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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-11-28 to 2019-07-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
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

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

OECD 422

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. Recovery animals were not involved in mating procedure.

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. 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. In addition, for five males and females randomly selected from each group and for recovery animals, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.

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 reduced mean body weight gain in the first two weeks of treatment resulted only in minor changes in the mean absolute body weight (≤ - 5% of the control) in male animals at 800 mg/kg bw/day and was thus considered to be not adverse and consequently 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 was transient – during the first week of the treatment only – and was of 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).

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. Since CPN is a finding commonly seen in male rats without a human counterpart it is judged as effect of no human relevance (Hard et al. 2013).

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 statistically significant differences between test item-treated groups or a dose –response relationship detectable. The increased α2-microglobulin accumulation in the kidneys of male rats is commonly known to be a sex and species-specific phenomenon and as such not of human relevance (Swenberg, 1993).

The offspring’s development was undisturbed at 50, 200 and 800 mg/kg bw/day from birth to post-natal day 13. No effects 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.

These findings are known to be sex and species specific and are of no human relevance.

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 (without human relevance): 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

 

Based on the observations of an OECD 422 compliant study with the test item the NOAEL for F1 Offspring was 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

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 reproduction toxicity 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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Pyrolysis Reaction Product of Pinane
EC Number:
947-964-7
Molecular formula:
n. a. for UVCB
IUPAC Name:
Pyrolysis Reaction Product of Pinane

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 or test system 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 oral 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
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

Observations and examinations performed and frequency:
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
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, after examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites was counted and recorded.
The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, and all organs showing macroscopic lesions of all adult animals will be preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
All organs showing macroscopic lesions and the organs in table No 5 were preserved for five male and five female animals randomly selected from each group.

HISTOPATHOLOGY: Yes
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".
Other examinations:
Kidneys of male animals showing macroscopic alterations or weight changes compared to control animals were examined for an increase of alpha 2 µ globulin. The objective of this immunohistocemical evaluation was to evaluate the possible reason for the renal changes observed in male rats.
Statistics:
The statistical evaluation of appropriate data was be 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 were significant, Duncan Multiple Range test was used to access the significance of inter-group differences.

Getting significant results at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons was performed using Mann-Whitney U-test. Chi2 test will be performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Results and discussion

Results of examinations

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.
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
Description (incidence and severity):
Evaluation of absolute and relative organ weights revealed higher weights of liver at 800 mg/kg bw/day (male and female) and kidneys at 200 mg/kg bw/day (male) and at 800 mg/kg bw/day (male and female). Changes in liver and kidneys weight were indicative of test item influence both in male and female groups.

Treatment period
In male animals at 50 mg/kg bw/day, the mean kidneys weight relative to body weight exceeded the control.
The weights of all examined organs were comparable with their control in female animals at 50 mg/kg bw/day.
At 200 mg/kg bw/day, statistically significant difference with respect to the control was detected at the higher mean liver weight relative to body weight and at the higher mean kidney weights (absolute and relative to body and brain weights).
The weights of all examined organs were comparable to their control in female animals at 200 mg/kg bw/day.
At 800 mg/kg bw/day, the mean fasted body weight of male animals was lower than in the control, the mean liver and kidney weights (absolute and relative to body and brain weights) of male animals were significantly higher than in the control group.
Statistical significance with respect to the control was observed at the higher mean weights of liver (absolute and relative to body and brain weights) and kidneys relative to body weight in female animals at 800 mg/kg bw/day. The absolute kidneys weight and kidneys weight relative to brain weight were also higher than in the control group in female animals at 800 mg/kg bw/day without statistical significance.

Recovery period
Statistical significance with respect to the control was detected at the higher mean weight of liver and adrenal glands (absolute and relative to body and brain weights), at the higher mean kidney and heart weights (relative to body and brain weights) and at the lower mean testes weights (absolute and relative to brain weight) at the end of the recovery period.
In the female animals of recovery group at 800 mg/kg bw/day, the mean liver weights (absolute and relative to body weight) were higher than in the control group. The weight of all other examined organs was comparable in the control and high dose treated female animals at the end of the post-treatment observation period.
The changes in kidneys weights of male animals were related to doses in full correlation with necropsy and histopathological findings at 200 and 800 mg/kg bw/day referring to test item influence.
Slight elevation of kidney weights in female animals at 800 mg/kg bw/day were probably due to the adaptation function of the organ in the lack of related macroscopic or histological alterations.
Similarly, the liver weight changes in male and female animals were related to a test item influence on the hepatic function. However, there were no supporting findings in the clinical chemistry parameters or histopathological examinations therefore, changes in liver weight were considered to be indication of the adaptation process and the toxicological significance is equivocal.
The 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. test item influence was not fully reversible after 14-day post-observation period. Necropsy and histopathology findings supported changes in the kidney weights in male animals.
Minor changes in the weights of testes, adrenal glands and heart of high dose treated male animals were considered to have no or little toxicological relevance as similar findings were not detected at the end of the treatment period and there were no accompanying histopathological lesions in the se organs.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
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 necropsy being in full accordance with the histopathological findings.

