Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-11-28 to 2019-06-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference

Based on the observations of an OECD 422 compliant study with the test item the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male rats:               50 mg/kg bw/day based on a sex and species specific effect, not relevant for human risk assessment

NOAEL for systemic toxicity female rats:              800 mg/kg bw/day

NOAEL for reproductive performance of male/female rats:     800 mg/kg bw/day

NOAEL for F1 Offspring:               800 mg/kg bw/day

Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
800 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and Guideline study
Endpoint conclusion:
no study available
Endpoint conclusion:
no study available
Endpoint conclusion:
no study available
Endpoint conclusion:
no study available

Key study, OECD 422

The purpose of a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was to obtain initial information on the toxic potential of the test item and its possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as development of the F1 offspring from conception to day 13 post-partum when repeatedly administered orally (by gavage) to parental animals at doses of 50, 200 and 800 mg/kg bw/day compared to control animals.

Four groups of Han:WIST rats (n=17/sex/group in the control and high dose groups; n=12 animals/sex in the low and middle dose groups) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 50, 200 and 800 mg/kg bw/day doses corresponding to concentrations of 0, 10, 40 and 160 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, sunflower oil. 5 animals/ sex in the control and high dose groups were observed for two weeks post-treatment (recovery) period.

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front. The concentration of the test item in the dosing formulations was checked two times during the study. The test item concentrations in the dosing formulations varied in the acceptable range – between 102 % and 108 % of the nominal values – confirming the proper dosing.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 62 days). Dams were additionally exposed through the gestation period and up to lactation days 13-16 (altogether for 51-65 days). One control dam was administered up to and including lactation day 12 due to over-anesthesia at the blood sampling for thyroid hormone determination. Animals of the recovery group were administered for 62 days and non-pregnant or not mated female animals were administered for 42 days.

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Five dams and the male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating. Vaginal smear was also prepared on the day of the necropsy. Recovery animals were observed for mortality, clinical signs, body weight, food consumption (during the treatment and post-treatment periods), hematology, clinical chemistry, gross necropsy, organ weighing and histopathology (at the termination of the recovery period). The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter.

Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4, TSH) from 2-7 pups per litter (where it was feasible) on post-natal day 4, from all dams and 1-6 pups per litter at termination on post-partum/post-natal day 13 – or shortly thereafter – and from all parent male animals at termination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined. Histopathology examination was performed on reproductive organs (testes, epididymides, prostate and seminal vesicles with coagulating glands, uterus with cervix, vagina, ovaries) in the control and high dose groups and in one infertile pair (male and female) at 50 mg/kg bw/day. Additionally, full histopathology was performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations and in the recovery animals in the control and high dose groups. Based on macroscopic findings in male animals at 200 and 800 mg/kg bw/day (enlargement, uneven surface, paleness), the kidneys were also evaluated histologically in selected male animals of the low and mid dose groups as well as in male animals with macroscopic renal findings at 200 and 800 mg/kg bw/day. Immunohistochemistry examinations were also performed for determining α2µ-globulin in these kidneys – in selected male animals and animals with macroscopic renal changes.

Organs showing macroscopic findings at necropsy (thymus, liver, female kidneys, skin, submandibular lymph nodes) were also processed and evaluated histologically in animals of control, 50, 200 and 800 mg/kg bw/day).

The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only. Historical control data were also considered.

 

There was no mortality at 50, 200 or 800 mg/kg bw/day groups during the course of study. Adverse clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily or detailed weekly clinical observations or at the terminal functional observations. The behavior and physical condition of animals was not affected by the test item at any dose level (50, 200 or 800 mg/kg bw/day) during the entire observation period. Salivation and nuzzling up the bedding material noted for 800 mg/kg bw/day dose treated animals (male and female) were with short duration after the daily treatment and transient occurrence. Therefore, these signs were considered to be toxicologically not relevant.

The body weight development was not adversely affected by the test item in male or female animals at 50, 200 or 800 mg/kg bw/day during the entire treatment period. A slightly reduced mean body weight gain resulted in minor changes only in the mean body weight (≥ - 5% of the control) in male animals at 800 mg/kg bw/day. Therefore, the slight changes in body weight and body weight gain at 800 mg/kg bw/day were considered to be of little or no toxicological relevance.

There were no test item related adverse changes in the mean daily food consumption of male and female animals at any dose level (50, 200 and 800 mg/kg bw/day). Slight reduction in the daily mean food consumption of male and female animals at 800 mg/kg bw/day during was transient – during the first week of the treatment only – and was with minor degree. Therefore, this finding was judged to be toxicologically not relevant.

A test item influence on the estrous cycle was not found at any dose level (50, 200 and 800 mg/kg bw/day). Delivery data of dams. There were no toxicologically relevant differences between the control and test item treated groups (50, 200 and 800 mg/kg bw/day) in the evaluated delivery parameters of dams.

The examined parameters of reproductive performance were not affected by the treatment with the test item in male or female animals at 50, 200 or 800 mg/kg bw/day.

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 50, 200 or 800 mg/kg bw/day. Specific alterations related to test item effect were not detected in the examined clinical chemistry parameters at any dose level (male or female; 50, 200 or 800 mg/kg bw/day).

