactation period).
Satellite females
The statistical analysis was performed for a body weight and necropsy body weight. Statistically significant differences were not found.
Body weight of satellite treated females was comparable to the control group during the whole study.

Mean Body Weight Increment
Males
The body weight increments were variable in males of treated and control groups during the study. Negative effect on the body weigh increments was recorded in males at the dose level 750 mg/kg/day at the 6th week of application period.
Satellite males
Body weight increments in males of treated group were variable to control during the whole application period and recovery period. Negative effect on the body weigh increments was recorded in treated group at the 4th and 6th week of application period and 1st week of recovery period.
Females
Weight increments in treated females were variable to the control females within the 1st and 2nd week of application. Negative effect on the body weigh increments was recorded in females at the dose level 80 mg/kg/day at the 1st week of application period.
The evaluation of weight increments during pregnancy and lactation period is included in the reproduction part of study.
Satellite females
No marked differences in weight increments were recorded between satellite treated females and satellite control females. Negative body weight increment was recorded in control as well as in treated animals.

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 control males during the application period.
Satellite males
The food consumption of satellite treated males was comparable to the control group during the application and recovery period.
Females
During the pre-mating period the food consumption of treated females was similar in comparison with control females.
During the pregnancy period the food consumption of treated females was comparable to the control group.
During the lactation period the food consumption of treated females was comparable to the control group (except day 4-12 of lactation period at the dose level 80 mg/kg/day when food consumption was decreased, but without effects).
Satellite females
The food consumption of satellite treated females was comparable to the control group during the application and recovery period.

Food efficiency:

no effects observed

Description (incidence and severity):

Food Conversion
Males
The food conversion of treated males was variable during application period compared to the control group.
Satellite males
The food conversion of satellite treated males was variable in comparison with the satellite control.
Females
The food conversion of treated females in the pre-mating, pregnancy and lactation period periods was variable.
Satellite females
The food conversion of satellite treated females was variable compared to satellite control females.

Description (incidence and severity):

Satellite males
Water consumption of satellite treated males was increased compared to water consumption of satellite control males during the whole application period. During the recovery period water consumption of satellite treated males was variable compared to water consumption of satellite control males.
Satellite females
Water consumption of satellite treated females was increased compared to water consumption of satellite control females during the whole application and recovery period.

Description (incidence and severity):

Males
Statistically significantly increased value of total leucocyte count in males of dose level 750 mg/kg/day was detected. The portion of eosinophiles was statistically significantly decreased at the dose level 750 mg/kg/day and portion of monocytes was statistically significantly decreased in all treated groups in comparison with the control group. Statistically significantly increased value of platelet count in males at the dose level 750 mg/kg/day was detected.
The value of fibrinogen was statistically significantly increased at the dose levels 250 and 750 mg/kg/day. Other haematological parameters of treated males were similar with the control males. The mean value of total leucocyte count (14.32) in males at the dose level 750 mg/kg/day was above the upper limit of historical control (historical control parameters: 5.71 – 12.14). The mean value of fibrinogen (2.38) in males at the dose level 750 mg/kg/day was above the upper
limit of historical control (historical control parameters 1.42 – 2.20).
Satellite males
The value of mean corpuscular volume was statistically significantly decreased in satellite treated males in comparison with the satellite control group (in a range of historical control). The value of platelet count was statistically significantly increased in satellite treated males compared to control group. The portion of eosinophiles was statistically significantly decreased in satellite treated males in comparison with the satellite control group.
Other values were similar with satellite control males and in range of historical control limits.
Females
Statistically significantly increased value of total leucocyte count in females of all treated groups was detected. The percentual portion of reticulocytes was statistically significantly increased at the dose levels 250 and 750 mg/kg/day.
Other haematological parameters of treated females were similar with the control females. All haematological parameters were in a range of historical control limits.
Satellite females
The values of total erythrocyte count, haemoglobin and haematocrite were statistically significantly decreased in satellite treated females in comparison with the satellite control group (in a range of historical control). The portion of eosinophiles was statistically significantly decreased in satellite treated females in comparison with the satellite control group.
Other values were similar with satellite control males and in range of historical control limits.

