Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-20 to 2018-09-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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 OPPTS 870.3650 (Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening)
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Batch No.: D1608206

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for toxicity and reproduction toxicity studies and the test guidelines were designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in toxicity and reproduction toxicity studies and well-known fertility parameters.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90., H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 12 wks
- Weight at study initiation: (P) Males: 338 - 408 g; Females: 190 - 238 g
- Housing:
Before mating: 2 animals of the same sex/cage
Mating hours: 1 male and 1 female/cage
Pregnant females were housed individually
Males after mating: 2 animals/cage

- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2018-06-20 To: 2018-08-26

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle in concentrations of 2, 6 and 20 mg/mL. The pH of the formulations was adjusted to pH= 7-8 with 10 N HCl. Formulations were prepared in the formulation laboratory of the Test Facility daily not longer than for four hours before the use.

VEHICLE
- Concentration in vehicle: 2, 6, 20 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug / 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.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Pregnant females were housed individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulations was performed in the Analytical Laboratory of Test Facility. Five samples were taken from different places from each concentration (Groups 2, 3 and 4) and at least three replicates were measured on 2 occasions, during the sixth and eighth treatment week. Similarly, five samples were taken from the control solution (Group 1) from different places and analyzed.
Concentration of the test item in the dosing formulations varied in the range of 93 and 109% of the nominal values at both analytical occasions.
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Recovery of CeTePox® 0214 H from distilled water was 105% and 103% of nominal concentrations at ~1 mg/mL and ~25 mg/mL, respectively.
CeTePox® 0214 H was stable in distilled water at the intended concentrations for at least three days at room temperature or in a refrigerator. A separate analytical report provided these results.
Duration of treatment / exposure:
Males: 49 days (14 days pre-mating and 14 days mating plus an optional extended post-mating period)
Females: 50 - 67 days depending on day of mating (14 days pre-mating, during mating period, through gestation and up to lactation days 13-17)
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day
Remarks:
2 mg/mL
Dose / conc.:
30 mg/kg bw/day
Remarks:
6 mg/mL
Dose / conc.:
100 mg/kg bw/day
Remarks:
20 mg/mL
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting was based on findings obtained in a 14-day dose range finding study performed with CeTePox® 0214 H (study no. 902-400-2833, non-GLP) and in agreement with the Sponsor. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily (twice daily for morbidity; mortality)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing (Day 0), weekly thereafter and on the day of necropsy
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on post-partum day 0 (within 24 hours after parturition), 4 and 13. Additionally, female animals were weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not evaluated statistically.

FOOD CONSUMPTION:
- Time schedule: once weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the administration period, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus.
- Anaesthetic used for blood collection: Isoflourane
- Animals fasted: Yes, over night (16 hours)
- How many animals: 5 /sex/dose (randomly selected from each dosing group)
- Parameters checked: White Blood Cell (leukocyte) count (WBC), Red Blood Cell (erythrocyte) count (RBC), Hemoglobin concentration (HGB), Hematocrit (relative volume of erythrocytes) (HCT), Mean Corpuscular (erythrocyte) Volume (MCV), Mean Corpuscular (erythrocyte) Hemoglobin Concentration (MCHC), Mean Corpuscular (erythrocyte) Hemoglobin (MCH), Platelet (thrombocyte) count (PLT), Reticulocytes (RET), Differential white blood cell count (NEU, EOS, BASO, LYM, MONO), Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the administration period, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus.
- Anaesthetic used for blood collection: Isoflourane
- Animals fasted: Yes, over night (16 hours)
- How many animals: 5 /sex/dose (randomly selected from each dosing group)
- Parameters checked: Alanine Aminotransferase activity (ALT), Aspartate Aminotransferase activity (AST), Gamma Glutamyltransferase activity (GGT), Alkaline Phosphatase activity (ALP), Total Bilirubin concentration (TBIL), Creatinine concentration (CREA), Glucose concentration (GLUC), Cholesterol concentration (CHOL), Urea concentration (UREA), Calcium concentration (Ca++), Sodium concentration (Na+), Potassium concentration (K+), Chloride concentration (Cl-), Total Protein concentration (TPROT), Albumin concentration (ALB), Albumin/globulin ratio (A/G)