There were no test item related changes in the serum thyroid hormone (FT4, TSH) levels at any dose (parental male animals or PN13 offspring). Macroscopic alterations related to the effect of the test item were not detected in the reproductive organs of male or female animals at 50, 200 or 800 mg/kg bw/day at the necropsy. Test item related changes were detected in the kidneys in male animals at 200 mg/kg bw/day (enlargement, paleness, uneven surface) and at 800 mg/kg bw/day (enlargement, paleness) at the terminal necropsy being in full accordance with the organ weights and histopathological findings. These renal findings (enlargement and paleness) were observed in male animals at 800 mg/kg bw/day with a lower incidence at the end of the recovery period.

Significant elevation of the liver weights (at 800 mg/kg bw/day, male and female) and kidneys weights (at 200 mg/kg bw/day, male and at 800 mg/kg bw/day, male and female) were observed in selected animals at the termination of the treatment. Liver weight changes were considered to be sign of adaptation process of the organ in the lack of related clinical chemistry or histopathological alterations. Changes in weights of liver (male and female) and kidneys (male) were also observed in a lower degree at the end of the recovery period i.e. the organ weight changes were not fully reversible.

Histopathological examinations of the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 800 mg/kg bw/day.

Chronic progressive nephropathy (CPN) was revealed in male animals at 200 and 800 mg/kg bw/day by histological investigations in compliance with macroscopic findings at the necropsy. Chronic progressive nephropathy is an important spontaneous renal disease of the commonly used strains of laboratory rat. However, regarding the significantly higher incidence of CPN in the male animals, a nephrotoxic effect of the test item might be supposed at 200 and 800 mg/kg bw/day and the test item could be considered as a predisposing factor in the pathogenesis of renal lesion in the investigated male animals. This renal change was not reversible as CPN was also observed in all male animals at 800 mg/kg bw/day at the end of the post-treatment observation period.

Immunohistochemistry investigation revealed a statistically significant increase of α2-microglobulin in male animals at 50, 200 and 800 mg/kg bw/day when compared to their control. There were no differences between test item-treated groups.

The offspring’s development was undisturbed at 50, 200 and 800 mg/kg bw/day from birth to post-natal day 13. No effect on the mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

 

Under the conditions of the present study, the test item did not adversely influence the fertility or reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats at 50, 200 and 800 mg/kg bw/day doses administered by oral gavage. 200 and 800 mg/kg bw/day induced chronic progressive nephropathy in male animals associated with accumulation of α-2μ-globulin in the kidneys, macroscopic (enlargement, uneven surface, paleness) and organ weight changes, which were not reversible after two weeks post-treatment observation period. At 50 mg/kg bw/day, elevated level of α2-microglobulin was revealed in male animals.

The development of the F1 offspring was not impaired at any dose level from birth to post-natal day 13 after repeated oral administration of dams.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male rats:             50 mg/kg bw/day, based on a sex and species specific effect, not relevant for human risk assessment

NOAEL for systemic toxicity female rats:              800 mg/kg bw/day

NOAEL for reproductive performance of male/female rats:     800 mg/kg bw/day

NOAEL for F1 Offspring:               800 mg/kg bw/day

The alpha2 -microglobulin accumulation and corresponding CPN in male rat kidneys is known to be a species and sex specific effect. Thus, the male rat NOAEL of 50 mg/kg bw/d is regarded as not relevant for human health risk assessment and the NOAEL of 800 mg/kg bw/d is taken into account as key value for DNEL derivation.

Supporting

A 14-Day toxicity study was conducted to obtain initial information on the toxic potential of the test item after subacute repeated application in rats at three dose levels and to allow dose-setting for a subsequent Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (main study). The test item was administered orally (by gavage) to Han:WIST rats (n=5 animals/sex/group) once a day at 0 (vehicle control), 100 300 and 1000 mg/kg bw/day corresponding to concentrations of 20, 60 and 200 mg/mL for 14 days. The body weight development of the male and female animals was not affected in any test item treated group (100, 300 or 1000 mg/kg bw/day) throughout the entire observation period. There were no test item related toxic changes in the examined hematological or blood coagulation parameters in male or female animals administered with 100, 300 or 1000 mg/kg bw/day dose of the test item. Clinical chemistry observations revealed minor changes in the ALT activity and cholesterol level might be indicative of altered hepatic function in female animals at 1000 mg/kg bw/day. Macroscopic changes related to treatment with test item were not detected in the organs or tissues of male or female animals at 100, 300 or 1000 mg/kg bw/day. Slight elevation in the weights of liver and kidneys with respect to their control in male animals at 1000 mg/kg bw/day and in female animals at 100, 300 and 1000 mg/kg bw/day were probably indicative of enhanced function of these organs. Based on these observations, the doses for the subsequent Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test (main study) are selected as follows:

Group 1 Vehicle control

Group 2 50 mg/kg bw/day

Group 3 200 mg/kg bw/day

Group 4 800 mg/kg bw/day

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item does not require classification for specific target organ toxicity after repeated exposure (STOT RE) according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) No 2019/521.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in reproduction toxicity studies and well-known fertility parameters.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
Young adult rats, approximately 13 weeks old at the beginning of the pre-mating phase and 15 weeks at mating.
- Weight at study initiation:
The weight variation in animals involved at the starting point of the study did not exceed ± 20 % of the mean group weight of each sex.
- Housing:

Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage
Pregnant females will be housed individually.
Males after mating: 2 animals/cage
Animals in recovery group: 2 or 3 animals/sex/ cage
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum
- Water: tap water from municipal supply, as for human consumption, from 500 mL bottles ad libitum
- Acclimation period:
20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): above 10 air-exchanges / hour by central air-condition system
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sunflower oil (Helianthi annui oleum raffinatum)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared in the formulation laboratory of the Test Facility not longer than for five days beforehand and stored at room temperature until use.

