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Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From February 26 to November 30, 2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study was run according to OECD Guideline 422. Information on foetuses development recorded during the study are reported as preliminary information on developmental toxicity
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 26 to November 30, 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
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: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): relative humidity 30-70 %
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Aqua pro iniectione
Details on exposure:
- Concentration in vehicle: 150 mg, 300 mg and 600 mg in 10 ml
Details on mating procedure:
- M/F ratio per cage: 1 to 1
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually
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 beaker 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 unstability 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 350 rpm for 10 min.
The beaker with test item was during dissolving and homogenisation covered by aluminium foil due to possible light unstability of test item.

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 350 rpm.

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 (350 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 the both application forms (10 mg and 1000 mg /10 mL) of the test item, FAT 65037/G TE, 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 aluminium foil due to possible light unstability of test item.
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
Details on study schedule:
Not applicable
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control of the basic group and satellite
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
In 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 vehicle
Details on study design:
- Dose selection rationale: The doses have been selected based on the results of the DRFE
- 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
Parental animals: Observations and examinations:
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

- Mortality control: twice daily


Oestrous cyclicity (parental animals):
Vaginal smears were made from the 1st till the 14th day of study (pre-exposure period) for monitoring of oestrous cycle of females. 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.
Sperm parameters (parental animals):
In all males (except the satellite group) the following sperm parameters were examined: sperm motility and sperm morphology.

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.
Litter observations:
All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 1 post-partum) and on the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.

Anogenital Distance (AGD) Measurement
The AGD of each pup was measured on day 4 of lactation. For measuring digital calliper was used. The AGD was normalised to a measure of pup size. Corrected AGD was calculated according to the formula: AGD divided by the cube root of body weight.

Nipples Examination
The presence and number of nipples in male pups were counted on day 13 of lactation. The presence of nipples in male pup on day 13 of lactation is undesirable (non-physiological).
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.
SACRIFICE
At the end of study, the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

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 (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.
Full histopathology of the preserved reproductive organs and tissues was performed for all high dose and control animals.
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 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:
Male mating index = (number of males with confirmed mating / number of males cohabited) x 100
Female mating index = (number of sperm-positive females / number of females cohabited) x 100
Male fertility index = (number of males impregnating a female / number of males cohabited) x 100
Female fertility index = (number of pregnant females / number of sperm-positive females ) x 100
Gestation index = (number of females with live born pups / number of pregnant females) x 100
Offspring viability indices:
number of pups surviving on PND4 x 100 / number of pups born alive
Clinical signs:
no effects observed
Description (incidence and severity):
Males
No signs of disease, clinical changes and clinical signs of intoxication were recorded during the application period in all treated males. One male from the group of 150 mg/kg/day had overgrown teeth which were adjusted during the study (as needed).

Satellite males
No signs of disease, clinical changes 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, clinical changes and clinical signs of intoxication were recorded during the application period in treated females.
Female No.124 of the dose level 150 mg/kg/daywas found dead on day 25 of application (this female was in a group designated for the reproduction part of study). Piloerection and apathy were recorded during the observation a day before the death. The cause of death was unknown.

Satellite females
No signs of disease, clinical changes and clinical signs of intoxication were recorded during the application period in all treated females. During the observation period (recovery; weeks 8 and 9) no changes of health status were noted in satellite treated females.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Males
There were no unscheduled deaths during the study.

Females
Female No.124 of the dose level 150 mg/kg/day was found dead on day 25 of application There were no other unscheduled deaths during the study.
Description (incidence and severity):
Males
At the beginning of study, mean body weight of all groups of animals was similar. Slightly lower body weight was recorded in animals at the dose level 600 mg/kg/day in comparison with the control group during the whole application period. Statistically significant differences in necropsy body weight were not found in treated males.

Satellite males
Body weights of satellite treated males were 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
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 slightly lower in comparison with the control group from the second week of administration. 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.


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 and recovery period.

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

Satellite females
The food consumption of satellite treated females was similar in comparison with the control group during the entire application and recovery periods.
Food efficiency:
no effects observed
Description (incidence and severity):
Food Conversion

Males
The food conversion of treated males was comparable with the control males in the pre-mating period and variable but not negatively influenced by the test item treatment during the period after mating.

Satellite males
The food conversion of satellite treated males was similar or slightly lower compared to the satellite control.

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 similar 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 slightly lower compared to the satellite control group till the 6th week of study, then the water consumption of treated satellite animals was similar to the control animals.