DETERMINATION OF SERUM LEVELS OF THYROID HORMONES: Yes
- Anaesthetic used for blood collection: Isoflourane
- Animals fasted: Yes, over night (16 hours)
- Time schedule and animl number:
- from at least two pups per litter on post-natal day 4, if it was feasible;
- from all dams and at least two pups per litter on day 13;
- from all parent male animals at termination.

- Parameters checked: thyroid hormones T4 (Thyroxine – free Tetra-iodothyronine) and TSH (Thyroid-stimulating hormone)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At the end of the administration period
- Dose groups that were examined: 5 parental males and females per group
- Battery of functions tested: a functional observational battery was performed and motor activity was measured (modified Irwin test (1968) was performed)
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. 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. Vaginal smear was also prepared on the day of the necropsy.
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, runts, presence of gross anomalies, 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 determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [after the optionally extended post-mating period on Day 49]
- Maternal animals: All surviving animals
- Non-pregnant female animals: on Day 49.
- Dams not selected for toxicology examinations: on post-partum day 13 or shortly thereafter (Days 49, 50, 51, 55, 56 or 67).
- Dams selected for toxicology examinations: shortly after post-partum day 13 (Day 55).


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

HISTOPATHOLOGY
The tissues indicated in Table [#1] were prepared for microscopic examination and weighed, respectively. Detailed histological examinations 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 non-pregnant females (no. 226 and 330), respective male mating partners of these females in the low (no. 206) and mid (no. 310) dose groups and one not mated male (no. 312) in the mid dose and its change male for the same group (no. 311).
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals (n=5 animals/sex/group) in the control and high dose groups.
The kidneys of one male animal (no. 301) and of one female animal (no. 323) at 30 mg/kg bw/day and the uterus of one female animal (no. 222) at 10 mg/kg bw/day were also processed and examined histologically due to the necropsy observations: pyelectasia and hydrometra, respectively.

ORGAN WEIGHTS
At the time of termination, fasted body weight (all male animals) and weight of the testes, epididymides as well as prostate and seminal vesicles with coagulating glands as a whole of adult male animals were determined. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 13 days of age or shortly thereafter.
- These animals were subjected to postmortem examinations as follows:
Dead pups and pups euthanized on post-natal day 13 or shortly thereafter, were carefully examined for gross abnormalities at least externally. Particular attention was paid to the external reproductive genitals. Any pups showing abnormalities in structure or behavior were subjected to necropsy by macroscopic examination.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid gland was preserved from one male and one female pup per litter (at day 13) for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.
Statistics:
The statistical evaluation of appropriate data (marked with † above) 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, Fertility Index
Female: Copulatory Index, Fertility Index, Gestation Index, Live Birth Index, Postimplatation mortality
Offspring viability indices:
Viability index, Survival Index, Sex Ratio, Anogenital Index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
Adverse signs of systemic toxicity related to the test item were not detected at any dose level at the daily clinical observations (10, 30 and 100 mg/kg bw/day, male or female).
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development was reduced in male and female animals administered with 100 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption was reduced in male and female animals at 100 mg/kg bw/day in compliance with the body weight changes.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
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 10, 30 or 100 mg/kg bw/day.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly elevated enzyme activity of alanin aminotransferase and aspartate aminotransferase was detected in male animals at 100 mg/kg bw/day presumably in accordance with the altered hepatic function.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observations did not demonstrate any test item related adverse 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 (control, 10, 30 and 100 mg/kg bw/day; n=5 animals/sex/group).
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 revealed test item related changes (centrilobular vacuolation in the hepatocytes) in the liver at 100 mg/kg bw/day in male animals.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
The thyroid hormone (free T4 and TSH) levels were not affected in the parental male animals or in PN13 offspring at any dose levels.