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water therefore sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil is a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle:
The test item was formulated in the vehicle in concentrations of 10 mg/mL, 40 mg/mL and 160 mg/mL.
- Lot/batch no.: 1804-4239
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: Females remained with the same male until copulation occurs or 14 days have elapsed.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulations was performed by the Analytical Laboratory of the Test Facility (concentration and homogeneity) twice during the study.
Five samples were taken (from different places) from each concentration (Groups 2, 3 and 4) and at least three parallels were measured. Similarly, five samples were taken from the control solution (Group 1).
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front.
Duration of treatment / exposure:
62 days (males)
51 - 65 days (females)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 animals/sex in the control and dose groups (at least 8 pregnant female animals per group are necessary according to guideline OECD 422/OPPTS 870.3650).
5 animals /sex in the control and high dose group for recovery groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose setting is based on findings obtained in a 14-day dose range finding study (non-GLP; study no. 561-400-3972). The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals
- Randomization:

All parental (P) male and female animals were sorted according to body weight and divided to weight groups aided by a computerized calculation. There were an equal number of animals from each weight group in each of the experimental groups assigned by randomization to ensure that the mean weight of animals from all test groups were as uniformly as practicable. Grouping was aided by SPSS/PC+ software, verifying the homogeneity and variability between the groups according to the actual body weight.
Besides, estrous cycle of female animals was also considered at the randomization. Animals exhibiting typical 4-5 days cycles were included in the study preferably
- Fasting period before blood sampling for clinical biochemistry:
Animals were food deprived for approximately 16 hours (overnight) prior to blood collection.
Positive control:
NA

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing. Pertinent behavioral changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration of signs.
More detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations:
weekly
Parental males were weighed on the first day of dosing (Day 0), at least weekly thereafter and at termination.
Parental females were weighed on the first day of dosing (Day 0), then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals were additionally measured on gestation days 10 in order to give accurate treatment volumes, but these data were evaluated statistically. Body weight data were reported individually for adult animals.
Animals allocated to recovery groups were weighed weekly during the treatment and recovery periods.
Body weights were measured on the day of necropsy for animals subjected to organ weighing (all male animals and females selected for further examinations).

FOOD CONSUMPTION AND COMPOUND INTAKE
The food consumption was determined weekly by weighing the given and non-consumed diet with a precision of 1 g during the treatment period – except mating phase – as follows:
- premating Days 0, 7 and 13 and by weekly interval during post-mating period for male animals;
- premating Days 0, 7 and 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals.
The food consumption was determined by a weekly interval for animals allocated to recovery groups during the treatment and recovery periods.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
one day after the last treatment (i.e. on the day of necropsy), animals of the recovery group were subjected to clinical pathology examinations at the end of the 14 days post-treatment period (on recovery day 14)
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes, animals will be food deprived for approximately 16 hours (overnight) prior to blood collection
- How many animals:
5 male and 5 ffemale animals randomly selected from each group
- Parameters checked in table No.1 and 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
one day after the last treatment (i.e. on the day of necropsy), animals of the recovery group were subjected to clinical pathology examinations at the end of the 14 days post-treatment period (on recovery day 14)
- Animals fasted: Yes, animals will be food deprived for approximately 16 hours (overnight) prior to blood collection
- How many animals:
5 male and 5 ffemale animals randomly selected from each group
- Parameters checked in table No.3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
once during the last exposure week but before the blood sampling
- Dose groups that were examined:
five male and five female animals randomly selected from each group
- Battery of functions tested: sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), grip strength motor activity

IMMUNOLOGY: Yes, Determination of Serum Levels of Thyroid Hormones (T4 and TSH)
- How many animals:
from all dams on day 13 if feasible, from all parent male animals and not mated, non-pregnant or not delivered females at termination
- Parameters checked in table No.4 were examined.

OTHER:
Estrous cycle was monitored by examining vaginal smears before the treatment starts from each animal being considered for study for two weeks
Oestrous cyclicity (parental animals):
Estrous cycle was monitored by examining vaginal smears before the treatment starts from each animal being considered for study for two weeks. Animals exhibiting typical 4-5 days cycles were included in the main groups of the study preferably. Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of copulation (main groups only). Vaginal smear was also prepared on the day of the necropsy. Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear will be examined with a light microscope.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight, sperm count in testes, sperm count in epididymides
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not performed

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: not performed
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals: after 62 days treatment
- Maternal animals: All surviving animals: up to 51 - 65 (up to lactation day 14)

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

HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examination were performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in the control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure; on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary as well as the epithelial capsule and ovarian stroma.
In addition, these organs were processed and examined histologically in not mated, non-pregnant or not delivered females and males these females were cohabited with in the low and middle dose groups.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose groups and in animals in the recovery groups.
All gross lesions were also examined histologically. Additionally, alpha 2u microglobulin examinations were performed in the kidneys of 5 male animals/ dose group. Please refer to "Other examinations".
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring sacrificed at PND 13
- 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 : yes, kidney only
Statistics:
The statistical evaluation of appropriate data were performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. Frequency of toxic response, pathological and histopathological findings by sex and dose were calculated. Results were evaluated in comparison with values of control group (i.e. control value).
Reproductive indices:
Male Copulatory index (%) = (number of males with confirmed mating / total number of males cohabited with females) x 100
Female Copulatory index (%) = (number of females with confirmed mating / total number of females cohabited with females) x 100
Male fertility index (%) = (number of males impregnating a female / number of males with confirmed mating) x 100
Female fertility index (%) = (number of pregnant females / number of sperm positive females) x 100
Gestation index (%) = (number of females with live pups / number of pregnant females) x 100
Offspring viability indices:
Post-implantation loss (%) = [(number of implantations - number of live borns) / number of implantations] x 100
Post-natal mortality (%) = [(number of live borns - number of live pups on PND 13) / number of live borns] x 100
Survival index (%) = (number of live pups on PND 13 / number of live borns) x 100
Sex ratio (%) = [(number pups examined - number of males (females)) / number of pups examined] x 100
Normalized anogenital distance = Anogenital distance divided by the cube root body weight

