Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 22, 2019 to February 14, 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

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

Constituent 1
Chemical structure
Reference substance name:
Disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate
EC Number:
224-073-5
EC Name:
Disodium 4,4'-bis[6-anilino-[4-[bis(2-hydroxyethyl)amino]-1,3,5-triazin-2-yl]amino]stilbene-2,2'-disulphonate
Cas Number:
4193-55-9
Molecular formula:
C40H42N12Na2O10S2
IUPAC Name:
disodium 5-({4-[bis(2-hydroxyethyl)amino]-6-(phenylamino)-1,3,5-triazin-2-yl}amino)-2-{2-[4-({4-[bis(2-hydroxyethyl)amino]-6-(phenylamino)-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]ethenyl}benzene-1-sulfonate
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Remarks:
SPF quality
Details on species / strain selection:
Laboratory rat has been chosen because our testing laboratory has long experience with this species and because rat is recommended according to the test guideline
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.,
- Age at study initiation: males, females: sexually adult; males - 8 weeks on arrival, females – 10 weeks on arrival
- Fasting period before study: animals were without feed two hours before application
- Housing: SPF conditions according to internal SOP No.12
- Diet (e.g. ad libitum): maintenance pelleted diet for rats and mice - Altromin for rats/mice ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 13 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:
water for injection
Details on 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 one hour after preparation of application form. The stirring of solutions continued during administration.

Details on mating procedure:
During the acclimatisation period the health condition of all animals was controlled daily. Normal course of the oestrus cycle of all females was controlled during 14 days before start of application.
Females with abnormal oestrus cycle were removed from mating. Then the animals were randomly divided into the control and test groups and they were marked individually.
Animals were mated from the 29th day of study. Mating 1:1 (one male to one female) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears.
Day 0 of pregnancy was defined as the day the sperms were found.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and the homogeneity of application form were determined in CETA analytical laboratories (Analytical Group I).

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 with wider end of glass Pasteur pipette into a 150mL glass beaker calibrated to 100 mL and the beaker was slowly replenished by the vehicle to mark. 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 unstability.
Concentration Level 1000 mg/10 mL
Ca 10.0 g of the test item was weighed with laboratory spoon 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 390 rpm for 10 min.
The beaker with test item was during dissolving and homogenisation covered by aluminium foil due to possible light unstability.

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 5 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 390 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 unstability 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 5 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 10 minutes of mixing by magnetic stirrer (390 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
It follows from the results of analyses (homogeneity and stability) that both application forms (10 mg and 1000 mg /10 mL) of the test item, 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.
The beaker with test item was during dissolving, homogenisation and stability measuring covered by alluminium foil due to possible light unstability of test item.This measure was recommended for further test item application form preparation
Duration of treatment / exposure:
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:
7 days per week at the same time (7.00 – 10.00 am)
Doses / concentrationsopen allclose all
Dose / conc.:
80 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results obtained in the Dose Range Finding Experiment (DRFE) the doses for the main study were selected
- Rationale for animal assignment (if not random): random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex should not exceed ± 20% of the mean weight
- Fasting period before blood sampling for clinical biochemistry: the animals were fasted approximately for 18 hours before blood collection but they were supplied drinking water ad libitum.

Examinations

Parental animals: Observations and examinations:
Health condition control: daily - during the acclimatisation and the experimental part

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

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

Water consumption: satellite males and females – twice a week

Clinical observations: males and females - daily during the administration period

Mortality control: twice daily

Detailed clinical observation: before the first application and then weekly (except the mating period)

Functional observation: at the end of administration/observation period

Laboratory examinations:
- vaginal smears: daily – 1st – 14th day of study
daily in mating period (max. 14 days)
on necropsy day
- urinalysis: the last day of administration/observation period – only males
- haematology: males – after the end of application period
parental females – on the 13th day of lactation
satellite animals – after the end of observation period
- biochemistry: males and nonpregnant females – after the end of application period
parental females – on the 13th day of lactation
satellite animals – after the end of observation period
- pathological examination: males and nonpregnant females – after the end of application period
satellite animals – after the end of observation period
- weight of organs: during necropsy
- sperm observation: parental males – during and after necropsy (not performed in satellite males)
- histopathological examination: after necropsy
Oestrous cyclicity (parental animals):
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. Vaginal smears were prepared and examined according to internal SOP No. M/74.
Sperm parameters (parental animals):
Observation of Sperm
In all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology. Sperm specimens were prepared and examined according to internal SOP No. M/74 and M/82.