Description (incidence and severity):

Males
Statistically significantly decreased values of total protein, albumin and bile acid in males at the dose level 750 mg/kg/day were recorded. Statistically significantly increased activity of AST in male s at the dose level 750 mg/kg/day was detected. The activity of ALP was statistically significantly decreased in all treated males in comparison with the control group (dose dependently). Statistically significantly increased value of creatinine in males at the dose levels 250 and 750 mg/kg/day was recorded. Statistically significantly decreased value of bilirubin total in males at the dose levels 250 and 750 mg/kg/day was recorded.
Differences in values of other biochemical parameters did not show any significant changes.
The mean value of creatinine (38.27) in males at the dose level 750 mg/kg/day was above the upper limit of historical control (historical control parameters 21.2 – 35.9).
Satellite males
Statistically significantly increased activity of AST, values of creatinine and cholinesterase were recorded in treated males.
Other biochemical parameters of satellite treated males were similar with the satellite control males.
All values of biochemical parameters were in historical control limits.
Females
Statistically significantly increased values of cholesterol total, triglyceride and inorganic phosphorus in females at the dose levels 80, 250 and 750 mg/kg/day were recorded. Statistically significantly increased value of glucose in females at the dose levels 250 and 750 mg/kg/day was detected. The activity of AST was statistically significantly increased in females at the dose level 750 mg/kg/day in comparison with the control group. The concentration of potassium ions was statistically significantly decreased in females at the dose level 80 mg/kg/day.
Differences in values of other biochemical parameters did not show any significant changes.
The mean values of triglyceride (3.65/ 3.22/ 1.73) in females of all treated groups (80/ 250/ 750 mg/kg/day) were above the upper limit of historical control (historical control parameters 0.28 – 1.59).
Satellite females
Statistically significantly increased values of calcium ions, inorganic phosphorus and bile acid in satellite treated females were detected.
Other biochemical parameters of satellite treated females were similar with the satellite control females.
The mean values of inorganic phosphorus (2.47) were above the upper limit of historical control in satellite treated females (historical control parameters 1.39 – 2.40).

Description (incidence and severity):

Males
The volume of urine was statistically significantly decreased in males at the dose levels 80 and 750 mg/kg/day but the water consumption was increased. Presence of proteins in 2-3-0-5 males, presence of blood in 0-0-3-3 males and presence of leucocytes in 1-4-4-5 males were found out.
Satellite males
The volume of urine was statistically significantly decreased in satellite treated males. Presence of proteins in 2-1 males, presence of blood in 1-0 male and presence of leucocytes in 3-6 males were recorded.

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 at the dose level 80 mg/kg/day was decreased compared to control males. The variability was adequate to species, sex and age of animals used in experiments. Emiction and defecation of treated animals was without important changes. The time of grip of pectoral legs was slightly decreased at the dose level 250 mg/kg/day but without toxicological importance.
Satellite males
Reactions to touch, noise, pain and pupillary reflex of satellite treated males were the same as in the control group. The number of upstanding, emiction, defecation and examination of grip strength of satellite treated animals was without important changes.
Females
Reactions to touch, noise, pain and pupillary reflex of treated females were the same as in the control group. The number of upstanding of treated females compared to control females was slightly decreased. The variability was adequate to species, sex and age of animals used in experiments. Emiction, defecation and examination of grip strength of treated animals was without important changes.
Satellite females
Reactions to touch, noise, pain and pupillary reflex of satellite treated females were the same as in the control group. The number of upstanding of satellite treated females was slightly decreased compared to control females. The variability was adequate to species, sex and age of animals used in experiments. Emiction, defecation and examination of grip strength of satellite treated animals was without important changes.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute Organ Weight
Males
The absolute weight of the prostate gland with seminal vesicles was statistically significantly decreased in males at the dose level 250 mg/kg/day in comparison with control males. Absolute weights of other organs of treated males were balanced with control males.
Satellite males
The absolute weights of the kidneys, spleen and pituitary gland were statistically significantly increased in satellite treated males in comparison with satellite control males. Absolute weights of other organs of satellite treated males were balanced with satellite control males.
Females
The absolute weight of the kidneys was statistically significantly increased in females at the dose levels 250 and 750 mg/kg/day in comparison with control females. Statistically significantly increased absolute weight of adrenal glands in females at the dose level 250 mg/kg/day was detected.
Absolute weights of other organs of treated females were balanced with control females.
Satellite females
The absolute weights of the kidneys and spleen were statistically significantly increased in satellite treated females in comparison with satellite control females.Absolute weights of other organs of satellite treated females were balanced with satellite control females.