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Daily Observations
Adverse signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations (50, 200 and 800 mg/kg bw/day, male or female). Salivation and nuzzling up the bedding material noted for 800 mg/kg bw/day dose treated animals (male and female) were with short duration after the daily treatment and were transient. Therefore, these signs were considered to be toxicologically not relevant.
No clinical signs were detected in male animals of control group during the treatment period.
Alopecia was observed on the chest or on the forelimbs in two control dams (2/11) from gestation day 21 or 6 up to lactation day 3 or up to the termination of the study on lactation day13. Alopecia is a common observation in experimental rats of this strain with similar age without toxicological relevance. There were no clinical signs in male and female animals of 50 or 200 mg/kg bw/day groups, i.e. these animals exhibited normal behavior and physical condition with no abnormalities during the entire treatment period. Salivation and nuzzling up the bedding material were observed with variable incidence at 800 mg/kg bw/day during the pre-mating, mating and post-mating period in male animals (14/17 and 17/17, respectively). In the female animals, salivation and nuzzling up the bedding material were observed during the premating, mating and gestation periods as follows:
- pre-mating and mating periods: salivation 4/12 and nuzzling up the bedding material 8/12;
- post-mating: salivation and nuzzling up the bedding material 1/1, both;
- gestation period: salivation 2/11 and nuzzling up the bedding material 8/11;
- during the treatment period of recovery animals: salivation 5/5 and nuzzling up the bedding material 5/5;
These signs appeared immediately after the administration and ceased shortly thereafter and were considered to be toxicologically not relevant.

Recovery period
The behavior and physical condition of animals – male and female –were normal in the control and high dose (800 mg/kg bw/day) groups during the post-treatment observation period.

Detailed weekly observation
The behavior and physical condition of animals was not affected by the test item at any dose level (50, 200 or 800 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period. Alopecia on the chest or forelimbs – as described above – were also recorded at the detailed weekly observation in control dams (2/11) on gestation day 21 and lactation day 0; as well as on gestation days 7, 14, 21 and on lactation days 0, 4 and 13, respectively.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no mortality in the control, 50, 200 or 800 mg/kg bw/day groups (parental male and female) during the course of this study. One control dam and its offspring were subjected to early necropsy because of over anesthesia at the blood sampling for thyroid hormone determination on lactation day 13.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight development was not adversely affected by the test item in male or female animals at 50, 200 or 800 mg/kg bw/day during the entire treatment period.
The body weight development was slightly depressed in male animals at 800 mg/kg bw/day. However, the reduced body weight gain resulted in minor changes only in the body weight (≥ - 5% of the control). Therefore, the slight changes in body weight and body weight gain were considered to be of no or small toxicological relevance.

Treatment period
The body weight and body weight gain were similar in the control and dosed groups of male and female animals at 50 and 200 mg/kg bw/day during the entire treatment period – pre-mating, mating and post-mating periods for males and pre-mating, gestation and lactation periods for females. Slight but statistically significant difference with respect to the control at the lower mean body weight gain of male animals at 50 mg/kg bw/day (low dose) between Days 41 and 48 was transient and had no influence on the body weight value. Therefore, this minor change in low dose treated animals was considered to be toxicologically not relevant.
At 800 mg/kg bw/day, statistical significances with respect to the control were detected at the slightly lower mean body weight of male animals on days 13, 20, 41, 48, 55 and 61 as well as at the lower mean body weight gain between Days 0-7, 7-13, 55-61 and for the study overall (between Days 0-61).
The body weight development of female animals was comparable in the control and test item treated groups during the entire observation period. Statistically significant difference at the higher mean body weight gain with respect to their control was transient and with minor degree in female animals at 800 mg/kg bw/day in recovery group between Days 34 and 41 and between gestation days 0 and 7.

Recovery period
There were no statistically significant differences between the control and 800 mg/kg w/day dose treated animals in the body weight or body weight gain during the recovery period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item related adverse changes in the mean daily food consumption of male and female animals at any dose level (50, 200 and 800 mg/kg bw/day).
Slight reduction in the daily mean food consumption of male and female animals at 800 mg/kg bw/day during was transient – during the first week of the treatment only – and was with minor degree. Therefore, this finding was judged to be toxicologically not relevant.

Treatment period
The mean food consumption was similar in male and female animals in the control and 50 and 200 mg/kg bw/day groups during the entire observation period. Slightly – but statistically significantly – lower mean daily food intake of female animals at 200 mg/kg bw/day between gestation days 0 and 7 was not related to the test item as similar finding was not detected at the high dose treated animals.
Compared to their control, statistical significances were observed at the slightly lower mean food consumption between Days 0-7 in male and female animals at 800 mg/kg bw/day. The mean daily food consumption slightly exceeded the control value in male animals from Day 34 up to the termination of the treatment. These slight differences in the mean daily food consumption were with minor degree and were considered to be of little or no toxicological relevance.