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 (10, 30 or 100 mg/kg bw/day).
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 10, 30 or 100 mg/kg bw/day.

Details on results (P0)

MORTALITY
There was no mortality in the control, 10, 30 or 100 mg/kg bw/day groups (male or female) during the course of study.

CLINICAL OBSERVATIONS
Daily Observations:
Some transient clinical signs – activity decrease and piloerection – were noted for some animals at 100 mg/kg bw/day. Test item influence was supposed in development of these signs. However, due to the minor degree, short duration and low incidence, these findings were judged to be not adverse. There were no clinical signs in animals in the control (12/12 males) group. In one control female animal (1/12), alopecia was observed on the fore limbs from gestation day 16 until termination. At 10 mg/kg bw/day, red colored hair around the right eye (1/12) was detected in one male animal (1/12) between Days 38 and 48. There were no clinical signs in female animals at 10 mg/kg bw/day (12/12) during the pre-mating, mating, gestation, lactation or post-mating periods. Male animals administered with 30 mg/kg bw/day (12/12) were symptom-free during the entire observation period. Alopecia was observed on the fore limbs of one dam at 30 mg/kg bw/day (1/11) between lactation days 2 and 13. In the male animals at 100 mg/kg bw/day, slightly decreased activity and piloerection was observed (4/12) for some days (started on Day 7 and ceased between Days 9 and 20. In two female animals at 100 mg/kg bw/day, piloerection was observed between Days 7 and 9 (1/12) as well as between gestation days 20 and 21 (1/12) Alopecia and red colored hairs around the eye are species specific findings, which are also observed in untreated experimental rats of this strain with similar age. These were individual findings with low incidence in animals of control or lower dose groups and were not related to the treatment. Red colored hair around the eye probably was indicative of enhanced production of porphyrin in lachrymal/Harderian gland of this animal.

Detailed weekly observations:
The behavior and physical condition of animals was not adversely affected by the test item at any dose level (10, 30 or 100 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period. The above described clinical signs were also detected at the detailed weekly clinical observations as follows:
- Alopecia on the forelimbs on one control dam (1/12) on gestation day 21, on lactation days 0, 4 and 13;
- Red colored hair around the right eye at 10 mg/kg bw/day (1/12 male) on Days 41 and 48;
- Alopecia on the forelimbs in one dam at 30 mg/kg bw/day on lactation days 4 and 13;
- Slightly decreased activity and piloerection in male animals at 100 mg/kg bw/day on Day 7 (4/12) and on Day 13 (2/12);
- Piloerection in two dams at 100 mg/kg bw/day on Day 7 and on gestation day 21, respectively;

FUNCTIONAL OBSERVATIONS
Functional observations did not demonstrate any test item related adverse 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 (control, 10, 30 and 100 mg/kg bw/day; n=5 animals/sex/group).
BODY WEIGHT
The mean body weight and body weight gain were comparable to their control in male and female animals at 10 mg/kg bw/day and in male animals at 30 mg/kg bw/day groups during the entire treatment period. In the female animals at 30 mg/kg bw/day, statistical significance was observed with respect to the control at the lower mean body weight on gestation days 0 and 7 and on lactation days 0, 4 and 13. The mean body weight gain of this group was slightly lower than the control – no statistical significance – during the premating and gestation periods and was comparable to the control during the lactation period.
In the male animals at 100 mg/kg bw/day, the mean body weight was slightly lower than in their control from Day 7 up to the termination of the study. The mean body weight gain was also significantly reduced in this group on weeks 1 and 2 and between Days 0 and 48.
In the female animals at 100 mg/kg bw/day, the mean body weight was lower than in the control group on Day 7 and during the entire gestation and lactation period (on gestation days 0, 7, 14 and 21 and on lactation days 0, 4 and 13). The mean body weight gain of this group was significantly lower than in the control group during the first week of the treatment period resulting in significantly lower summarized mean body weight gain during the pre-mating period (between Days 0 and 13). The mean body weight gain was also lower than in the control during the gestation and lactation periods reaching statistical significance between gestation days 14 and 21 as well as for the entire gestation period (between gestation days 0 and 21).