Sperm Motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 - fast progressive motility, 2 - slow progressive motility, 3 - no progressive motility, 4 - non-motile sperm.

Sperm Morphology
Sperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination. All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, and abnormal form of neck were recorded.

The absolute weights of testes, prostate gland and seminal vesicles were recorded for Repeated Dose Toxicity part of study (6 males from each group + satellite groups); testes, prostate gland and seminal vesicles for Reproduction part of study (all animals).
Litter observations:
Body weight: pups (litters) - 1st, 4th day, 12th day and 13th day
pups – individually – 4th day of lactation
Clinical observations: pups - as soon as possible after delivery and then daily
Laboratory examinations:
- biochemistry: 2 pups per litter - on the 4th day of lactation
- anogenital distance measurement: pups – 4th day of lactation
- pathological examination: 2 pups per litter - on the 4th day of lactation
Postmortem examinations (parental animals):
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 x 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.
Postmortem examinations (offspring):
Blood samples from the day 13 pups (pooled sample, one male + one female pup) were assessed for serum levels of thyroid hormone thyroxine (T4 total) and rat thyroid stimulating hormone (TSH) by ELISA kit (Biovendor, Brno, Czech Republic).
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.

In general:
The parametric tests were used for statistical evaluation of
• body weight of males and females
• mean pup weight
• litter weight
• anogenital distance of pups
• 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 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 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).
Reproductive indices:
See table 7 in " Any other information on materials and methods incl. tables"
Offspring viability indices:
See table 7 in " Any other information on materials and methods incl. tables"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
The clinical observation results were noted for all 12 animals per group and satellite animals.
Males
No clinical changes were recorded in control or treated males during the application period.
Satellite males
No clinical changes were recorded in satellite control or satellite treated males during the application period.
Females
In all control and treated females no clinical changes were recorded during the entire study.
Satellite females
No clinical changes were recorded in satellite control or satellite treated females during the application period.

Health Condition Control
The health condition control results were noted for all 12 animals per group and satellite animals.
The males and females were not distributed into groups during the check-in, acclimatisation and the pre-exposure period with monitoring of oestrous cycle in females. No signs of diseases were recorded during that time.
Males
No signs of disease and clinical signs of intoxication were recorded during the application period in all treated males.
Satellite males
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 males.
Females
No signs of disease and clinical signs of intoxication were recorded during the application period in all treated females.
Satellite females
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 males.


Mortality:
mortality observed, non-treatment-related
Description (incidence):
Males
Male No. 46 (the dose level 250 mg/kg/day) was found dead on day 30 of application. Due to partial cannibalism, the cause of death was not established.