Relative Organ Weight
Males
The relative weights of the kidneys and brain were statistically significantly increased in males at the dose level 750 mg/kg/day in comparison with control males. Relative weights of other organs of treated males were balanced with control males.
Satellite males
The relative weights of the brain, spleen, kidneys and pituitary gland and thyroid gland were statistically significantly increased in satellite treated males in comparison with satellite control males. Relative weights of other organs of satellite treated males were balanced with satellite control males.
Females
The relative weight of the spleen was statistically significantly increased in females of all treated groups in comparison with control females. Statistically significantly increased relative weights of kidneys, liver and thyroid gland in females at the dose levels 250 and 750 mg/kg/day were recorded (dose dependently). The relative weight of the brain was statistically significantly increased in females at the dose level 750 mg/kg/day.
Relative weights of other organs of treated females were balanced with control females.
Satellite females
The relative weights of the spleen, kidneys and liver were statistically significantly increased in treated females in comparison with control females.
Relative weights of other organs of satellite treated females were balanced with satellite control females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Males
Control: no pathological findings were recorded in all 6 males.
80 mg/kg/day: no pathological findings were recorded in all 6 males.
250 mg/kg/day: light coloured kidneys in 2 males were found out. No pathological findings were recorded in 4 males.
750 mg/kg/day: light coloured kidneys in all 6 males were found out.
Satellite males
Control satellite: no pathological findings were recorded in all 6 males.
750 mg/kg/day satellite: light coloured kidneys in 5 males were found out. Green-grey body on caput epididymis in one male was found out. No pathological findings were recorded in 1 male.
Females
Control: no pathological findings were recorded in all 6 females.
80 mg/kg/day: no pathological findings were recorded in all 6 females.
250 mg/kg/day: light coloured kidneys in 5 females were found out. No pathological findings were recorded in 1 female.
750 mg/kg/day: light coloured kidneys in all 6 females were found out.
Satellite females
Control satellite: dilatation of uterus was recorded in 2 of 6 females. This finding is not related to the test item treatment, it relates to the oestrus cycle of females. No pathological findings were recorded in all 6 females.
750 mg/kg/day satellite: dilatation of uterus was recorded in 2 of 6 females. This finding is not related to the test item treatment, it relates to the oestrus cycle of females. Light coloured kidneys in 2 females were found out. No pathological findings were recorded in 4 females.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males
Full histopathology of the preserved organs and tissues was performed for first six males of high dose and control animals and for all satellite males. In the middle and lowest dose levels the organ (only kidneys) with macroscopical findings was examined.
The incidence of affected animals is expressed in numeric form and ranged in sequence of dose levels 0-80-250-750 mg/kg/day and 0S-750S in satellite groups. In kidneys, hydronephrosis in 0-3-0-2 males, hyaline casts in 0-1-1-0 males and tubular necrosis in 0-0-2-6 males were found out.
Siderosis in spleen in 0-/-/-2 males was found out. Blood aspiration into lungs and/or trachea in 4-/-/-6 males was found out - this is a terminal lesion related to the mode of euthanasia. Spontaneous lesion of the prostate gland, focal chronic inflammation in 1-/-/-1 males was found out.
Other microscopical changes occurred in males only sporadically.
Satellite males
In kidneys, hydronephrosis in 0-1 male and tubular necrosis in 0-6 males were found out. In spleen, extramedullary hemopoiesis in 2-0 males and siderosis in 3-2 males were observed. Blood aspiration into lungs and/or trachea in 4-4 males was found out - this is a terminal lesion related to the mode of euthanasia.
Spontaneous lesion of the prostate gland, focal chronic inflammation in 2-0 males was found out.
Other microscopical changes occurred in males only sporadically.
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. 102,103,104,107,108,111) and high dose group (Nos. 162,163,165,166,167,168) in the first instance. In the middle and lowest dose levels the organ (only kidneys) with macroscopical findings was examined.
Full histopathology of the preserved organs and tissues was also performed for satellite females. The incidence of affected animals is expressed in numeric form and ranged in sequence of dose levels 0-80-250-750 mg/kg/day and 0S-750S in satellite groups. In kidneys, hydronephrosis in 0-1-1-1 females and tubular necrosis in 0-0-3-6 females were found out. Siderosis in spleen in 3-/-/-1 females was found out. In uterus presence of the hemosiderin in mucosa in 5-/-/-6 females and focal accumulation of lipophages and siderophages in the mesometrium in 5-/-/-6 females were found out, these findings related to previous gravidity. The mammary gland of six control and six administered females showed marked lobular hyperplasia related to its gravidity.
Blood aspiration into lungs and trachea in 6-/-/-2 females was found out - this is a terminal lesion related to the mode of euthanasia.
Other microscopical changes occurred in females only sporadically.
Satellite females
In kidneys, hyaline casts in 1-0 female and tubular necrosis in 0-2 females were found out. Blood aspiration into lungs and trachea in 1-3 females was found out this is a terminal lesion related to the mode of euthanasia.
Hydrometra of uterus was observed in three recovered control and four recovered high dose females in relation to the status of their reproduction cycle.
Siderosis in spleen in 2-4 females was detected.
Other microscopical changes occurred in females only sporadically.