Treatment period
In the control group, one or both sides pyelectasia (1/12 male, 2/11 dam) and alopecia on the skin of chest and legs (1/11 dam) were observed. There were no macroscopic findings in not mated (1/1) control female animal.
At 50 mg/kg bw/day, one or both sides pyelectasia (1/12 male, 1/11 dam), thymic hemorrhage (1/12 male) and Hernia diaphragmatica (1/11 dam) were detected. There were no macroscopic findings in non-pregnant female animal at 50 mg/kg bw/day.
At 200 mg/kg bw/day, one or both side pyelectasia (5/12 male, 5/12 dam), enlarged kidneys (3/12 male), uneven surface of the kidneys (2/12), pale kidneys (3/12 male), pale spotted liver (1/12 dam), hemorrhage in the submandibular lymph nodes (2/12) were seen at the terminal necropsy.
At 800 mg/kg bw/day, gross necropsy revealed renal changes in male animals – 2/12 one side pyelectasia, 12/12 enlargement, 9/12 paleness – and in female animals – 5/11 one or both sides pyelectasia, 1/11 paleness. In non-pregnant female animal (1/1) at 800 mg/kg bw/day, marked hydrometra was observed.

Recovery period
Renal pyelectasia (1/5 control), enlarged kidneys (2/5 at 800 mg/kg bw/day), pale kidneys (1/5 at 800 mg/kg bw/day) and pale liver (1/5 at 800 mg/kg bw/day) were observed at the necropsy of male animal at the end of the recovery period.
In the female animals, one or both sides pyelectasia (1/5 control, 1/5 at 800 mg/kg bw/day) and slight or marked hydrometra (4/5 control and 4/5 at 800 mg/kg bw/day) were detected at the termination of the post-treatment observation period.
Renal pyelectasia occurred with high incidence in this study – especially at 200 mg/kg bw/day, male and female – but without dose dependency and histopathologic lesion. Therefore, this finding was considered as a common one without toxicological significance.
Hemorrhage in the thymus or lymph nodes was probably due to circulatory disturbance developed during exsanguination procedure.

Pale spotted liver and pale kidneys in female animals were considered as an individual finding in the lack of supporting histopathological changes and not caused by test item treatment.
Hernia diaphragmatica and dermal alopecia are common findings in untreated experimental rats of this strain of and are toxicologically not relevant.
Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. In the lack of related histopathological alterations (inflammatory or another pathological lesion) these were judged not to be toxicologically relevant.
Neuropathological findings:
not examined
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
Description (incidence and severity):
Estrus cycle
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.

Serum levels of Thyroid Hormones
There were no differences with respect to the control in the FT4 and TSH hormone levels in parental male animals or in offspring sampled on postnatal day 13 at any dose levels.

Effect levels

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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:
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: sex and species specific finding with no human relevance

Target system / organ toxicity

Key result
Critical 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. Recovery animals were not involved in mating procedure.

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. 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. In addition, for five males and females randomly selected from each group and for recovery animals, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.

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 reduced mean body weight gain in the first two weeks of treatment resulted only in minor changes in the mean absolute body weight (≤ - 5% of the control) in male animals at 800 mg/kg bw/day and was thus considered to be not adverse and consequently 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 was transient – during the first week of the treatment only – and was of 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).

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. Since CPN is a finding commonly seen in male rats without a human counterpart it is judged as effect of no human relevance (Hard et al. 2013).

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 statistically significant differences between test item-treated groups or a dose –response relationship detectable. The increased α2-microglobulin accumulation in the kidneys of male rats is commonly known to be a sex and species-specific phenomenon and as such not of human relevance (Swenberg, 1993).

The offspring’s development was undisturbed at 50, 200 and 800 mg/kg bw/day from birth to post-natal day 13. No effects 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.

These findings are known to be sex and species specific and are of no human relevance.

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 (without human relevance): 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