Females
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Males
In the beginning of study, mean body weight of all groups of animals was similar. Slightly higher body weight was recorded in animals at the dose levels 80 and 250 mg/kg/day in comparison with the control group during the whole application period. Comparable body weight was recorded in animals at the dose level 750 mg/kg/day and control group. Statistically significant differences in necropsy body weight were not found in treated males.
Satellite males
Body weights of satellite treated males were slightly lower in comparison with the satellite control males during the whole study. Statistically significant differences in necropsy body weight were not found in satellite treated males.
Females
Different body weight on the 1st day of administration was caused by sequential inclusion of animal groups to the study. Before the mating period, body weights of females in all treated groups was similar to the control group. During the pregnancy and lactation periods, body weights of treated females in all groups was comparable with the control females.
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 not adversely influenced by the test item treatment.
Satellite males
Weight increments of satellite treated males in application and recovery period were variable and not adversely influenced by the test item administration.
Females
Weight increments in treated females were variable in comparison with the control females and not affected by the test item treatment.
Satellite females
Weight increments in treated females were variable and not affected by the test item treatment.
Pregnancy
Mean body weight of all treated groups was similar with controls.
Weight increments of treated females was comparable with the control females
Lactation
Only mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period. Mean body weight of females at the dose level 750 mg/kg/day was insignificantly decreased on day 4 and 12 of lactation in comparison with the control females.
Weight increments of treated mothers at tall dose levels were lower at the end of lactation period in comparison with the control 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 or slightly higher 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 application period and slightly lower during the recovery period (8th – 9th week).
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 or slightly higher in comparison with the control group during the entire application and recovery periods.
Description (incidence and severity):
Males
The food conversion of treated males was comparable with the control males in the pre-mating period and not negatively influenced by the test item treatment.During the period after mating, the food conversion of males in the treated group was similar with the control group of males (except the conversion at the dose level 250 mg/kg/day at the 6th week of study).
Satellite males
The food conversion of satellite treated males was quite similar to the satellite control (exc. the 9th and the 9th week of study).
Females
The food conversion of treated females in the pre-mating period was variable but not adversely influenced by the test item treatment.
During the pregnancy period, the food conversion of treated females was similar with the control group.
During the lactation period, the food conversion of treated females at all dose levels was decreased in comparison with the control females.
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.


Description (incidence and severity):
Satellite males
The water consumption of satellite treated males was lower compared to the satellite control group during the entire application and recovery period.
Satellite females
The water consumption of satellite treated females was higher compared to the satellite control group during the entire application and recovery period.
Ophthalmological findings:
not examined
Description (incidence and severity):
Males
The value of the total leucocyte count was not affected by the test item treatment. The five-population differential of white blood cells were not affected by the test item treatment. The count of lymphocytes, monocytes, neutrophils, eosinophils and basophiles were similar in treated group of males and control group of males.
The red blood components were changed mainly in males at the dose level 80 mg/kg /day. An increase in the total value of erythrocytes (RBC) (p ≤ 0.05) was detected. This increase is related to the increased concentration of haemoglobin (p ≤ 0.05, out of historical control range) and value of haematocrit (p ≤ 0.05). There was an increase in the percentage value of reticulocytes in males at the dose level 750 mg/kg – even out of historical control range, but not statistically significant. The values of haemocoagulation parameters were not significantly affected by the test item treatment.
Values over the historical range (HGB concentration in the lowest dose level and reticulocytes in the highest dose level) were isolated values very close to the upper limit, without dose-dependent and biological significance. These findings were reversible (not observed in satellite animals).
Satellite males
The APTT value recorded in satellite treated males was statistically significantly decreased in comparison with the satellite control group of males. Other values were similar with satellite control males. All values were in a range of historical control.
Females
The value of the total leucocyte count was not affected by the test item treatment. The five-population differential of white blood cells were not affected by the test item treatment except the value of monocytes. The percentage value of monocytes was significantly decreased (p ≤ 0.05) in females at the dose level 750 mg/kg /day compared to control females.
The red blood components in treated females were not changed in comparison with the control females except significantly increased value of MCV (p ≤ 0.05) at the dose level 250 mg/kg/day.
Haematocoagulation parameter were not affected by the test item treatment.
The value over the historical range (MCV in the middle dose level) was isolated value close to the upper limit without dose dependence.
Satellite females
A decreased value of RBC (p ≤ 0.05) and associated increased value of MCV (p ≤ 0.05) were recorded in satellite treated females in comparison with the satellite control females. Decreased value of neutrophils (p ≤ 0.05) was also recorded in satellite treated females.
Other values were similar with satellite control females and in a range of historical control.