Description (incidence and severity):

Thyroid hormones
Blood (serum) samples from all adult parental males were assessed for thyroid hormones (T4 and TSH total). Mean concentrations of T4 and TSH hormones did not show any significant changes in males.

Effect levels

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Any other information on results incl. tables

Results of main study

	dose (mg/kg)				satellite - dose (mg/kg)		notes
	0	80	250	750	0S	750S
Body Weight (M) - at necropsy	502.07 ± 41.35	498.18 ± 35.22	475.77 ± 26.32	455.30 ± 30.86	519.14 ± 26.25	467.08 ± 18.40	no statistically significant differences. satellite: decrease of body weight was statistically significant from the 5th week to the end of the study.
Body Weight (F) - at necropsy	340.26 ± 38.10	320.43 ± 21.89	305.33 ± 11.45	286.94 ± 30.60	273.69 ± 10.63	260.61 ± 24.07	during pregnancy (day 0, 7, 14, 20) and lactation (day 1, 4, 12, 13) periods, at the dose levels 250 and 750 mg/kg/day a decrease of body weight was statistically significant (except for day 0 of pregnancy at 250 mg/kg/day and day 13 of lactation at 250 and 750 mg/kg/day). satellite: no statistically significant differences.
Food Consumption (M) g/animal/day at 7 w (9 w for satellite)	28.00	30.40	27.86	25.87	26.93	25.92	no effects
Food Consumption (F) g/animal/day at 12 day lactation (9 w for satellite)	57.17	36.50	55.90	50.97	19.74	20.30	comparable to control groups (except day 4-12 of lactation period at the dose level 80 mg/kg/day when food consumption was decreased, but without effects)
Water Consumption (M) ml/animal/day at 9 w	not examined	not examined	not examined	not examined	50.00	57.38	water consumption of treated males was increased compared to control males during the whole application period. During the recovery period water consumption of satellite treated males was variable compared to water consumption of satellite control males.
Water Consumption (F)ml/animal/day at 9 w	not examined	not examined	not examined	not examined	44.05	65.71	water consumption of satellite treated females was increased compared to water consumption of satellite control females during the whole application and recovery period.
Mortality	no unscheduled deaths	no unscheduled deaths	no unscheduled deaths	no unscheduled deaths	no unscheduled deaths	no unscheduled deaths
Signs of toxicity	no signs of disease; no changes of health status.	no signs of disease; no changes of health status.	no signs of disease; no changes of health status.	no signs of disease; no changes of health status.	no signs of disease; no changes of health status.	no signs of disease; no changes of health status.
Nature, severity and duration of clinical observations (whether reversible or not)	no clinical changes	no clinical changes	no clinical changes	no clinical changes	no clinical changes	no clinical changes
Haematological tests (M) - parameters with statistically significant changes
WBC -Total leucocyte count 10^3/µl	8.75	9.12	9.54	14.32	8.48	10.25	Statistically significantly increased in dose level 750 mg/kg/day.
platelet count 10^6/µl	880.33	895.17	984.33	1035.50	871.83	1039.67	Statistically significantly increased in dose level 750 mg/kg/day. Statistically significantly increased in satellite treated males compared to control.
portion of eosinophiles %	1.89	1.74	2.12	1.14	2.21	1.08	Statistically significantly decreased at the dose level 750 mg/kg/day. Statistically significantly decreased in satellite treated males in comparison with the satellite control group.
portion of monocytes %	9.18	6.66	5.79	6.34	6.32	5.87	Statistically significantly decreased in all treated groups in comparison with the control group.
value of fibrinogen g/l	2.12	2.12	2.24	2.38	1.94	2.01	Statistically significantly increased at the dose levels 250 and 750 mg/kg/day.
MCV - Mean corpuscular volume Fl	51.08	50.80	49.97	50.20	50.97	47.58	Statistically significantly decreased in satellite treated males in comparison with the satellite control group (in a range of historical control).
Haematological tests (F) - parameters with statistically significant changes		increase of value of total leucocyte count (5.38 vs. 3.94 10^3/µl)	increase of value of total leucocyte count (6.47 vs. 3.94 10^3/µl) ; increase of percentual portion of reticulocytes	increase of value of total leucocyte count (5.92 vs 3.94 10^3/µl); increase of percentual portion of reticulocytes		decrease of total erythrocyte count, haemoglobin, haematocrite; portion of eosinophiles.	The mean value of total leucocyte count (14.32) in males at the dose level 750 mg/kg/day was above the upper limit of historical control (historical control parameters: 5.71 – 12.14). The mean value of fibrinogen (2.38) in males at the dose level 750 mg/kg/day was above the upper limit of historical control (historical control parameters 1.42 – 2.20).