Satellite females
The water consumption of satellite treated males was similar or higher compared to the satellite control group during the entire application and recovery period.
Description (incidence and severity):
Males
Changes in the values of white blood components were recorded in treated groups of males. The WBC value of treated groups of males was comparable to the control group and was not affected by the test item treatment. Some differences were observed in the five-population differential of white blood cells. Statistically significantly increased value of lymphocytes (p ≤ 0.05) and decreased values of neutrophils (p ≤ 0.05) were recorded in dose groups 150 and 300 mg/kg/day. Statistically significantly decreased values of eosinophils (p ≤ 0.05) was recorded in dose groups 300 mg/kg/day.
The red blood components were not affected by the test item treatment. The values of RBC, Hgb, Hct, MCV and Plt were similar in treated groups of males in comparison with the control group of males.
The values of haemocoagulation parameters were not significantly affected by the test item treatment except the value of fibrinogen concentration (p ≤ 0.05) in males of the dose level 600 mg/kg/day. This value was significantly increased in comparison with the control group of males.
All values were in a historical control range, without dose-dependency and reversible (not observed in satellite animals).

Satellite males
No significantly changed parameters were recorded except the value of reticulocytes in satellite treated males.
All values were in a range of historical control.

Females
The white blood components were not affected by the test item treatment. No significantly changed values of total leucocyte count (WBC) and five-population differential of white blood cells in treated groups of females were recorded in comparison with the control group.
The changes in values of red blood components were recorded in treated groups of females. Statistically significantly increased value of RBC (p ≤ 0.05) associated with increased value of Hct (p ≤ 0.05) and decreased MCV (p ≤ 0.05) were recorded in females at the dose level 600 mg/kg/day in comparison with the control group of females.
The values of haemocoagulation parameters were not significantly affected by the test item treatment.
All values were in a historical control range.

Satellite females
A decreased value of RBC (p ≤ 0.05) and associated decreased value of Hct (p ≤ 0.05) and Hgb (p ≤ 0.05) were recorded in satellite treated females in comparison with the satellite control 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) as ALT and BUN – decreased in all dosed groups of males, GLU decreased in males at the dose level 300 and 600 mg/kg/day and Ca in males at the dose level 150 and 300 mg/kg/day were recorded in comparison with the control group of males. Significantly changed value (p ≤ 0.05) of Crea – increased in males at the dose level 600 mg/kg/day was recorded.
Decreasing of ALT value was irreversible (recorded also in satellite treated group).
Increased value of Crea in high dosed males persisted in satellite treated group of males.
Values of other biochemical parameters of treated males were not significantly altered in comparison with the controls. All values are in a historical control range.


Satellite males
A statistically significant increased value (p ≤ 0.05) of Crea and Cl were recorded in satellite treated males compared to control males. Significantly decreased value (p ≤ 0.05) of ALT and CHE were recorded in satellite treated males.
Values of other biochemical parameters of satellite treated males were similar to the satellite control group. All values are in a historical control range.

Females
Significantly changed biochemical values were recorded only sporadically in treated females in comparison with the control females.
Significantly (p ≤ 0.05) increased value of T-Chol was reported in females at the dose level 150 and 600 mg/kg/day/day. Significantly (p ≤ 0.05) increased value of TG was recorded in females at the dose level 150 mg/kg/day/day. Significantly (p ≤ 0.05) decreased value of GLU and Na was recorded in females at the dose level 150 mg/kg/day/day only.
Increased value of Triglycerides (TG) were out of historical control range in all dosed groups of females.
Although the value of Crea was not increased statistically significantly, dose dependence and elevated values in treated groups of females compared to control were recorded. This trend was confirmed by a statistically significantly increased value of creatinine in satellite treated females.
Values of other biochemical parameters of treated females were comparable to the control group. All values except the value of TG are in a historical control range.

Satellite females
Statistically significantly (p ≤ 0.05) increased values of Crea and Cl and decreased (p ≤ 0.05) value of ALT were noted in satellite treated females in comparison with the satellite control females.
Values of other biochemical parameters of treated satellite females were comparable to the control group. All values are in a historical control range.