FOOD CONSUMPTION
There was no significant difference between the control and 10 mg/kg bw/day groups (male and female animals) in the mean daily food consumption. At 30 mg/kg bw/day, the mean daily food consumption was similar to their control in male animals. In female animals at 30 mg/kg bw/day, lower mean daily food consumption was noted when compared to the control on the first week of gestation period. The daily food mean consumption was lower than in the control in male animals at 100 mg/kg bw/day during the entire treatment period attaining statistical significance on weeks 1, 2, 5 and 6 (between Days 0-7, 7-13, 34-41 and between Days 41 and 48). The daily mean food consumption of female animals at 100 mg/kg bw/day was also lower than in the control during the entire observation period attaining statistical significance during the first week of the pre-mating period, during the first week of the gestation period and between lactation days 4 and 13.

ESTROUS CYCLE
There were no statistically significant differences between the control and test item 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-estrus, estrous or diestrus during the pre-experimental or pre-mating periods.

DELIVERY DATA OF DAMS
Delivery data of dams was affected at 100 mg/kg bw/day dose level. Lower mean number of implantation sites and lower mean birth (total birth, live born and viable pups per litter) were observed at the high dose treated animals.
Statistical significance was noted with respect to the control for the slightly shorter mean duration of pregnancy at 30 mg/kg bw/day. This slight difference was considered to have no toxicological relevance.
Statistically significant difference was observed at 100 mg/kg bw/day when compared to their control in the lower mean number of implantations, mean number of total birth, liveborns and consequently in the lower mean viable pups on day 0. All these values were well below the historical control data and seem to correlate with the lower mean body weight of the female animals during the entire gestation period.
The number of pregnant females and dams delivered, the live birth index was comparable in all groups and the differences between control and test item treated doses did not obtain any statistical significance.

REPRODUCTIVE PERFORMANCE
Statistical significance was observed at the lower copulatory index of male animals at 30 mg/kg bw/day because one male animal (1/12) in this group failed to mate within 14 days. The fertility index of male animals at 10 and 30 mg/kg bw/day was statistically significantly lower than in the control group as one mated male animal in each group (1/12 at 10 mg/kg bw/day and 1/12 at 30 mg/kg bw/day) did not fertilize its female partner. In the female animals at 10 and 30 mg/kg bw/day, the fertility index was slightly lower than in the control group as one sperm positive female at 10 and 30 mg/kg bw/day group (1/12, each) was not pregnant. These minor changes in the copulatory or fertility indices (male or female) at 10 and/or 30 mg/kg bw/day were considered to be not related to the test item because similar incidences also occur in non-treated experimental rats of this strain, moreover there was no dose relevance.

HEMATOLOGY AND BLOOD COAGULATION
The examined hematological parameters were comparable in male animals in the control and 10 mg/kg bw/day, in female animals in the control and 10 and 30 mg/kg bw/day groups. In the male animals at 30 mg/kg bw/day, statistical significances were detected for the lower mean percentage of eosinophil granulocytes (EOS), hemoglobin concentration (HGB), mean corpuscular (erythrocyte) volume (MCV) and for the slightly shorter mean activated partial thromboplastin time (APTT). In male animals at 100 mg/kg bw/day, the mean percentage of eosinophil granulocytes, mean hemoglobin concentration, mean corpuscular (erythrocyte) volume were lower and mean percentage of reticulocytes (RET) was higher at 100 mg/kg bw/day when compared to the control. In female animals at 100 mg/kg bw/day, statistically significantly higher mean percentage of reticulocytes was detected. The differences with respect to control were judged to be toxicologically not relevant due to the minor degree (HGB, MCV) and in the lack of dose relevance (HGB, APTT). The individual values of EOS were within or marginal to the historical control range while the control mean was well above the historical control mean, therefore the differences in this parameter were considered to have little or no toxicological relevance. The mean and individual values of RET in male animals at 100 mg/kg bw/day were within the historical control range however the values were below the range in several cases in the control group. In female animals, the individual values of RET at 100 mg/kg bw/day exceeded the upper limit of the historical control range in 4/5 cases. As there were no related findings (signs of anemia, hemolysis or hemorrhage, etc.) these changes in male and female animals were considered to be non-adverse.