Description (incidence and severity):
Males
Significantly changed values (p ≤ 0.05) of T-Pro (increased), Crea (decreased, under the historical control range), T-Bil (decreased), Na (increased) and Cl (increased) were detected in males at the dose level 750 mg/kg/day compared to control animals. The changed values (p ≤ 0.05) of T-Bil (decreased) and Na (increased) were also recorded in males at the dose level 250 mg/kg/day. In males at the dose level 80 mg/kg/day was detected changed values (p ≤ 0.05) of T-Bil (decreased) and Cl (increased).
The value altered at all dose levels (statistically significantly) was bilirubin (T-Bil) - decreased at all treated levels compared to control animals.
The value of creatinine recorded under the historical control range was very close to the lower limit, without dose dependency and reversible change (not observed in satellite animals)
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
A statistically significant increased value of bile acid (BA) only was 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
Significantly changed biochemical values were recorded only sporadically in treated females in comparison with the control females.
Significantly (p ≤ 0.05, over the historical control range) increased value of TG only was reported in females at the dose level 750 mg/kg/day. Significantly (p ≤ 0.05) decreased value of GLU was recorded in females at the dose level 80 mg/kg/day only.
The value of triglyceride recorded over the historical control range was very close to the upper limit, without dose dependency and reversible change (not observed in satellite animals).
Values of other biochemical parameters of treated females were comparable to the control group.

Satellite females
Statistically significantly (p ≤ 0.05) increased values of Ca and BA were noted in satellite treated females in comparison with the satellite control females. Values of other biochemical parameters of treated satellite females were in a historical control range and comparable to the control group.

Description (incidence and severity):
Urinalysis was performed only in males (six of each group) during the last week of the study. Statistical evaluation was performed for pH and volume of urine.
Males
A statistically significantly increased pH of urine was detected in males at the dose level 750 mg/kg/day. The volume of urine was insignificantly decreased in males at the dose level 750 mg/kg/day. The presence of proteins was recorded in treated males as well as in control males. The presence of blood and leucocytes were recorded in males at the dose levels 80 and 250 mg/kg/day.
Satellite males
The presence of leucocytes was recorded in satellite treated males as well as in satellite control males.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Males
Reactions to touch, noise, pain and pupillary reflex of treated males were the same as in the control group. The number of upstanding in treated males was comparable 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 treated females was similar 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
Description (incidence and severity):
Full histopathology of the preserved organs and tissues was performed for high dose and control animals and satellite animals.
Histopathological examination of the preserved organs and tissues was performed for males Nos. 1-6 and the first six mothers that delivered pups in the repeated dose toxicity part of the study.
Because no treatment related histological findings were recorded in animals at the highest dose level (750 mg/kg/day), histological examination of organs was not performed for the animals from the dose level 80 and 250 mg/kg/day.
The incidence of affected males is expressed in numeric form and ranged in sequence of dose levels 0-750 mg/kg /day and 0S-750S in satellite groups.

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).
Only sporadic findings were recorded in the control group of males as well as in dosed group of males.
Hydronephrosis in kidneys was detected in 1-0 males. Focal chronic inflammation in liver was recorded in 0-1 male, slight focal chronic inflammation of prostate gland in 1-1 male and extramedullary hemopoiesis in spleen in 0-1 male.
The incidence of other microscopical findings was very sporadic and these findings are mentioned in individual tables
No treatment-related changes were found in male genital tract.

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.
Only sporadic findings were recorded.
Hydronephrosis in kidneys in 1-1 males, extramedullary hemopoiesis in spleen in 1-2 male, tubular atrophy in testes in 0-1 male were recorded. The incidence of other microscopical findings was very sporadic and these findings are mentioned in individual tables

Test item orally administered to male rats in the dose of 750 mg/kg/day did not cause any pathological changes in the male genital tract organs, in the pituitary gland, did not cause gross or histopathological changes in the kidneys of rats indicative of a toxic effect and did not cause gross or histopathological changes in the gastrointestinal tract.