WBC -Total leucocyte count 10^3/µl	3.94	5.38	6.47	5.92	5.15	6.42	Statistically significantly increased value in all treated groups.
RBC - Total erythrocyte count 10^6/µl	7.16	7.27	6.71	6.67	7.66	6.91	Statistically significantly decreased in satellite treated females in comparison with the satellite control group (in a range of historical control).
Haemoglobin /100 ml	14.30	14.38	13.63	13.40	14.92	13.60	Statistically significantly decreased in satellite treated females in comparison with the satellite control group (in a range of historical control).
HCT Haematocrite %	39.67	40.22	38.02	37.62	40.70	36.55	Statistically significantly decreased in satellite treated females in comparison with the satellite control group (in a range of historical control).
Eosinophiles %	1.50	1.76	1.03	0.72	1.96	1.30	Statistically significantly decreased in satellite treated females in comparison with the satellite control group.
Reticulocytes %	1.95	2.05	2.90	2.78	2.67	2.28	Statistically significantly increased at the dose levels 250 and 750 mg/kg/day.
Urinalysis (only in 6 M per group) - volume m/l			8.17	6.67	8.50	6.00	Statistically significantly decreased volume at dose levels 80 and 750 mg/kg/day but water consumption was increased. Presence of proteins in 2-3-0-5 males, presence of blood in 0-0-3-3 males and presence of leucocytes in 1-4-4-5 males. Statistically significantly decreased volume in satellite treated males. Presence of proteins in 2-1 males, presence of blood in 1-0 male and presence of leucocytes in 3-6 males.
Clinical biochemistry tests with relevant baseline values - M
T-Pro g/l	66.18	64.87	64.56	62.80	67.17	66.72	Statistically significantly decreased values at the dose level 750 mg/kg/day
ALP µkat/l	1.75	1.46	1.41	1.36	1.62	1.44	Statistically significantly decreased values at the dose level 750 mg/kg/day
AST µkat/l	1.74	1.86	1.84	2.90	1.64	4.15	Statistically significantly increased values at the dose level 750 mg/kg/day and in satellite treated group.
Crea µmol/l	23.95	24.17	28.97	38.27 (above upper limit of historical control)	24.28	31.47	Statistically significantly increased values at the dose level 250 and 750 mg/kg/day and in satellite treated group.
ALB g/l	42.35	42.28	42.02	40.53	43.87	43.42	Statistically significantly decreased values at the dose level 750 mg/kg/day
T-BIL µmol/l	2.70	2.68	2.19	2.08	2.53	3.46	Statistically significantly decreased values at the dose level 250 and 750 mg/kg/day
CHE µkat/l	7.85	7.49	7.23	6.49	7.76	10.36	Statistically significantly increased values in satellite treated group.
BA µmol/l	64.64	58.56	51.19	30.55	50.04	89.57	Statistically significantly decreased values at the dose level 750 mg/kg/day
Clinical biochemistry tests with relevant baseline values - F
T-Chol mmol/l	1.87	2.67	2.53	2.75	2.37	2.58	Statistically significantly increased values at the dose levels 80, 250 and 750 mg/kg/day
TG mmol/l	0.67	3.65 (above upper limit of historical control)	3.22 (above upper limit of historical control)	1.73 (above upper limit of historical control)	0.63	0.84	Statistically significantly increased values at the dose levels 80, 250 and 750 mg/kg/day. In all treated groups above the upper limit of historical control (historical control parameters 0.28 – 1.59).
AST µkat/l	2.00	1.98	2.09	2.74	1.91	2.13	Statistically significantly increased at the dose level 750 mg/kg/day.
GLU mmol/l	4.66	4.26	5.20	5.47	5.87	5.91	Statistically significantly increased value at the dose levels 250 and 750 mg/kg/day.
Ca mmol/l	2.43	2.51	2.48	2.53	2.73	2.81	Statistically significantly increased values in satellite group.
IP mmol/l	2.47	3.28	3.44	3.40	1.97	2.47	Statistically significantly increased values at the dose levels 80, 250 and 750 mg/kg/day and in satellite group (above the upper limit of historical control in satellite treated females (historical control parameters 1.39 – 2.40)).
BA µmol/l	119.21	138.09	189.92	149.23	43.53	93.41	Statistically significantly increased values in satellite group.
K mmol/l	3.90	3.42	3.67	3.90	3.15	3.23	Statistically significantly decreased at the dose level 80 mg/kg/day.
Organ weights - M