Description (incidence and severity):
Males
A statistically significantly increased pH of urine was detected in males at the dose level 150 mg/kg/day/day.
The presence of proteins was recorded sporadically - only in males at the dose level 150 mg/kg/day. Specific gravity was increased in males at the dose levels 300 and 600 mg/kg/day in comparison with the control group of males. Also presence of blood was recorded in males at the dose level 300 and 600 mg/kg/day.
The presence of leucocytes was recorded in treated males as well as in control males. In males at the dose level 300 and 600 was presence of leucocytes in the urine more frequent.

Satellite males
The volume and pH of urine was statistically significantly decreased in treated males.
The presence of leucocytes was recorded in satellite treated males as well as in satellite control males. The blood in urine was recorded in one satellite treated male only.

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

Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males
For examination of the repeated toxicity of the test item, the full histopathology was performed for the first six males of control (No 1-6) and high dose (No 61-66).
Histological examination of kidneys only was also performed in males Nos. 21-26 (150 mg/kg/day) and 41-46 (300 mg/kg/day).
Kidneys were probably target organ. Only sporadic findings on other examined organs were recorded in the control group of males as well as in dosed group of males.
Hydronephrosis in kidneys was detected in 2-4-1-2 males. Tubular necrosis in kidneys was recorded in 0-0-2-6 males.
The incidence of other microscopical findings was very sporadic and these findings are mentioned in individual tables. Mild to marked hydronephrosis was revealed as a spontaneous lesion.
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.
Histological findings on kidneys - tubular necrosis was found out in all satellite treated males 0-6 males).
Mild to marked hydronephrosis was revealed as a spontaneous lesion. The incidence of other microscopical findings was very sporadic and these findings are mentioned in individual tables.
Histological examination revealed minimal to marked signs of tubular necrosis in kidneys in all high dose males as a direct effect of the test item administered. This lesion was histologically characterized by a variety of findings: dilatation of cortical tubules lined by flattened epithelium, sometimes was epithelium of basophilic color as a sign of reparation. Further, amorphous eosinophilic material or cellular debris were present in the lumen of some tubules, and in some cases chronic interstitial inflammation was found. This basic pattern was accompanied also by tubular vacuolation and presence of hyaline casts. Subsequent examination of animals from the middle dose group found this lesion in minimal extent, mainly vacuolation of tubular epithelium in two males. All recovered high dose males showed also this lesion.

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. 103,105,106,109,111,112) and high dose group (Nos. 161,162,165,166,167,171).
Histological examination of kidneys as probably target organ was also performed in females Nos. 121,122,123,126,128,131 (150 mg/kg/day) and 141,143,146,147,149,150 (300 mg/kg/day).
Only sporadic findings were recorded in the control group of females as well as in dosed group of females.
Kidneys were probably target organ. Only sporadic findings on other examined organs were recorded in the control group of males as well as in dosed group of females.
Hydronephrosis in kidneys was detected in 0-3-1-0 females. Mild to marked hydronephrosis was revealed as a spontaneous lesion. Tubular necrosis in kidneys was recorded in 0-5-5-6 females.
The changes related to pregnancy were found in both control and high treated females: focal accumulation of lipophages and siderophages in mesometrium of uterus in 6-/-/-4 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
No treatment-related changes were found in female genital tract.

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.
Histological findings on kidneys - tubular necrosis was found out in all satellite treated females (0-6 females).
The incidence of other microscopical findings was very sporadic and these findings are mentioned in individual tables
Histological examination revealed minimal to marked signs of tubular necrosis in kidneys in all high dose females as a direct effect of the test item administered. This lesion was histologically characterized by a variety of findings: dilatation of cortical tubules lined by flattened epithelium, sometimes was epithelium of basophilic color as a sign of reparation. Further, amorphous eosinophilic material or cellular debris were present in the lumen of some tubules, and in some cases chronic interstitial inflammation was found. This basic pattern was accompanied also by tubular vacuolation and presence of hyaline casts. Subsequent examination of animals from the middle dose group found this lesion in minimal extent, mainly vacuolation of tubular epithelium in five females and also in the low dose in five females. All recovered high dose females showed also this lesion.

FOR REPRODUCTIVE TOXICITY
Males
Microscopical examination of reproductive organs, thyroid gland and the pituitary gland
Microscopical examination 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 4-2 males, spermatogranuloma in 0-1 males. In the prostate gland of 5-1 males, chronic inflammation was observed. Chronic inflammation in epididymides was recorded in 0-1 males. No treatment-related changes were found in male genital tract.
Pituitary and Thyroid glands of both control and treated rats were without any pathological lesions.