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. 103,104,105,106,107,108) and high dose group (Nos. 161,165,166,167,168,169).
In one female from the dose level 250 mg/kg/day (No.150) was examined uterus (macroscopical finding – dilatation). Marked hydrometra was detected.

The incidence of affected females is expressed in numeric form and ranged in sequence of dose levels 0-750 mg/kg /day and 0S-750S in satellite groups.
Only sporadic findings were recorded in the control group of females as well as in dosed group of females.
Hydronephrosis in kidneys was detected in 1-3 females.
The changes related to pregnancy were found in both control and high treated females: accumulation of siderophages in mesometrium of uterus in 6-6 females, hemosiderin in mucosa in 6-6 females and lobular hyperplasia of mammary gland in 6-6 females. The incidence of other microscopical findings was very sporadic and these findings are mentioned in individual tables

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.

Hyaline casts in kidneys in 0-1 female and hydropherosis in kidneys in 0-1 female were detected. Hydrometra of iterus was observed in 2-3 females. The incidence of other microscopical findings was very sporadic and these findings are mentioned in individual tables.

Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Neoplastic tissues were not observed during the histopathology
Other effects:
no effects observed
Description (incidence and severity):
Biochemical Examination – Thyroid hormones
Blood (serum) samples from all adult parental males were assessed for thyroid hormones thyroxine (T4 total) and rat thyroid stimulating hormone (TSH).
Mean concentrations of T4 and TSH hormones at all dosed groups were not significantly changed in comparison with the control group of males.

.


Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Assessment of Oestrous Cycle
Oestrous cycles were monitored before treatment started to select females for the study with regular cyclicity. Vaginal smears of all females were monitored daily for two weeks. Two females were placed into the satellite group for irregular oestrus cycle.

Pre-mating period
Different body weights on the 1st day of administration was caused by sequential inclusion of animal groups to the study. Body weights of treated females were comparable with the control group of females. Weight increments were variable but not affected by the test item treatment.

Examination of absolute weight of reproductive organs
Absolute weight of ovaries and uterus were not affected by the test item treatment. Insignificant decreasing of absolute weight of pituitary gland was recorded in females at the dose level 250 and 750 mg/kg/day and in thyroid gland in females at the dose level 80 and 750 mg/kg/day.
Examination of relative weight of reproductive organs – the relative weight of uterus was increased in group of females at the dose levels 250 and 750 mg/kg/day compared to the control females but it was not statistically significant. The relative weight of the pituitary gland of treated females was insignificantly decreased in females at the dose level 250 mg/kg/day.
No statistically significant differences in absolute and relative weight of organs were recorded when comparing the treated females with the control females.

Histopathological Examination
For examination of the reproduction toxicity of the test item, microscopic examination of the reproductive organs, pituitary and thyroid gland was performed for high dose females (Nos. 161-172) and control females (Nos. 101-112).
As treatment-related changes at the highest dose level in reproductive organs were not found, histopathological examination of macroscopically changed organs only was performed. Macroscopical changes on the uterus (dialatation) was recorded only in female No. 150. Histologically was detected hydrometra of uterus.
The incidence of affected females is expressed in numeric form and ranges in sequence for the dose levels 0-750 mg/kg/day.
Microscopic examination of reproductive organs, thyroid gland and the pituitary gland did not reveal the presence of treatment-related changes. The changes related to pregnancy were found in both control and treated females: accumulation of lipophages and siderophages in mesometrium in 10-10 females and hemosiderin in mucosa of uterus in 10-11 females. Lobular hyperplasia of mammary glands was recorded in 6-6 females. Hydrometra of uterus (non-pathological finding related to the oestrous cycle) occurred in 1-0 females.
The vagina of both control and administered females was without pathological findings except for mild purulent inflammation in one control female.
Ovaries of all control and high dose females were without pathological lesions. No treatment-related changes were found in female genital tract.
Pituitary gland of both control and treated rats was without any pathological lesions.