Spleen	0.9426	0.9165	0.9074	0.9385	0.9211	1.0258	Statistically significantly increased in satellite treated males in comparison with satellite control males.
Kidneys	3.3336	3.4158	3.3167	3.7963	3.2937	4.1430	Statistically significantly increased in satellite treated males in comparison with satellite control males.
Prostate gland+ Seminal vesicles	4.3703	4.2503	3.6690	3.7418	4.1513	4.1842	Statistically significantly decreased at the dose level 250 mg/kg/day.
Pituitary gland	0.0150	0.0142	0.0128	0.0149	0.0144	0.0166	Statistically significantly increased in satellite treated males in comparison with satellite control males.
Organ weights - F
Adrenal glands	0.0918	0.1058	0.1111	0.0873	0.0963	0.1045	Statistically significantly increased at the dose level 250 mg/kg/day.
Spleen	0.5909	0.6977	0.7690	0.6861	0.5490	0.6803	Statistically significantly increased in satellite treated group.
Kidneys	2.0111	2.0190	2.2853	2.4548	1.9065	2.5539	Statistically significantly increased at the dose levels 250 and 750 mg/kg/day and in satellite treated group.
Organ/body weight ratios - M
Brain	0.4331	0.4437	0.4523	0.4855	0.4259	0.4809	Statistically significantly increased at the dose level 750 mg/kg/day and in satellite treated group.
Kidneys	0.6660	0.6872	0.6964	0.8340	0.6344	0.8902	Statistically significantly increased at the dose level 750 mg/kg/day and in satellite treated group.
Pituitary gland	0.0030	0.0029	0.0027	0.0033	0.0028	0.0036	Statistically significantly increased in satellite treated group.
Thyroid gland	0.0065	0.0066	0.0072	0.0071	0.0064	0.0073	Statistically significantly increased in satellite treated group.
Spleen	0.1866	0.1835	0.1908	0.2058	0.1776	0.2199	Statistically significantly increased in satellite treated group.
Organ/body weight ratios - F
Brain	0.5814	0.6098	0.6371	0.6740	0.7415	0.7788	Statistically significantly increased at the dose level 750 mg/kg/day.
Spleen	0.1753	0.2183	0.2518	0.2388	0.2012	0.2610	Statistically significantly increased in all treated groups and in satellite treated group.
Kidneys	0.5956	0.6314	0.7478	0.8610	0.6984	0.9823	Statistically significantly increased at the dose levels 250 and 750 mg/kg/day and in satellite treated group.
Liver	3.5925	3.8622	4.4445	4.6569	3.0686	3.6705	Statistically significantly increased at the dose levels 250 and 750 mg/kg/day and in satellite treated group.
Thyroid gland	0.0090	0.0099	0.0104	0.0111	0.0115	0.0120	Statistically significantly increased at the dose levels 250 and 750 mg/kg/day.
Necropsy findings - M	no pathological findings in 6/6	no pathological findings in 6/6	light coloured kidneys in 2/6. No pathological findings in 4/6	light coloured kidneys in 6/6.	no pathological findings in 6/6	light coloured kidneys in 5/6. Green-grey body on caput epididymis in 1/6. No pathological findings were recorded in 1/6.
Necropsy findings - F	no pathological findings in 6/6	no pathological findings in 6/6	light coloured kidneys in 5/6. No pathological findings in 1/6	light coloured kidneys in 6/6.	no pathological findings in 6/6	light coloured kidneys in 2/6. No pathological findings in 4/6.
Histopathological findings -M
Number of examined animals	6	6	6	6	6	6
Epididymides: spermatogranuloma	0	/	/	0	0	1
Heart: myocardial scar	1	/	/	0	0	0
Kidneys: hyaline casts	0	1	1	0	0	0
Kidneys: hydronephrosis	0	3	0	2	0	1
Kidneys: tubular necrosis	0	0	2	6	0	6
Prostate gland: focal chron. inflammation	1	/	/	1	2	0
Spleen: extramedullary hemopoiesis	1	/	/	0	2	0
Spleen: siderosis	0	/	/	2	3	2
Stomach: submucosal oedema with chronic inflammation	0	/	/	0	2	1
Testes: atrophy	1	/	/	0	0	0
Trachea: blood aspiration	3	/	/	6	3	3
Histopathological findings -F
Number of examined animals	6	6	6	6	6	6
Kidneys: focal calcification	0	0	1	0	0	0
Kidneys: hyaline casts	0	0	0	0	1	0
Kidneys: tubular necrosis	0	0	3	6	0	2
Kidneys: hydronephrosis	0	1	1	1	0	0
Liver: brown pigment	0	/	/	0	0	1
Liver: focal acute inflammation	0	/	/	0	0	1
Liver: vacuolation	0	/	/	0	0	1
Lymph nodes (cervical): hyperplasia	0	/	/	2	0	0
Mammary gland: lobular hyperplasia	6	/	/	6	0	0
Spleen: siderosis	3	/	/	1	2	4
Stomach: submucosal edema with chronic inflammation	1	/	/	0	0	0
Thymus: cysts	0	/	/	0	2	0
Trachea: blood aspiration	4	/	/	0	0	1
Urinary bladder: focal chronic inflammation	0	/	/	0	0	2
Uterus: focal accumulation of lipophages and siderophages in mesometrium	5	/	/	6	0	0
Uterus: focal squamous cell metaplasia	0	/	/	0	0	1
Uterus: hemosiderin in mucosa	5	/	/	6	0	0
Uterus: hydrometra	0	/	/	0	3	4