Females
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 12-9 females and hemosiderin in mucosa of uterus in 12-11 females.
The vagina of both control and administered females was without pathological findings except cyst 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.
Description (incidence and severity):
No neoplastic tissues were observed during the hystopathology
Description (incidence and severity):
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 except statistically significantly decreased value in T4 concentration in males at the dose level 150 mg/kg/day. No dose-dependence was observed, therefore this decreasing was probably not due to application of the test item.
Description (incidence and severity):
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.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
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.
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 dose level 150 mg/k (due to the death of one female during the study) as well as at the dose level 600 mg/kg/day/day (due to the one non-pregnant female). 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 higher at all dosed groups of females in comparison with the control group.
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 mean pup weight at PND4 was lower in all treated groups compared to control group however 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 slightly lower in high dosed groups in comparison with the control group due to one non-pregnant female. The gestation index was the same in the control and all treated groups; the viability index on PND 4 was the best in the middle and high dosed groups.
Post-implantation and post-natal losses were the highest in females from the control group in comparison with the dosed females.

Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
< 150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
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: 151 of 153 born pups were examined.
Female No. 106: one male pup of ten pups disappeared due to cannibalism.
Female No. 110: one female pup of fifteen pups disappeared due to cannibalism.
No macroscopical findings were recorded in other pups/litters.


150 mg/kg/day : 169 of 170 born pups were examined.
Female No. 131: one female pup of nineteen pups disappeared due to cannibalism
No macroscopical findings were recorded in other pups/litters.

300 mg/kg/day : 166 of 166 born pups were examined.
No macroscopical findings were recorded in pups/litters.

600 mg/kg/day : 156 of 156 born pups were examined.
No macroscopical findings were recorded in pups/litters.
Mortality / viability:
no mortality observed
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 higher in all dosed groups of females in comparison with the control group of females.
No stillborn pups were recorded in any group of females.
The mean number of live pups among the dosed groups was higher at all checking intervals in comparison with the control group. Statistically significant increasing of mean number of pups was recorded in all checking intervals at the dose level 150 mg/kg/day compared to control group.
Description (incidence and severity):
Statistically significant differences were recorded. The mean body weight of pup was lower on 4th day of lactation in all dosed groups compared to control group. In next checking intervals the mean body weight of pup was comparable to the control group in all treatment groups.
Description (incidence and severity):
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.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The corrected anogenital distance did not show significant changes among the treated groups and control group
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The presence of nipples in male pups was checked on day 13 of lactation and none were present (nipple presence in male pups on day 13 of lactation is undesirable)
Description (incidence and severity):
Thyroid Gland Weight and Histopathological Examination
The weight of the thyroid gland in pups of the treated group of mothers was comparable to the control group of mothers. Histological examination of thyroid glands did not reveal pathological findings in pups of mothers dosed by the test item.
Histopathological examination of thyroid glands of pups of females from the high dose group did not reveal any pathological finding.
Other effects:
no effects observed
Description (incidence and severity):
Sex Ratio of Pups
Number and sex ration of pups was not affected by the test item treatment.

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
NOAEL for REPRODUCTION and DEVELOPMENT = 600 mg/kg/day body weight/day
Executive summary:

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

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

 

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 150, 300, 600 mg/kg/day) and one control group (vehicle only) and two satellite groups of animals (one control group (vehicle only) and one treated group (600 mg/kg/day). Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females.

 

The first six males and six mothers who delivered pups per group and a satellite groups of animals (control and treated) are part of therepeated dose toxicity studyand 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 thereproduction studyand examined with respect to reproduction parameters.

 

The treated groups were administered daily. 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 did not cause the death of animals except one female from the dose level 150 mg/kg/day/day, who was found dead on day 25 of application; the cause of death was not determined due to partial autolysis of organs. This death was accidental and not treatment-related.

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

 

Repeated Dose Toxicity part of study

The haematological examination did not reveal toxic effect of the test item on administered animals.

The biochemical examination of treated animals showed a toxicologically significant effect of the test item on the treated animals. Changes of Creatinine concentration in serum (irreversibly increased values in males and females) and increased concentration of chloride ions in satellite animals can be associated with the findings reported during the histological examination of kidneys.

The examination of urine parameters showed the presence of blood and leucocytes in urine of animals from middle and highest dose level.

The test item had toxicology significant effect on weight of organs of treated animals. The weight of kidneys (absolute and relative) was significantly increased in all dose groups of females and this increase was irreversible. Insignificant increase of relative weight of kidneys was also reported in males. This was confirmed by pathological and histopathological examination, where the findings would indicate a toxic effect of the test item on kidneys of dosed animals.