Recovery period
The mean daily food consumption was similar in the control and high dose treated animals (male and female) during the recovery period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no test item related adverse changes in the examined hematological parameters in male or female animals at 50, 200 or 800 mg/kg bw/day.

Treatment period
In male animals, statistical significances were detected at the slightly lower mean percentage of lymphocytes (LYM) at 50 mg/kg bw/day and at lower mean hemoglobin concentration (HGB) at 200 mg/kg bw/day.
In female animals, slightly but statistically significantly lower mean percentage of eosinophil granulocytes (EOS) and higher mean corpuscular hemoglobin concentration (MCHC) was observed at 800 and 200 mg/kg bw/day, respectively, when compared to their control.
The statistically significant differences between the control and high dose treated groups for some parameters (LYM, HGB, EOS, MCHC) were considered to have no toxicological relevance due to the minor degree and lack of any changes in other related parameters. Furthermore, the values are still well within the historical control ranges.

Recovery period
Comparing the control, statistical significance was detected at the lower mean corpuscular volume (MCV) in male animal and at the higher mean percentages of neutrophil granulocytes (NEU) in female animals at 800 mg/kg bw/day at the end of the recovery period. The differences were with small degree, values met well the historical control and similar findings were not detected at the termination of the treatment. Therefore, the minor changes in MCV and NEU were considered to have no toxicological relevance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item related adverse alterations were detected in the examined clinical chemistry parameters in male or female animals at 50, 200 or 800 mg/kg bw/day.

Treatment period
In the male animals, statistical significance was detected with respect to their control at the lower mean alkaline phosphatase activity (ALP) at 50 and 800 mg/kg bw/day and at the higher mean potassium concentration (K+) at 200 mg/kg bw/day.
In the female animals, statistically significant difference with respect to the control was noted for the slightly higher mean concentration of total bilirubin (TBIL) at 200 and 800 mg/kg bw/day. Higher mean concentrations of cholesterol (CHOL), calcium (Ca2+), albumin (ALB) and total protein (TPROT) and lower mean concentration of chloride (Cl-) were also observed in the female animals at 800 mg/kg bw/day.

Recovery period
At the end of the recovery period, higher men concentration of urea was observed in male animal at 800 mg/kg bw/day.
In the female animals of high dose recovery group, higher mean concentrations of cholesterol and lower mean concentration of sodium, chloride and lower mean albumin: globulin ratio (A/G) were observed at the end of the post-treatment observation period.
Statistically significant differences in these parameters were considered to be of little or no biological significance as the profile of change has no biological significance (ALP in male animals), or the mean values correlated well with the historical control values (UREA, K+, in male animals, TBIL, CHOL, ALB, TPROT, Na+ in female animals) or the minor degree of change (Ca2+, Cl-, A/G).

Moreover, there were no supporting histopathological findings.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observations did not demonstrate any test item related changes. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in all groups (50, 200 and 800 mg/kg bw/day, control).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examinations did not reveal any test item related adverse alterations in the reproductive organs or tissues of male or female animals at 800 mg/kg/bw/day (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland).
Chronic progressive nephropathy (CPN) was observed in male animals at 200 and 800 mg/kg bw/day by histological investigations in compliance with macroscopic findings at the necropsy. Chronic progressive spontaneous nephropathy is an important spontaneous renal disease of the commonly used strains of laboratory rat. However, test item could be considered as a predisposing factor in the pathogenesis of renal lesion in the investigated male animals regarding the higher incidence of CPN in the animals administered with 200 or 800 mg/kg bw/day dose.

Treatment period
In all male animals (12/12 control, 12/12 at 800 mg/kg bw/day), the investigated organs of reproductive system (testes, epididymides, prostate and seminal vesicles with coagulating gland) were histologically normal and characteristic on the sexually mature organism in all cases including not mated male animal (1/12 control) and males, which did not fertilize their pairs (1/1 at 50 mg/kg bw/day and 1/12 at 800 mg/kg bw/day).
The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and high dose treated animals.

The histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all animals as well.
In the female animals (12/12 control, 12/12 at 800 mg/kg bw/day) the investigated organs of reproductive system (ovaries, uterus with cervix, vagina) had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well (including in not mated (1/12 control) and non-pregnant females (1/1 at 50 mg/kg bw/day and 1/12 at 800mg/kg bw/day).
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male and female animals.
Acute hemorrhage in the thymus (1/1 male at 50 mg/kg bw/day; 1/1 male at 200 mg/kg bw/day; 1/5 male at 800 mg/kg bw/day) or in the lymph nodes (2/2 female at 200 mg/kg bw/day) occurred sporadically and is considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.
Chronic progressive nephropathy was noted for several male animals as follows: 1/5 at 50 mg/kg bw/day; 9/10 at 200 mg/kg bw/day; 12/12 at 800 mg/kg bw/day.
One or both sides pyelectasia was observed in the kidneys in several animals:
- control: 1/5 male, 2/7 female;
- 50 mg/kg bw/day: 1/5 male and 1/1 female;
- 200 mg/kg bw/day: 5/10 male and 5/5 female;
- 800 mg/kg bw/day: 2/12 male and 5/8 female
Renal pyelectasia – without degenerative, inflammatory or other histological (fibrotic etc.) lesion – is a common finding without toxicological significance. This phenomenon was observed in male and female animals (in contrast with the CPN) and no dose dependence was detected.
Renal cyst was noted for one male (1/12) and for one female (1/8) animal at 800 mg/kg bw/day. Cyst in the kidney is an individual disorder and has no toxicological significance.
Focal fibrosis in the Glisson’s capsule of the liver (1/1 female at 50 mg/kg bw/day) was a consequence of the mechanical irritation due to the diaphragmatic hernia.
Focal atrophy of hair follicles (1/6 control female), without inflammatory agent (Trichophyton, Microsporon or Demodex bodies) was in connection with the focal alopecia. Abnormal hair follicle atrophy could be due to dermal ischemia or autoimmune conditions as well.
Histological examination did not reveal any lesions in the liver of female animal at 200 mg/kg bw/day showing macroscopic finding (1/1).