The incidence of other microscopical findings was very sporadic and unrelated to treatment.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
No statistically significant differences in absolute and/or relative weight of organs of treated animals was recorded.
Absolute weight of reproductive organs – testes, epididymis and prostate gland with seminal vesicles in dosed males was comparable with the control males. Absolute weight of pituitary and thyroid gland of administered animals were quit well-balanced with the control animals.
Relative weight of reproductive organs – testes, epididymis and prostate gland with seminal of treated groups of males was comparable to the control males. The relative weight of the pituitary gland and thyroid gland of treated males was similar to the control males.

Observation of Sperm
A comparison of sperm motility in the control males and males from treated groups did not show differences. The test item treatment did not affect sperm morphology. Male rats ability to produce sperm that can fertilise eggs was not affected by the test item administration.

Hystopathology - reproductive organs
For examination of the reproduction toxicity of the test item, microscopic examination with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure was performed for all males of control (No 1-12) and high dose (No 61-72).
Microscopical examination of reproductive organs did not reveal presence of treatment-related changes (therefore examination of reproductive organs was performed only in control and high dosed males); only spontaneous changes were noted in males. In the testes, the following microscopic change was recorded: tubular atrophy in 1-0 males, spermatogranuloma in 1-0 males. In the prostate gland of 2-1 males, chronic inflammation was observed. Findings in epididymides were recorded only in control males. Azoospermia in 1-0 male and focal chronic inflammation in 2-0 males were detected.
No treatment-related changes were found in male genital tract.


Reproductive performance:
no effects observed
Description (incidence and severity):
Reproduction Parameters
Evidence of copulation was found out in all females. A decreased number of females achieving pregnancy was recorded at the control dose level as well as at the dose level 750 mg/kg/day. No abortions were recorded. The mean duration of pregnancy was similar in treated and control groups. The mean number of implantations was comparable in groups of treated females with the control females.
The mean number of live pups at the first litter check after parturition was slightly decreased in females at the dose level 80 mg/kg/day; in females at the dose levels 250 and 750 was the mean number of pups comparable with the control group of females.
The stillborn were not found at any group of treated females as well as in females at the control group.
The mean weights of litters of treated groups at birth, at PND 4 (postnatal day) and PND 13 were comparable with the weights of control group. The measurement of the AGD (corrected) in pups showed no difference between males and females of control and treated groups and was similar in all groups.
Macroscopic examination did not reveal pathological findings caused by the test item application.
Fertility Parameters
The values of mating indexes showed that mating was not negatively affected by the test item treatment. Fertility indexes were the same in control and high dosed groups. The gestation index was the same in the control and all treated groups; the viability index on PND 4 was comparable in treated and control group.
Post-implantation losses were the highest in females from the group 80 mg/kg/day.
Post-natal losses were similar in females from control group and group 750 mg/kg/day.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The clinical observation of pups was made daily during the study. The macroscopic examination was performed on all surviving pups and/or pups found dead. The examination could not be performed on pups that disappeared due to cannibalism and/or in dead pups with autolysed organs.
Control: 157 of 159 born pups were examined.
Female No. 103: one pup of the seventeen pups were found dead on the second day after delivery (autolysis of organs was recorded).
Female No. 105: one pup of fourteen disappeared because of cannibalism.
No macroscopical findings were recorded in other pups/litters.

80 mg/kg /day: 173 of 174 born pups were examined.
Female No. 128: one pup of fourteen disappeared because of cannibalism.
No macroscopical findings were recorded in other pups/litters.

250 mg/kg /day: 169 of 169 born pups were examined.
No macroscopical findings were recorded in pups/litters.