During the macroscopic examination, findings related to the test item treatment were found out. Changed colour of kidneys in the mid and high dose groups. 

Histological examinationrevealedminimal to marked signs of tubular necrosis in kidneys of rats administered by highest dose of test item (600 mg/kg).Thislesion was subsequently revealed in some rats from the middle and low dose groups (300, respective 150 mg/kg). This lesion was irreversible during recovery period. All recovered high dose males and females showed also this lesion.

 

Reproduction Toxicity part of study

Test item treatmentdid 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. Decreased concentration of T4 hormone in males at the dose level 150 mg/kg was considered toxicologically insignificant.

Evaluation of the absolute and relative weight of reproductive organs of male and female, as well as the weight of pituitary and thyroid gland revealed only sporadic findings. The absolute weight of testes and epididymis was significantly decreased in males at the dose level 600 mg/kg/day (also reduced absolute weight of testes was noted in males at the dose level 150 mg/kg/day). Evaluation of relative weight of reproductive organs in males, absolute and relative weights of reproductive organs in treated females did not reveal any statistically significant differences compared to control.

Histological examination did not reveal negative effect of the test item on collected reproductive organs, pituitary and thyroid glands.

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.

Examination of sperm motility and morphology in treated parental males did not show any differences in comparison with the control males.

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

The calculated parameters (mating and fertility indexes) were similar in all treated groups in comparison with the control groups. Fertility indexes were slightly lower in high dosed groups in comparison with the control group due to one non-pregnant female. Gestation indexes in treated groups were identical with the control group. The viability index was the best in the highest dosed group of females.

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

The total number of pups,mean number of pups per litter and mean litter weight at first litter check after parturition and during the next intervals were similar or higher in treated groups of mothers compared to control group. The mean pup body weights at all checking intervals was slightly lower in all treated groups compared to control group due to higher number of pups per group of treated females. Macroscopical examinationof pups did not show negative effect of the test item administration on development of pups. 

No adverse effect of the test item treatment was observed during examination of thyroxine and rat thyroid stimulating hormone blood concentration in pups. Male nipple retention at day 13 was not recorded in any male pup.Corrected anogenital distance in treated pups was comparable to the control pups. 

 

CONCLUSION

According to the study results the NOAEL (No Observed Adverse Effect Level) value for REPEATED DOSE TOXICITY in MALES was established as 150mg/kg/day and in FEMALES was established to be less than 150 mg/kg/day.

This value is based on toxicologically significant changes recorded in treated males and females. Damage of kidneysconfirmed by the histological examination, evaluation of biochemical parameters and biometry of organs was observed. Histopathological finding -tubular necrosis in the kidneys –was reported in a middle and high dose level in males and in all dose levels of females. This finding was irreversible. All satellite high dose males and females showed also this lesion at the end of recovery period.

 

According to the study results the NOAEL(No Observed Adverse Effect Level) value for REPRODUCTION and DEVELOPMENT was established as 600 mg/kg/day body weight/day.No biologically significant changes relevant to reproduction and development were observed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD guideline 422
Version / remarks:
29th July 2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
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)
EC Number:
257-827-7
EC Name:
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)
Cas Number:
52301-70-9
Molecular formula:
C40H40N12O20S6.6Na
IUPAC Name:
hexasodium 2-{[4-({4-[(E)-2-[4-({4-[(2,5-disulfophenyl)amino]-6-(morpholin-4-yl)-1,3,5-triazin-2-yl}amino)-2-sulfophenyl]ethenyl]-3-sulfophenyl}amino)-6-(morpholin-4-yl)-1,3,5-triazin-2-yl]amino}benzene-1,4-disulfonate

Test animals

Species:
rat
Strain:
Wistar
Remarks:
SPF quality

Administration / exposure

Route of administration:
oral: gavage
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 beaker 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 unstability 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 350 rpm for 10 min.
The beaker with test item was during dissolving and homogenisation covered by aluminium foil due to possible light unstability of test item.
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 350 rpm.
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 (350 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 the both application forms (10 mg and 1000 mg /10 mL) of the test item, FAT 65037/G TE, 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 aluminium foil due to possible light unstability of test item.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: one male and one female in one cage
- Length of cohabitation: max. 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: 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
- After successful mating each pregnant female was caged (how): individually
Duration of treatment / exposure:
Parental females 42 days + gestation → lactation → day 12 post-partum
Satellite females totally 49 days of administration + 14 days of observation
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control of the basic group and satellite
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
In the basic group and satellite
No. of animals per sex per dose:
Basic group: 12 females per group
Satellite groups: 6 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses have been selected based on the results of the DRFE
- 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

Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first application and then weekly (except the mating period)
BODY WEIGHT: Yes
- Time schedule for examinations: 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 females – three times a week
- Mortality control: twice daily
SACRIFICE
At the end of study, the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
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,ovaries, uterus, thymus, spleen, brain, pituitary gland and heart were recorded (Repeated Dose Toxicity part of study – 6 females from each group + satellite groups);
ovaries, uterus, 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.
Full histopathology of the preserved reproductive organs and tissues was performed for all high dose and control animals.
Ovaries and uterine content:
Absolute weights ovaries, uterus, pituitary gland
Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ × 100/ body weight.
Statistics:
For statistical evaluation the software Statgraphic ® Centurion (version XVII, USA) was used.
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).
Indices:
Gestation index = (number of females with live born pups / number of pregnant females) x 100
Offspring viability indices = number of pups surviving on PND4 x 100 / number of pups born alive

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Females
No signs of disease, clinical changes and clinical signs of intoxication were recorded during the application period in treated females.
Female No.124 of the dose level 150 mg/kg/daywas found dead on day 25 of application (this female was in a group designated for the reproduction part of study). Piloerection and apathy were recorded during the observation a day before the death. The cause of death was unknown.
Satellite females
No signs of disease, clinical changes and clinical signs of intoxication were recorded during the application period in all treated females. During the observation period (recovery; weeks 8 and 9) no changes of health status were noted in satellite treated females.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female No.124 of the dose level 150 mg/kg/day was found dead on day 25 of application There were no other unscheduled deaths during the study.
Description (incidence and severity):
Females
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 slightly lower in comparison with the control group from the second week of administration. Statistically significant differences in necropsy body weight were not found in satellite treated females.

Mean Body Weight Increment
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.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Females
During the pre-mating period, pregnancy and lactation period the food consumption of treated females was similar or higher in comparison with the control females.
Satellite females
The food consumption of satellite treated females was similar in comparison with the control group
during the entire application and recovery periods.
Food efficiency:
no effects observed
Description (incidence and severity):
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 similar 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):
The water consumption of satellite treated males was similar or higher compared to the satellite control group during the entire application and recovery period.
Description (incidence and severity):
Females
The white blood components were not affected by the test item treatment. No significantly changed values of total leucocyte count (WBC) and five-population differential of white blood cells in treated groups of females were recorded in comparison with the control group.
The changes in values of red blood components were recorded in treated groups of females. Statistically significantly increased value of RBC (p ≤ 0.05) associated with increased value of Hct (p ≤ 0.05)
and decreased MCV (p ≤ 0.05) were recorded in females at the dose level 600 mg/kg/day in comparison with the control group of females.
The values of haemocoagulation parameters were not significantly affected by the test item treatment.
All values were in a historical control range.
Satellite females
A decreased value of RBC (p ≤ 0.05) and associated decreased value of Hct (p ≤ 0.05) and Hgb (p ≤ 0.05) were recorded in satellite treated females in comparison with the satellite control females.
Other values were similar with satellite control females and in a range of historical control.
Description (incidence and severity):
Females
Significantly changed biochemical values were recorded only sporadically in treated females in comparison with the control females.
Significantly (p ≤ 0.05) increased value of T-Chol was reported in females at the dose level 150 and 600 mg/kg/day/day. Significantly (p ≤ 0.05) increased value of TG was recorded in females at the dose level 150 mg/kg/day/day. Significantly (p ≤ 0.05) decreased value of GLU and Na was recorded in females at the dose level 150 mg/kg/day/day only.
Increased value of Triglycerides (TG) were out of historical control range in all dosed groups of females.
Although the value of Crea was not increased statistically significantly, dose dependence and elevated values in treated groups of females compared to control were recorded. This trend was confirmed by a statistically significantly increased value of creatinine in satellite treated females.
Values of other biochemical parameters of treated females were comparable to the control group. All values except the value of TG are in a historical control range.
Satellite females
Statistically significantly (p ≤ 0.05) increased values of Crea and Cl and decreased (p ≤ 0.05) value of ALT were noted in satellite treated females in comparison with the satellite control females.
Values of other biochemical parameters of treated satellite females were comparable to the control group. All values are in a historical control range.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute Organ Weight
Females
Statistically significantly (p ≤ 0.05) increased absolute weight of kidneys was recorded in females at all dose levels in comparison with the control females (irreversible). Statistically significantly (p ≤ 0.05) increased absolute weight of liver was recorded in females at dose levels 150 and 300 mg/kg/day in comparison with the control females. A significant (p ≤ 0.05) decrease in the weight of the ovaries was recorded in females at the dose level 600 mg/kg/day/day in comparison with the control group of females. Absolute weights of other organs were in dosed females comparable with the control females.
Satellite females
Statistically significant differences were recorded in the absolute weight of kidneys (p ≤ 0.05) in satellite females. Absolute weight of kidneys was increased in treated females in comparison with the control females.