Recovery period
In the male animals, chronic progressive nephropathy was observed in one control (1/5) and in all male animals (5/5) of high dose group at the end of the recovery period.
One or both sides pyelectasia was also detected in male (1/5 control) and female (1/5 control and 1/5 at 800 mg/kg bw/day) animals.
Dilatation of uterine horns was observed in female animals of control (4/5) and high dose (4/5) groups at the end of the post-treatment observation period. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and could be in connection with the normal sexual cycle (proestrus phase) of uterus without pathological significance.
There was no morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver, the cardiovascular system, the respiratory system, the hematopoietic system, the lymphoid system, the skeleton, the muscular system, the central, or peripheral nervous system, the eyes, the integumentary system, the reproductive system in the selected animals.
The cyto-morphology of the endocrine glands was the same in the control and the treated animals.

Immunohistochemistry investigation revealed a statistically significant increase of α2-microglobulin in all male animals at 200, 800 mg/kg bw/day with respect to their control. There were no differences between test item-treated groups. The increased α2-microglobulin is deemed to be the explanation for renal changes noted in hematoxylin & eosin stained sections. Although, histological changes (CPN) were not detected at 50 mg/kg bw/day.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The estrous cycle was not affected by the test item at any dose level (50, 200 or 800 mg/kg bw/day).
Statistical difference was detected at the slightly shorter mean length of estrous cycle of female animals assigned for 50 mg/kg bw/day group during the pre-experimental period. This finding was indicative of biological variation and values were well within the historical control range. There were no significant differences between the control and treated groups in the number or percentage of animals with regular cycles, in the mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrus during the pre-mating period.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
The examined parameters of reproductive performance were not affected by the treatment with the test item in male or female animals at 50, 200 or 800 mg/kg bw/day.
Statistical significances were detected at the lower fertility indices (percentage of fertile animals) at 50 and 800 mg/kg bw/day and at the higher copulatory indices at 50, 200 and 800 mg/kg bw/day in male and female animals. The slight differences were due to the failure of mating of one control pair (copulatory index) and lack of conceiving in single females at 50 and 800 mg/kg bw/day. However, values met well the historical control and changes were independent from the test item.
The percentage of pregnant females, dams delivered, pregnants with liveborn(s) and the pre-coital interval and the mean number of conceiving days were comparable in female animals of control and test item treated animals (50, 200 and 800 mg/kg bw/day).

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed at the highest dose tested
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: sex and species specific finding with no human relevance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no adverse clinical signs in the offspring between post-natal days 0 and 13.
The percentage of cold and not suckled offspring was slightly higher at 800 mg/kg bw/day than in the control group on post-natal day 0 signs. However, these findings were not considered to be toxicologically relevant as the signs were transient – detected shortly after the delivery. Moreover, these observations were not associated with any influence on the development of the offspring. In the control group, pale and smaller than normal pup (1%, both) were also observed.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality.
Statistical significance was detected at the higher mean number of viable pups at 200 and 800 mg/kg bw/day on postnatal 13 and at the lower mean number of dead pups at 800 mg/kg bw/day between postnatal days 0 and 13. These minor differences were indicative of biological variation and was not related to the test item. The mean number of dead offspring (including missing pups) per litter was comparable in the control and test item treated groups on post-natal day 0.
The survival indices were similar in the control and test item treated (50, 200 or 800 mg/kg bw/day) groups. The mean number of live pups per litter and the mean number of viable pups per litter were comparable in all groups on postnatal days 0, 4 and 13.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight development of the offspring was undisturbed at 50, 200 and 800 mg/kg bw/day from birth up to post-natal day 13. Statistical significance with respect to the control was observed at the higher mean litter weight at 50 mg/kg bw/day on post-natal day 13.
The mean pup weight was higher than in the control group at 50 mg/kg bw/day on post-natal days 0 and 4 and at 200 mg/kg bw/day on post-natal day 0. The mean pup weight gain exceeded the control at 50 mg/kg bw/day gain between post-natal day 0 and 4.
There were no statistical significances between the control and 800 mg/kg bw/day groups in the mean litter weight or litter weight gain or in the mean pup weight or pup weight gain during the entire observation period.
Considering the offspring’s body weight in males and females, separately, statistically significant difference with respect to the control was detected for the higher mean pup weight (male and female) at 50 mg/kg bw/day on post-natal day 4.
The mentioned differences in the pup’s weight have no toxicological relevance. The slightly higher mean body weight and body weight gain was indicative of biological variation at the lower doses.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex distribution
There were no test item related differences between the control and test item treated groups in the ratio or in the litter means of genders on post-natal days 0, 4 or 13.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distances in male or female offspring or nipple retention in male offspring were not affected by the test item at 50, 200 or 800 mg/kg bw/day).
The anogenital distances, both absolute and normalized, were comparable in male pups in the control and test item treated groups.
Statistical significance was detected at the slightly longer anogenital distance of female pups at 50 mg/kg bw/day with respect to their control.
The normalized anogenital distance was similar in female pups in the control and 50 and 800 mg/kg bw/day groups. At 200 mg/kg bw/day, the normalized anogenital distance was slightly shorter than in the control group. This small difference with respect to the control was considered to be toxicologically not relevant as similar findings was not detected at the high dose group.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Nipples/areoles were not visible in any of the examined male offspring in the control or 50, 200 or 800 mg/kg bw/day groups on post-natal day 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Test item related macroscopic alterations were not found in offspring subjected to gross necropsy examination.
In the control group, empty stomach (1/3), milk in the stomach (2/3) and hemorrhage on the head (1/3) were noted for dead pups subjected to necropsy on post-natal days 1, 3 or 13.
At 50 mg/kg bw/day, empty stomach (3/3) was observed in three dead pups of one dam. In two pups subjected to necropsy after euthanasia (2/2) on post-natal day 14 or 15, both sides pyelectasia was detected.
At 200 mg/kg bw/day, malformed head (absence of mouth – orifice – and eyes) was observed in stillborn pup (1/1). Empty stomach was noted for dead pup (1/1) on postnatal day 12. In pups euthanized terminally, one or both side pyelectasia was detected (5/24).
At 800 mg/kg bw/day, there were no macroscopic findings in stillborn pup (1/1) and one side pyelectasia was observed in one male pup (1/22).
Empty stomach, hemorrhage on head, malformed head, are frequently observed alterations in pups of this strain of animals. These occurred in the control and lower dose groups, therefore were considered to be not related to the test item treatment of dams.