CLINICAL CHEMISTRY
The examined clinical chemistry parameters were comparable in the control and test item treated male animals at 10 mg/kg bw/day. In female animals at 10 mg/kg bw/day, statistical significance was detected with respect to their control at the lower mean concentrations of total bilirubin (TBIL) and potassium (K+) and at the higher mean creatinine concentration (CREA). There were no significant differences between the control and 30 mg/kg bw/day treated animals (male and female) in the investigated clinical chemistry parameters. Statistical significance was observed in male animals at 100 mg/kg bw/day at the higher mean enzyme activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), at the lower mean glucose (GLUC) and potassium (K+) concentrations. There were no related findings, therefore these differences were judged to have no toxicological relevance. Moreover, the individual values were within (K+) or marginal (GLUC) to the historical control range. These differences were independent from doses therefore were judged to be indicative of biological variation and not related to the test item.

SERUM LEVELS OF THYROID HORMONES
Statistically significant difference with respect to the control was noted for the slightly lower free T4 level in parental animals at 100 mg/kg bw/day. However, this slight difference was considered to be toxicologically not relevant as individual values corresponded with the historical control (2.72±0.39 ng/dL; n=96; min: 1.91 ng/dL; max = 3.86 ng/dL). Moreover, there were no histological changes in the organs of hypothalamic-pituitary-thyroid axis of examined animals at 100 mg/kg bw/day.

NECROPSY
In one control male animal (1/12) left side pyelectasia was detected. Pyelectasia was noted for two dams (2/12, left or right side) and alopecia on the fore limbs for another one (1/12) in the control group. There were no macroscopic changes in the organs or tissues of male animals at 10 mg/kg bw/day (12/12) Moderate hydrometra was observed in one dam (1/11) and in one non-pregnant female animal at 10 mg/kg bw/day. At 30 mg/kg bw/day, pyelectasia was detected on the left side (1/12 male) or both sides (1/11 dam). In male animals at 100 mg/kg bw/day, pale liver (2/12), both sides pyelectasia (2/12) and alopecia on the forelimbs (1/12) were seen at the necropsy. The organs and tissues of all dams at 100 mg/kg bw/day (12/12) were judged to be normal. Pyelectasia and alopecia are common macroscopic finding in experimental rats of this strain with similar age. Pyelectasia was without degeneration, inflammation or fibrosis therefore this finding was considered as slight individual lesion without toxicological significance. 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) hydrometra was judged not to be toxicologically relevant.

ORGAN WEIGHT
In the male animals administered with 10 mg/kg bw/day, the weights of the examined organs (absolute, relative to body and brain weights) were comparable with their controls. In the female animals at 10 mg/kg bw/day, statistical significance was detected at the slightly lower mean brain weight and at the slightly higher mean spleen weights relative to body and brain weights with respect to the control. At 30 mg/kg bw/day, in male animals, the mean weights of epididymides (absolute and relative to body weight) and mean weight of seminal vesicles (absolute and relative to body and brain weight) were lower than in the control group. In the female animals at 30 mg/kg bw/day, the fasted mean body weight and the mean brain weights were below the control value. In the male animals at 100 mg/kg bw/day, the fasted mean body weights (absolute and relative to brain weight) were statistically significantly lower than their control. As a consequence, the main brain weight relative to body weight of these animals exceeded the control value group. Statistically significant difference with respect to the control was also detected at the higher mean liver weights (absolute and relative to body and brain weights), at the higher mean weights of kidneys (relative to body and brain weights) and higher mean testes weight relative to body weight, as well as at the lower mean weights of seminal vesicles (absolute and relative to brain weight) in the high dose treated male animals. In the female animals at 100 mg/kg bw/day, the fasted mean body weight, the mean brain weights and the mean weight of heart were slightly lower than their control. These changes were not considered to be toxicologically relevant in the lack of associated histopathological alterations.