Relative Organ Weight
Females
A statistically significantly changed relative weight of organs were recorded in dosed females in comparison with the control females. Significantly (p ≤ 0.05) increased relative weight of kidneys and liver was recorded in all dosed groups of females.
Satellite females
Statistically significant difference was recorded in the relative weight of kidneys (p ≤ 0.05) in satellite females. Relative weight of kidneys was increased in treated satellite females in comparison with the control satellite females.

FOR REPRODUCTIVE TOXICITY
Absolute weight
No statistically significant differences in absolute and/or relative weight of reproductive organs of treated females was recorded in comparison with the control females.
Relative weight
No statistically significant differences in absolute and/or relative weight of reproductive organs of treated females was recorded in comparison with the control females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Females
Control: no macroscopic findings were recorded in all 6 females.
150 mg/kg/day : no macroscopic findings were recorded in all 6 females.
300 mg/kg/day : no macroscopic findings were recorded in 2 females. Very light colour of kidneys was recorded in 4 females.
600 mg/kg/day : Very light colour of kidneys was recorded in all 6 females.
Satellite females
Control satellite: no macroscopic pathological findings were recorded in all 6 females (dilatation of uterus – non-pathological finding was recorded in two of six females).
600 mg/kg/day satellite: no macroscopic pathological findings were recorded in 2 females. Very light colour of kidneys was recorded in 4 females.
(dilatation of uterus – non-pathological finding was recorded in one of six females).
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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. 103,105,106,109,111,112) and high dose group (Nos. 161,162,165,166,167,171).
Histological examination of kidneys as probably target organ was also performed in females Nos.121,122,123,126,128,131 (150 mg/kg/day) and 141,143,146,147,149,150 (300 mg/kg/day).
Only sporadic findings were recorded in the control group of females as well as in dosed group of females.
Kidneys were probably target organ. Only sporadic findings on other examined organs were recorded in the control group of males as well as in dosed group of females.
Hydronephrosis in kidneys was detected in 0-3-1-0 females. Mild to marked hydronephrosis was revealed as a spontaneous lesion. Tubular necrosis in kidneys was recorded in 0-5-5-6 females.
The changes related to pregnancy were found in both control and high treated females: focal accumulation of lipophages and siderophages in mesometrium of uterus in 6-/-/-4 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.
No treatment-related changes were found in female genital tract.

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. Histological findings on kidneys - tubular necrosis was found out in all satellite treated females (0-6 females).
The incidence of other microscopical findings was very sporadic and these findings are mentioned in individual tables
Histological examination revealed minimal to marked signs of tubular necrosis in kidneys in all high dose females as a direct effect of the test item administered. This lesion was histologically characterized by a variety of findings: dilatation of cortical tubules lined by flattened epithelium, sometimes was epithelium of basophilic color as a sign of reparation. Further, amorphous eosinophilic material or cellular debris were present in the lumen of some tubules, and in some cases chronic interstitial inflammation was found. This basic pattern was accompanied also by tubular vacuolation and presence of hyaline casts. Subsequent examination of animals from the middle dose group found this lesion in minimal extent, mainly vacuolation of tubular epithelium in five females and also in
the low dose in five females. All recovered high dose females showed also this lesion.