Histopathological findings:
no effects observed
Description (incidence and severity):
Histopathological examinations revealed pyelectasia in euthanized animals (2/22 at 50 mg/kg bw/day; 5/24 at 200 mg/kg bw/day; 1/22 at 800 mg/kg bw/day) independently from doses. Pyelectasia is a common finding in animals of this strain and were without degenerative, inflammatory or other histological (fibrotic etc.) lesion in euthanized pups
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at the highest dose tested

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the observations of an OECD 422 compliant study with the test item the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity of male rats:             50 mg/kg bw/day
NOAEL for systemic toxicity female rats:              800 mg/kg bw/day
NOAEL for reproductive performance of male/female rats:     800 mg/kg bw/day
NOAEL for F1 Offspring:               800 mg/kg bw/day
Executive summary:

The purpose of this combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was to obtain initial information on the toxic potential of the test item and its possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as development of the F1 offspring from conception to day 13 post-partum when repeatedly administered orally (by gavage) to parental animals at doses of 50, 200 and 800 mg/kg bw/day compared to control animals.

Four groups of Han:WIST rats (n=17/sex/group in the control and high dose groups; n=12 animals/sex in the low and middle dose groups) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 50, 200 and 800 mg/kg bw/day doses corresponding to concentrations of 0, 10, 40 and 160 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, sunflower oil. 5 animals/ sex in the control and high dose groups were observed for two weeks post-treatment (recovery) period.

The suitability of the vehicle at the intended concentrations of the test item was analytically verified up front. The concentration of the test item in the dosing formulations was checked two times during the study. The test item concentrations in the dosing formulations varied in the acceptable range – between 102 % and 108 % of the nominal values – confirming the proper dosing.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 62 days). Dams were additionally exposed through the gestation period and up to lactation days 13-16 (altogether for 51-65 days). One control dam was administered up to and including lactation day 12 due to over-anesthesia at the blood sampling for thyroid hormone determination. Animals of the recovery group were administered for 62 days and non-pregnant or not mated female animals were administered for 42 days.

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offspring. Five dams and the male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. Estrous cycle was monitored by examining vaginal smears before the treatment for two weeks and for two weeks from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating. Vaginal smear was also prepared on the day of the necropsy. Recovery animals were observed for mortality, clinical signs, body weight, food consumption (during the treatment and post-treatment periods), hematology, clinical chemistry, gross necropsy, organ weighing and histopathology (at the termination of the recovery period). The dams were allowed to litter, and rear their offspring up to day 13 post-partum. Litters were weighed and offspring were observed for possible abnormalities and were euthanized on post-natal day 13 or shortly thereafter.

Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4, TSH) from 2-7 pups per litter (where it was feasible) on post-natal day 4, from all dams and 1-6 pups per litter at termination on post-partum/post-natal day 13 – or shortly thereafter – and from all parent male animals at termination.

All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all adult male animals were determined. Histopathology examination was performed on reproductive organs (testes, epididymides, prostate and seminal vesicles with coagulating glands, uterus with cervix, vagina, ovaries) in the control and high dose groups and in one infertile pair (male and female) at 50 mg/kg bw/day. Additionally, full histopathology was performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations and in the recovery animals in the control and high dose groups. Based on macroscopic findings in male animals at 200 and 800 mg/kg bw/day (enlargement, uneven surface, paleness), the kidneys were also evaluated histologically in selected male animals of the low and mid dose groups as well as in male animals with macroscopic renal findings at 200 and 800 mg/kg bw/day. Immunohistochemistry examinations were also performed for determining α2µ-globulin in these kidneys – in selected male animals and animals with macroscopic renal changes.