HISTOPATHOLOGY
There were no pathologic changes in the examined reproductive organs or tissues of male or female animals (control and 100 mg/kg bw/day). In selected animals, alveolar emphysema in minimal degree was observed in the lungs of one male animal in the control (1/5). This finding was considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure. Hyperplasia of bronchus associated lymphoid tissue (BALT) was noted in two animals in the control group (1/5 male, 1/5 dam). Hyperplasia of BALT is a physiological immune-morphological phenomenon, without toxicological significance. Atrophy of hair follicles was observed for one male animal at 100 mg/kg bw/day (1/5) which could be in connection with the macroscopically visible focal alopecia. Pyelectasia, without degenerative, inflammatory or other histopathological lesions, (one side or both sides) observed in some animals (1/5 male and 2/5 dams in the control, 1/1 male and 1/1 dam at 30 mg/kg bw/day, 2/5 males at 100 mg/kg bw/day) is a common finding in Wistar rats, without toxicological significance. Mild or moderate (grade 2 or 3) centrilobular vacuolation in the hepatocytes was observed in five male animals at 100 mg/kg bw/day (5/5). This phenomenon could be considered as hepatic lipidosis, and it cannot be excluded that the test item treatment played a role in the development of this lesion. Hepatic lipidosis could be considered as a light reversible liver injury and it could be in connection with a disturbance of energy metabolism of affected hepatocytes. In the male animals, 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 (12/12 control; 12/12 at 100 mg/kg bw/day; 1/1 not mated male at 30 mg/kg bw/day; two male pairs of the non-pregnant females: 1/1 at 10 mg/kg bw/day and 1/1 at 30 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 treated animals. The histological picture of epididymides, seminal vesicles, and coagulating glands was normal in all animals as well. In the female animals, 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 (12/12 control; 12/12 at 100 mg/kg bw/day, 1/1 non-pregnant female at 10 mg/kg bw/day and 1/1 non-pregnant female at 30 mg/kg bw/day). 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. Dilatation of uterine horns was observed in two females (1/11 dam at 10 mg/kg bw/day, 1/1 non-pregnant female at 10 mg/kg bw/day). This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance. There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis, etc.) of the stomach, the small and large intestines, the pancreas, the cardiovascular system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central or peripheral nervous system in the selected animals. The cyto-morphology of the endocrine glands was the same in the control and the treated animals.

Effect levels (P0)

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Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance

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.
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no test item related effect on offspring’s extra uterine mortality.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
A test item related effect on the body weight development of the offspring was not detected.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distances in male or female offspring were not affected by the test item at 10, 30 and 100 mg/kg bw/day.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The nipple retention in male offspring was not affected by the test item at 10, 30 and 100 mg/kg bw/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.

Details on results (F1)

EXTRA UTERINE MORTALITY
The mean number of dead offspring (including missing pups) per litter at 100 mg/kg bw/day was slightly but statistically significantly lower than in the control group between post-natal day 0 and 13.

SEX DISTRIBUTION AND SURVIVAL OF OFFSPRING

The mean number of male pups per litter was slightly lower than in the control group at 100 mg/kg bw/day on post-natal day 0. The mean number of liveborns per litter was statistically significantly lower at 100 mg/kg bw/day than in the control. Consequently, the mean number of viable pups per litter were lower at 100 mg/kg bw/day on post-natal days 0 and 4 and the mean number of pups euthanized on post-natal day 4 was lower when compared to the control. The mean number of viable pups per litter was comparable in all groups on post-natal day 13 after litter size adjustment.
There were no significant differences between the control and test item treated (10, 30 or 100 mg/kg bw/day) groups in the survival indices.