FOR REPRODUCTIVE TOXICITY
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 12-9 females and hemosiderin in mucosa of uterus in 12-11 females.
The vagina of both control and administered females was without pathological findings except cyst 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.
Description (incidence and severity):
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 except statistically significantly decreased value in T4 con centration in males at the dose level 150 mg/kg/day. No dose-dependence was observed, therefore this decreasing was probably not due to application of the test item.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were recorded. The mean duration of pregnancy was similar in treated and control groups.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean number of implantations was comparable in groups of treated females with the control females.
Dead fetuses:
no effects observed
Description (incidence and severity):
The stillborn were not found at any group of treated females as well as in females at the control group.
The mean number of live pups at the first litter check after parturition was higher at all dosed groups of females in comparison with the control group.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The mean duration of pregnancy was similar in treated and control groups
Description (incidence and severity):
A decreased number of females achieving pregnancy was recorded at the dose level 150 mg/k (due to the death of one female during the study) as well as at the dose level 600 mg/kg/day/day (due to the one non-pregnant female).

Effect levels (maternal animals)

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

Maternal abnormalities

Abnormalities:
no effects observed
Description (incidence and severity):
No effects observed on the maternal organs related to development of foetuses

Results (fetuses)

Description (incidence and severity):
Statistically significant differences were recorded. The mean body weight of pup was lower on 4th day of lactation in all dosed groups compared to control group. In next checking intervals the mean body weight of pup was comparable to the control group in all treatment groups.
Description (incidence and severity):
The total number of live pups at the first litter check after parturition was higher in all dosed groups of females in comparison with the control group of females.
No stillborn pups were recorded in any group of females.
The mean number of live pups among the dosed groups was higher at all checking intervals in comparison with the control group. Statistically significant increasing of mean number of pups was recorded in all checking intervals at the dose level 150 mg/kg/day compared to control group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Number and sex ration of pups was not affected by the test item treatment.
Description (incidence and severity):
The total number of live pups on the 4th day and 13th day of lactation was higher in all dosed groups of females in comparison with the control group of females.
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: 151 of 153 born pups were examined.
Female No. 106: one male pup of ten pups disappeared due to cannibalism.
Female No. 110: one female pup of fifteen pups disappeared due to cannibalism.
No macroscopical findings were recorded in other pups/litters.
150 mg/kg/day : 169 of 170 born pups were examined.
Female No. 131: one female pup of nineteen pups disappeared due to cannibalism
No macroscopical findings were recorded in other pups/litters.
300 mg/kg/day : 166 of 166 born pups were examined.
No macroscopical findings were recorded in pups/litters.
600 mg/kg/day : 156 of 156 born pups were examined.
No macroscopical findings were recorded in pups/litters.

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL for DEVELOPMENT = 600 mg/kg/day body weight/day
Executive summary:

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

Before the start of study with laboratory animals thestability and homogeneityof application form were determined at the test facility. A dose-range finding experiment was performed to determine the dose levels for the main study.

 

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 150, 300, 600 mg/kg/day) and one control group (vehicle only) and two satellite groups of animals (one control group (vehicle only) and one treated group (600 mg/kg/day). Each main group consisted of 12 males and 12 females; each satellite group consisted of 6 males and 6 females.

 

The first six males and six mothers who delivered pups per group and a satellite groups of animals (control and treated) are part of therepeated dose toxicity studyand 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 thereproduction studyand examined with respect to reproduction parameters.

 

The treated groups were administered daily. 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

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

Evaluation of the absolute and relative weight of reproductive organs of female, as well as the weight of pituitary and thyroid gland revealed only sporadic findings. Evaluation of relative weights of reproductive organs in treated females did not reveal any statistically significant differences compared to control.

Histological examination did not reveal negative effect of the test item on collected reproductive organs, pituitary and thyroid glands.

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

Gestation indexes in treated groups were identical with the control group. The viability index was the best in the highest dosed group of females.

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

The total number of pups,mean number of pups per litter and mean litter weight at first litter check after parturition and during the next intervals were similar or higher in treated groups of mothers compared to control group. The mean pup body weights at all checking intervals was slightly lower in all treated groups compared to control group due to higher number of pups per group of treated females. Macroscopical examinationof pups did not show negative effect of the test item administration on development of pups. 

No adverse effect of the test item treatment was observed during examination of thyroxine and rat thyroid stimulating hormone blood concentration in pups. Male nipple retention at day 13 was not recorded in any male pup.Corrected anogenital distance in treated pups was comparable to the control pups. 

 

CONCLUSION

According to the study results the NOAEL(No Observed Adverse Effect Level) value for DEVELOPMENT was established as 600 mg/kg/day body weight/day. No biologically significant changes relevant to reproduction and development were observed.