Organs showing macroscopic findings at necropsy (thymus, liver, female kidneys, skin, submandibular lymph nodes) were also processed and evaluated histologically in animals of control, 50, 200 and 800 mg/kg bw/day).

The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only. Historical control data were also considered.

 

There was no mortality at 50, 200 or 800 mg/kg bw/day groups during the course of study. Adverse clinical signs of systemic toxicity related to the test item were not detected at any dose level at the daily or detailed weekly clinical observations or at the terminal functional observations.

The behavior and physical condition of animals was not affected by the test item at any dose level (50, 200 or 800 mg/kg bw/day) during the entire observation period. Salivation and nuzzling up the bedding material noted for 800 mg/kg bw/day dose treated animals (male and female) were with short duration after the daily treatment and transient occurrence. Therefore, these signs were considered to be toxicologically not relevant.

The body weight development was not adversely affected by the test item in male or female animals at 50, 200 or 800 mg/kg bw/day during the entire treatment period. A slightly reduced mean body weight gain resulted in minor changes only in the mean body weight (≥ - 5% of the control) in male animals at 800 mg/kg bw/day. Therefore, the slight changes in body weight and body weight gain at 800 mg/kg bw/day were considered to be of little or no toxicological relevance.

There were no test item related adverse changes in the mean daily food consumption of male and female animals at any dose level (50, 200 and 800 mg/kg bw/day). Slight reduction in the daily mean food consumption of male and female animals at 800 mg/kg bw/day during was transient – during the first week of the treatment only – and was with minor degree. Therefore, this finding was judged to be toxicologically not relevant.

A test item influence on the estrous cycle was not found at any dose level (50, 200 and 800 mg/kg bw/day). Delivery data of dams. There were no toxicologically relevant differences between the control and test item treated groups (50, 200 and 800 mg/kg bw/day) in the evaluated delivery parameters of dams.

The examined parameters of reproductive performance were not affected by the treatment with the test item in male or female animals at 50, 200 or 800 mg/kg bw/day.

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 50, 200 or 800 mg/kg bw/day. Specific alterations related to test item effect were not detected in the examined clinical chemistry parameters at any dose level (male or female; 50, 200 or 800 mg/kg bw/day).

There were no test item related changes in the serum thyroid hormone (FT4, TSH) levels at any dose (parental male animals or PN13 offspring). Macroscopic alterations related to the effect of the test item were not detected in the reproductive organs of male or female animals at 50, 200 or 800 mg/kg bw/day at the necropsy. Test item related changes were detected in the kidneys in male animals at 200 mg/kg bw/day (enlargement, paleness, uneven surface) and at 800 mg/kg bw/day (enlargement, paleness) at the terminal necropsy being in full accordance with the organ weights and histopathological findings. These renal findings (enlargement and paleness) were observed in male animals at 800 mg/kg bw/day with a lower incidence at the end of the recovery period.

Significant elevation of the liver weights (at 800 mg/kg bw/day, male and female) and kidneys weights (at 200 mg/kg bw/day, male and at 800 mg/kg bw/day, male and female) were observed in selected animals at the termination of the treatment. Liver weight changes were considered to be sign of adaptation process of the organ in the lack of related clinical chemistry or histopathological alterations. Changes in weights of liver (male and female) and kidneys (male) were also observed in a lower degree at the end of the recovery period i.e. the organ weight changes were not fully reversible.

Histopathological examinations of the investigated reproductive organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 800 mg/kg bw/day.

Chronic progressive nephropathy (CPN) was revealed in male animals at 200 and 800 mg/kg bw/day by histological investigations in compliance with macroscopic findings at the necropsy. Chronic progressive nephropathy is an important spontaneous renal disease of the commonly used strains of laboratory rat. However, regarding the significantly higher incidence of CPN in the male animals, a nephrotoxic effect of the test item might be supposed at 200 and 800 mg/kg bw/day and the test item could be considered as a predisposing factor in the pathogenesis of renal lesion in the investigated male animals. This renal change was not reversible as CPN was also observed in all male animals at 800 mg/kg bw/day at the end of the post-treatment observation period.

Immunohistochemistry investigation revealed a statistically significant increase of α2-microglobulin in male animals at 50, 200 and 800 mg/kg bw/day when compared to their control. There were no differences between test item-treated groups.

The offspring’s development was undisturbed at 50, 200 and 800 mg/kg bw/day from birth to post-natal day 13. No effect on the mortality, clinical signs, body weight development, anogenital distance (male and female) or nipple retention (male) were detected.

 

Under the conditions of the present study, the test item did not adversely influence the fertility or reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female Han:WIST rats at 50, 200 and 800 mg/kg bw/day doses administered by oral gavage.

200 and 800 mg/kg bw/day induced chronic progressive nephropathy in male animals associated with accumulation of α-2μ-globulin in the kidneys, macroscopic (enlargement, uneven surface, paleness) and organ weight changes, which were not reversible after two weeks post-treatment observation period.

At 50 mg/kg bw/day, elevated level of α2-microglobulin was revealed in male animals.

The development of the F1 offspring was not impaired at any dose level from birth to post-natal day 13 after repeated oral administration of dams.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for systemic toxicity of male rats:             50 mg/kg bw/day

NOAEL for systemic toxicity female rats:              800 mg/kg bw/day

NOAEL for reproductive performance of male/female rats:     800 mg/kg bw/day

NOAEL for F1 Offspring:               800 mg/kg bw/day