Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: about 11-12 weeks (male animals); about 10 weeks (female animals)
- Housing: During pre-treatment: during pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany; during pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III; for motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany, with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material
- Diet (e.g. ad libitum): ad libitum, ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water (e.g. ad libitum): drinking water (ad libitum)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% sodium carboxymethyl cellulose in drinking water)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
C9-11 Methacrylate was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired
concentration. Then, 0.5% sodium carboxymethyl cellulose in drinking water was filled up to the desired volume and intensely mixed with a homogenizer. During administration, the preparations was kept homogeneous with a magnetic stirrer. The test substance preparations were produced weekly, at least.
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.
The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in 0.5% sodium carboxymethyl cellulose in drinking water over a period of 7 days at room temperature was proven
Duration of treatment / exposure:
The duration of treatment covered a 2-week premating period and a mating period in both sexes (mating pairs were from the same test group), two days post-mating in males, and the entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals (female: 65 days; male: 35 days)
Frequency of treatment:
daily at the same time in the morning
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
120 mg/kg bw/day (nominal)
Dose / conc.:
360 mg/kg bw/day (nominal)
Dose / conc.:
1 200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Parental animals
Mortality
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

Clinical observations
A cageside examination was conducted at least once daily for any signs of morbidity,
pertinent behavioral changes and/or signs of overt toxicity. All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal. The parturition and lactation behavior of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were documented on an individual dam basis. On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day.

Food consumption
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions: Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals). Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20. Food consumption of the females which gave birth to a litter was determined for PNDs
1-4, 4-7, 7-10 and 10-13. Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

Water consumption
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

Body weight data
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals: During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13. Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males (for the calculation of the administration volume). Females without litter and after weaning (PND 13) were weighed once a week (for the calculation of the administration volume).
Detailed clinical observations
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size.

Functional observational battery
A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Home cage observations:
The animals were observed in their closed home cages; during this period, any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: posture, tremors, convulsions, abnormal movements, gait, other findings

Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, gait, activity/arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/color), rearing within 2 minutes, other findings

Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), other findings, grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test

Motor activity assessment
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.

Clinical Pathology
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units. The parameters listed below were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, mean corpuscular volume, mean corpuscular Hemoglobin concentration, Platelet count, Differential blood, Reticulocytes, Prothrombin time.

Clinical chemistry
Parameters: Alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase), Aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase), Alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase), γ-Glutamyltransferase (GGT) (γ -glutamyl) peptide: aminoacid-γ- glutamyl-transferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, choloesterol, bile acids.

Thyroid Hormones
Blood samples were taken from all surplus pups pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling. All generated serum samples were frozen at -80°C until measurement. Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
Oestrous cyclicity (parental animals):
For all females of the pool estrous cycle normality was evaluated before the beginning of the administration. In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.
Litter observations:
Litter data
Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in “Pup Necropsy observations“. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying
between PNDs 1-4, 5-7 and 8-13 were determined. Pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13.

Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.

Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

Pup body weight data
The pups were weighed on the day after birth (PND 1) as well as on PND 4 (before
standardization), 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). In the summary tables pup body weights and pup body weight change are listed for males, females and males + females. „Runts“ were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
Postmortem examinations (parental animals):
Necropsy
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The
exsanguinated animals were necropsied and assessed by gross pathology, special
attention being given to the reproductive organs. Animal No. 26 was sacrificed moribund and was necropsied and assessed by gross pathology as soon as possible after their death.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
Anesthetized animals, Epididymides, Ovaries, Prostate, Seminal vesicles with coagulating glands, Testes, Thyroid glands (fixed) ,Uterus (with cervix).

The following weights were determined in 5 animals per sex/test group sacrificed on
schedule (females with litters only, same animals as used for clinical pathological
examinations): Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus.

Organ/tissue fixation
Parental animals
The following organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde solution or in modified Davidson`s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides (modified Davidson`s solution), Esophagus, Extraorbital lacrimal glands, Eyes with optic nerve (modified Davidson`s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer`s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson`s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson`s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained
according to Salewski E, 1964), Vagina.

The testes and epididymides of animal No 26, that was sacrificed moribund were fixed in 4% buffered formaldehyde solution.