Results of DRF study

	control	100	300	1000	notes
Number of pregnant females	4 (non-pregnant and no sperm)	6	6	5
Number of non-pregnant females	2	0	0	0
Mortality	0	0	0	1	died due to intubation error
Health condition and clinical observation	no effects	no effects	no effects	no effects
Body weight	no effects	no effects	no effects	effects	Decreased body weights of pregnant females at the dose level 1000 mg/kg/day during the whole study.
Haematological examination	no effects	no effects	no effects	no effects	WBC (10^3/µl), RBC (10^6/µl), HGB (g/dl), HCT (%), MCV (fl), PLT (10^3/µl)
Pathological examination of females	no effects	no effects	no effects	no effects
Implantations ± SD	17.50 ± 3.00	14.50 ± 4.23	17.83 ± 1.17	16.20 ± 2.39	Slightly reduced at the dose level 100 mg/kg/day.
Resorptions ± SD	0.25 ± 0.50	0.50 ± 0.55	0.67 ± 1.21	0.20 ± 0.45	Slightly increased at the dose levels 100 and 300 mg/kg/day.
Corpora lutea ± SD	17.75 ± 2.75	16.50 ± 1.87	18.33 ± 1.21	16.60 ± 1.52
Total number of live foetuses	69	84	103	80
Total number of dead foetuses	0	0	0	0
Average number of live foetuses ± SD	17.25 ± 2.87	14.00 ± 4.20	17.17 ± 1.47	16.00 ± 2.35	Slightly decreased at the dose level 100 mg/kg/day.
Average number of dead foetuses ± SD	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
Pathological examination of foetuses	no effects	no effects	no effects	no effects	No macroscopic changes of soft tissues and external alteration

Applicant's summary and conclusion

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY in MALES and FEMALES was established as 80 mg/kg body weight/day

Executive summary:

The test item was tested for effects on reproduction and subacute toxicity using the OECD Test Guideline No. 422.

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, after two hours of fasting. The study includes four main groups and two satellite groups of animals. Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females. Main groups contained 3 treated groups (doses 80, 250, 750 mg/kg/day) and one control group (vehicle only). The satellite groups contained one control group (vehicle only) and one treated group (750 mg/kg/day). The dose levels for the study were determined at the requestof the Study Monitor after evaluation of the results of the DRFE.

 

The first six males and six 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 twelve 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 period: males and females – 2 weeks prior to the mating period and during the mating period; pregnant females – during pregnancy and up to the 12^thday of lactation; males – after mating period – 49 days in total;

nonpregnant 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 period. 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 + 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 study

Repeated oral administration of the test item to rats by gavage at the dose levels of 80, 250 and 750 mg/kg/day did not cause any mortality.

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

 

The effect of the test item on the body weight was recorded. Decreased body weight was recorded in males in the middle (250 mg/kg/day), highest (750 mg/kg/day) dose levels and also in satellite treated males. Decreased body weight was observed in females in the middle and highest dose levels during the pregnancy and lactation period.

The decrease of body weightwas marked in the highest dose level in both sexes and can be considered as biologically significant.

 

No effect of the test item on the food consumption was recorded during the study. Slight changes of these parameters were without toxicological importance.

 

No clinical findings revealed influence of the test item on clinical status of treated animals and satellite treated animals were recorded.

 

The statistically significant differences were registered during the haematological examination in red blood components – irreversible increased value of platelet count and delayed decreased value of mean corpuscular volume in males, delayed decreased value of total erythrocyte count, accompanied with decreased values of haemoglobin and haematocrit in satellite treated females.

Haemocoagulation parameters – statistically significantly increased value of fibrinogen in males at the middle and highest dose levels was recorded. 