CLINICAL OBSERVATIONS
The percentage of offspring with signs (cold and not suckled) at 10, 30 and 100 mg/kg bw/day was lower than in the control group on post-natal day 0. These findings were transient – detected shortly after the delivery.
Occasionally, other clinical signs were also observed in the control group: pale (1%), smaller than normal pup (1%), hematoma on the head (1%), diffuse alopecia on the back (6%, within one litter). These observations were not associated with any influence on the development of the offspring.

ANOGENITAL DISTANCE AND NIPPLE RETENTION
In the male pups, statistical significances were observed at the shorter mean anogenital distances (absolute and normalized) at 100 mg/kg bw/day.
In female pups, statistical significances were observed at the shorter mean anogenital distances at 10 mg/kg bw/day (normalized only) and 30 mg/kg bw/day (absolute and normalized).
These differences were with minor degree or without dose relevance, therefore were considered to have no or little toxicological relevance.
Nipples/areoles were not visible in any of the examined male offspring in the control or 10, 30 or 100 mg/kg bw/day groups on post-natal day 13.

BODY WEIGHT
The mean litter weight and litter weight gain was statistically significantly lower than in the control group at 100 mg/kg bw/day on postnatal days 0, 4 and 13. These differences were due to the lower number of pups in this group.
There were no statistical significances between the control and test item treated (10, 30 and 100 mg/kg bw/day) groups in the mean pup weight and mean pup weight gain per litter on post-natal days 0, 4 and 13. Considering the offspring’s body weight in males and females, separately, statistically significant difference with respect to the control was detected for the slightly lower male and female pup weight at 30 mg/kg bw/day on post-natal day 4. These differences with respect to the control in the body weight of offspring only by genders were considered to be toxicologically not relevant due to the minor degree and lack of dose relevance.

NECROPSY
There were no macroscopic changes in the organs or tissues of stillborn offspring (2/2 in the control and 1/1 at 10 mg/kg bw/day) subjected to necropsy on post-natal day 0.
There were no macroscopic changes in the organs or tissues of the dead pup (1/2) in the control on post-natal day 0. Autolyzed visceral organs and stomach filled up with gas was observed for the other dead pup (1/2) in the control subjected to necropsy on post-natal day 1.
The determination of stillborn or liveborn was not feasible for two pups due to autolysis (1/1 in the control and 1/1 at 10 mg/kg bw/day) on post-natal day 0.
At termination on post-natal day 14, diffuse alopecia on the back was noted for two pups from one litter in the control group.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.

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:
Under the conditions of the present study, CeTePox® 0214 H caused reduced body weight and body weight gain, reduced food consumption in parental male and female Han:WIST rats at 100 mg/kg bw/day administered by oral gavage. In male animals at 100 mg/kg bw/day, test item influence on hepatic function was detected in clinical chemistry parameters, in necropsy findings and histopathological findings (centrilobular vacuolation in the hepatocytes). At 100 mg/kg bw/day, the delivery data of dams (mean number of implantation sites and lower mean birth (total birth, live born and viable pups per litter) was slightly depressed. There were no test item related changes in male or female animals at 10 or 30 mg/kg bw/day. 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/female rats: 30 mg/kg bw/day
 
NOAEL for reproductive performance of male rats: 100 mg/kg bw/day
 
NOAEL for reproductive performance of female rats: 30 mg/kg bw/day
 
NOAEL for F1 Offspring: 100 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 CeTePox® 0214 H 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 10 mg/kg bw/day, 30 mg/kg bw/day and 100 mg/kg bw/day compared to control animals.

Four groups of Han:WIST rats (n=12/sex/group) were administered with the test item orally (by gavage) once a day at 0 (vehicle), 10, 30 and 100 mg/kg bw/day doses corresponding to concentrations of 0, 2, 6 and 20 mg/mL. The application volume was 5 mL/kg bw. Control animals received the vehicle, distilled water.