750 mg/kg /day: 154 of 157 born pups were examined.
Female No. 163: one pup of fourteen disappeared because of cannibalism.
Female No. 166: one pup of fourteen disappeared because of cannibalism.
Female No. 167: one pup of seventeen disappeared because of cannibalism.
Female No. 169: one male pup (live) without tail was recorded in litter of eighteen pups.
No macroscopical findings were recorded in other pups/litters.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
see "clinical signs"
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Statistically significant differences were recorded. The mean body weight of litters and pups was comparable to the control group in all treatment groups at all examination intervals.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Biochemical Examination – Thyroid hormones
Blood samples from the 13-day old pups (pooled sample; one male and one female pup) were assessed for serum levels of thyroid hormone thyroxine (T4) and rat thyroid stimulating hormone (TSH). No statistically significant differences were recorded in the mean concentration of hormone T4 and TSH in pups from treated groups against control pups.
Description (incidence and severity):
Thyroid Gland Wight
The weight of the thyroid gland in female pups of the treated group of mothers was statistically significantly decreased in female pups in comparison with the control group of mothers.
Histopathological findings:
no effects observed
Description (incidence and severity):
Thyroid Gland - Histopathological Examination
Histopathological examination of thyroid glands of pups of females from the high dose group did not reveal any pathological finding.
Description (incidence and severity):
Number of pups
The total number of live pups at the first litter check after parturition, on the 4th day and 13th day of lactation was comparable in females of control group and females of the dose level 750 mg/kg/day; total number of pups was slightly increased in females at the dose level 80 and 250 mg/kg/day in comparison with the control group.
No stillborn pups were recorded in any group of females.
The mean number of live pups at the first litter check after parturition was slightly decreased in females at the dose level 80 mg/kg/day; in females at the dose levels 250 and 750 was the mean number of pups comparable with the control group of females.


Development of Pups
No differences in the postnatal development of pups were observed in the control and treated groups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL (No Observed Adverse Effect Level) value for REPRODUCTION and DEVELOPMENT was established as 750 mg/kg body weight/day.
Executive summary:

The test item ,was tested for effects on reproduction and subacute toxicity according to 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 water for injection. 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 four main groups (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)). The dose levels for the study were determined on the basis of the results of dose-range finding experiments. Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females.

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 periods: males and females – 2 weeks prior to the mating period and during the mating period; pregnant females – during pregnancy and up to the 12thday 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 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 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 (except one male from the dose level 250 mg/kg/day – male No.46, who was found dead on day 30 of application; the cause of death was not determined due to partial autolysis of organs). This death was accidental and not treatment-related.

All twelve males per group (Nos. 1-12, 21-32, 41-52, 61-72) and females (101-112, 121-132, 141-152, 161-172) were examined from the main groups.

Test item treatment did not affect male ability to produce sperm that can fertilise eggs and ability of females to become pregnant. 

No adverse effect of the test item treatment was observed during examination of thyroxine and rat thyroid stimulating hormone blood concentration in males. 

The body weight of parental males and females was not affected by the test item administration.

Evaluation of the absolute and relative weight of reproductive organs of male and female, as well as the weight of pituitary and thyroid gland, did not reveal any statistically significant differences in organ weight.

Histological examination did not reveal negative effect of the test item on collected organs of reproductive organs (+ pituitary and thyroid gland). Spermatogenesis in the testes of the high dose administered males was without any pathological findings. Epididymides of both control and high dose males were without any pathological findings. Sporadic findings were of spontaneous origin. The administration of test item did not cause pathological changes in the organs.Examination of sperm motility and morphology in treated parental males did not show any differences in comparison with the control males.The administration of test item did not cause damage of sperms.

The number of implantations was comparable between treated and control groups.

The test item did not affect the number of pups and their development.

The total number of pups and their mean body weights at first litter check after parturition and during the next intervals were comparable in treated groups of females to the control group of females. Macroscopical examination of pups did not show negative effect of the test item, administration on development of pups. 

Corrected anogenital distance in treated pups was comparable to the control pups. 

No adverse effect of the test item treatment was observed during examination of thyroxine and rat thyroid stimulating hormone blood concentration in pups.

 

Conclusions

According to the study results the NOAEL(No Observed Adverse Effect Level) value for REPRODUCTION and DEVELOPMENT was established as 750mg/kg body weight/day. No biologically significant changes were observed.