In white blood components - statistically significantly increased total leucocyte count in males in the highest dose level and in females in all treated groups was recorded. Decreased percentage portion of monocytes in males in all treated group (without dose dependence) and percentage portion of eosinophils in males in the highest dose level and in satellite treated males were observed. 

Statistically significantly increased value of reticulocytes in females in the middle and highest dose levels was recorded (in historical control limits).

The values of total leucocyte count (WBC) and fibrinogen in males were out of the historical control limits.

Increased values of WBC may be related to the organism´s reaction to the test item administration. This effect was reversible, no significant change of value of WBC was observed in satellite treated animals.

 

Biochemical examination showed following statistically significant differences in males: increased value of creatinine (dose-dependent, irreversible) in the middle and highest dose level; in males in the highest dose level this increasing was out of historical control limits, irreversible increased activity of AST, decreased activity of ALP in all treated dose levels, decreased value of bilirubin total in the middle and highest dose levels, decreased value of protein total, albumin and bile acid in the highest dose level and delayed increased value of cholinesterase.  

In females the values of cholesterol total, triglycerides and inorganic phosphorus (dose dependently) were statistically significantly increased in all treated groups. Increased concentration of inorganic phosphorus was recorded also in satellite treated females; this value was out of historical control limits. Statistically significantly increased value of glucose in middle and highest dose levels was detected (dose dependently). Activity of AST was statistically significantly increased in females in the highest dose level. Concentration of potassium ions in females in the lowest dose level (80 mg/kg/day) was statistically significantly decreased. Delayed increasing of values of calcium ions and bile acid were detected in satellite treated females.  

Irreversible increased concentration of creatinine in males and irreversible increased concentration of inorganic phosphorus in femalescan be considered as biologically significant and can be related to the histopathological findings of kidneys (tubular necrosis), that were found out during the histopathological examination.

 

The water consumption in satellite treated animals was increased during the application period.

The examination of urine parameters in males showed statistically significantly decreased urine volume in lowest and highest dose levels and in satellite treated males.

More frequent presence of protein in highest dose level and leucocyte in all treated groups were recorded. The presence of blood was recorded in males of middle and highest dose levels.[ml1] 

Changed urine parameterscan be related to histopathological findings of kidneys that were found out during the histopathological examination.

 

Biometry of organs showed statistically significantly delayed increased absolute weight of spleen in both sexes, statistically significantly increasedabsolute weight of kidneys in females at the middle and highest dose levels and in satellite treated animals of both sexes.

Relative weight of kidneys was increased in males in the highest dose level and in satellite treated males and also in females in the middle, highest dose levels and in satellite treated females. This increasing of kidneys was irreversible. In spleen, relative weight was irreversibly statistically significantly increased in satellite treated males and dose-dependently, irreversibly in females atall treated groups. Relative weight of liver was increased in females in the middle and highest dose levels (irreversibly).

Irreversible increased relative weight of kidneys in males and femalescan be related to histopathological findings of kidneys that were found out during the histopathological examination.

 

Histopathological examination showed minimal to marked signs of tubular necrosis in kidneys in two males in the middle dose level, six males in the highest dose level and in six males in the satellite treated males. The same finding was found out in three females in the middle dose level, six females in the highest dose level and two satellite treated females. 

This lesion was histologically characterized by variety of findings: dilatation of cortical tubules lined by flattened epithelium, sometimes was epithelium of basophilic colour as a sign of reparation. Further, amorphous eosinophilic material or cellular debris were present in the lumen of tubules, and in some cases chronic interstitial inflammation was found. This basic pattern was accompanied also by tubular vacuolation and presence of hyaline casts.

Histological examination of kidneys was expanded in the case of macroscopic findings in kidneys (very light colour) of test animals. Totally the tubular necrosis on kidneys was recorded in four males in the middle, twelve males in the highest dose level, in seven females in the middle dose levels and eight females in the highest dose level.

The test item, administered orally to rats caused minimal to marked tubular necrosis in the kidneys in rats in highest dose level (750 mg/kg/day). This lesion was subsequently revealed in some rats from the middle dose groups (respective 250 mg/kg). This lesion was irreversible during recovery period.

 

Conclusions

The NOAEL (No Observed Adverse Effect Level) for REPEATED DOSE TOXICITY in MALES and FEMALES was established as 80 mg/kg body weight/day. The NOAEL value is established on the base of overall decrease in body weight, changes in biometry of organs (increase of relative weight of kidneys, liver and spleen) and especially on the irreversible damage of kidneys confirmed by the histological examination and changes in biochemical parameters related to the kidney function observed in animals in the middle and highest dose levels