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. CeTePox® 0214 H concentrations in the dosing formulations varied in the acceptable range between 93 % and 109 % 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 49 days). Females were additionally exposed through the gestation period and up to lactation days 13-17 (altogether for 50-67 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.

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 (T4, TSH) from at least two pups per litter (where it was feasible) on post-natal day 4, from all dams and at least two pups per litter at termination on post-partum/post-natal day 13 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, uterus and ovaries) in the control and high dose groups and in non-pregnant females and the males these females cohabited with and not mated animals in the low and mid dose groups. Additionally, full histopathology was performed on the organs and tissues of parental animals (male and female) selected for general toxicological examinations in the control and high dose groups.

In addition, kidneys for one male and one female at 30 mg/kg bw/day and uterus for one female at 10 mg/kg bw/day were also processed and examined due to the necropsy observations (pyelectasia, hydrometra).

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

 

Mortality

There was no mortality at 10, 30 or 100 mg/kg bw/day groups during the course of study.

 

Clinical observation

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 (10, 30 or 100 mg/kg bw/day) during the entire observation period.

 

Body weight and body weight gain

The mean body weight gain and mean body weight were reduced in male and female animals at 100 mg/kg bw/day.

 

Food consumption

The mean food consumption was lower than in the control group in male and female animals at 100 mg/kg bw/day.

 

Estrous cycle

A test item influence on the estrous cycle was not found at any dose level (10, 30 and 100 mg/kg bw/day).

 

Reproductive performance

There were no test item related differences between the control and test item treated groups in the reproductive performance of male and female animals.

 

Delivery data of dams

Delivery data of dams was affected at 100 mg/kg bw/day dose level. Lower mean number of implantation sites and lower mean birth (total birth, live born and viable pups per litter) were observed at the high dose treated animals.

Hematology

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 10, 30 or 100 mg/kg bw/day.

 

 

Clinical chemistry

Elevated activity of aspartate aminotransferase and alkaline phosphatase was detected in male animals at 100 mg/kg bw/day referring to hepatic/hepatobiliary damage or alteration in hepatic function.

 

Serum thyroid hormones

There were no test item related changes in the serum thyroid hormone (T4, TSH) levels at any dose (parental male animals or PN13 offspring).

 

Necropsy

Macroscopic alterations were observed in the liver (pale) in some male animals at 100 mg/kg bw/day with low incidence.

 

Organ weight

There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes, epididymides and seminal vesicles of male animals at any dose level.

Test item related changes were observed in the weight of liver (higher absolute, relative to body and brain weights) of male animals at 100 mg/kg bw/day coinciding with changes in clinical chemistry parameters, necropsy and histopathology observations.

 

Histopathology

Histopathological examinations of the investigated sexual organs and accessory sex organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 100 mg/kg bw/day.

Hepatic lipidosis was detected in the male animals at 100 mg/kg bw/day. Hepatic lipidosis is considered as a slight reversible liver injury in connection with a disturbance of energy metabolism of affected hepatocytes.

 

The offspring’s development was not adversely influenced at any dose level.

 

Conclusion

Under the conditions of the present study, CeTePox® 0214 H caused reduced body weight and body weight gain, reduced food consumption in parental male and female Han:WIST rats at 100 mg/kg bw/day administered by oral gavage.

In male animals at 100 mg/kg bw/day, test item influence on hepatic function was detected in clinical chemistry parameters, in necropsy findings and histopathological findings (centrilobular vacuolation in the hepatocytes).

At 100 mg/kg bw/day, the delivery data of dams (mean number of implantation sites and lower mean birth (total birth, live born and viable pups per litter) was slightly depressed.

There were no test item related changes in male or female animals at 10 or 30 mg/kg bw/day.

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/female rats: 30 mg/kg bw/day

 

NOAEL for reproductive performance

of male rats: 100 mg/kg bw/day

 

NOAEL for reproductive performance

of female rats: 30 mg/kg bw/day

 

NOAEL for F1 Offspring: 100 mg/kg bw/day