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below. Following histotechnical processing, all histological slides, tables of individual macroscopic
findings as well as organ weight tables were sent to InSight Pathology BV, Chopinlaan 6, NL-5343EM Oss, The Netherlands. Light microscopical examination of the hematoxylineosin stained slides and assessment of findings was performed under the responsibility of the Principal Investigator Eric van Esch at the test site InSight Pathology BV under the test site phase number ISP 17211. All gross lesion, Adrenal glands, Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Eyes with optic nerve, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (axillary and mesenteric), Ovaries, Oviducts, Prostate gland, Peyer`s patches, Rectum, Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic, lumbar), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid , glands, Trachea, Urinary bladder, Uterus, Vagina
Postmortem examinations (offspring):
Pup necropsy observations
On PND 4, as a result of standardization, the surplus pups or 2 preferably female pups per litter, respectively, were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice,
the pups were examined externally and eviscerated, and the organs were assessed macroscopically. On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for further processing The remaining pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-bycase basis, depending on the type of finding noted.
Statistics:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.
Reproductive indices:
The following indeces were determined: mating and fertility indes for both males and females, gestation index, live birth index and postimplantation loss for female.
Offspring viability indices:
The following indeces were determined: viability index, survival index, sex ration, anogenital index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Summary clinical observations for males and females
(except gestation and lactation periods)
No treatment-related, adverse signs of toxicity were observed. During the mating period one male animal (No. 26) of test group 2 (360 mg/kg bw/d) was sacrificed in a moribund state due to a gavage error after moderate poor general condition and piloerection. Salivation within 2 hours after the administration was observed in 5 of 10 male and female animals during premating as well as in 6 male and 3 female animals during mating phase of test group 3 (1200 mg/kg bw/d) and two male animals of test group 2 (360 mg/kg bw/d). During postmating two male animals of test group 2 (360 mg/kg bw/d) and all male animals of test group 3 (1200 mg/kg bw/d) showed salivation within 2 hours after administration. These findings were considered to be related to treatment but not as adverse.

Summary clinical observations for females during gestation
Salivation within 2 hours after the administration was observed in 2 females of test group 2 (360 mg/kg bw/d) and in 8 of 10 females of test group 3 (1200 mg/kg bw/d).
These findings were considered to be related to treatment but not as adverse.

Clinical observations for females during lactation
Salivation within 2 hours after the administration was observed in 6 of 10 females of test group 3 (1200 mg/kg bw/d). These findings were considered to be related to treatment but not as adverse.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No treatment-related changes were observed in male animals during pre-mating and mating periods. Food consumption was significantly decreased during premating in female animals of test group 2 (360 mg/kg bw/d) between pre-mating days 0-7 and in females of test group 3 (1200 mg/kg bw/d) between pre-mating days 0-7, 7-13 and 0-13. In female animals of test group 3 (1200 mg/kg bw/d) food consumption was significantly decreased during gestation days 0-7 and 7-14. Based on the small deviation from corresponding current control values (maximum -11.4%), these findings were considered to be related to treatment but not as adverse.
Water consumption and compound intake (if drinking water study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
In males of test groups 1, 2 and 3 (120, 360 and 1200 mg/kg bw/d) sodium and chloride levels were significantly higher compared to controls. However, all values were within historical control ranges (males, sodium 140.4-145.8 mmol/L; chloride 97.9-104.8 mmol/L). In females of test group 3 (1200 mg/kg bw/d) creatinine levels were significantly increased. Again, this isolated changed value was within the historical control range (females, creatinine 27.4-35.4 µmol/L). Therefore, all mentioned alterations were regarded as incidental and not treatment-related.
Behaviour (functional findings):
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test group 3: 1200 mg/kg bw/d:
Increase in incidence and severity of single cell necrosis and accompanying
inflammatory cell infiltrates in the liver of males and females. Irritation of mucosa in forestomach of males and females (manifested in focal erosions, hyperplasia, squamous cell and/or hyperkeratosis, submucosal edema, focal vacuolar degeneration of the keratinocytes within the stratum granulosum, basophilic material within keratin of the stratum corneum).

Test group 2: 360 mg/kg bw/d:
Increase in incidence and severity of single cell necrosis and accompanying Inflammatory cell infiltrates in the liver in the liver of females. Irritation of mucosa in forestomach (manifested in focal ulceration, basophilic material within keratin of the stratum corneum).

Test group 1: 120 mg/kg bw/d:
No treatment-related, adverse effects were observed.

Concerning pathology, microscopically, in the liver of females treated with 360 mg/kg bw/d and of males and females treated with 1200 mg/kg/bw/d C9-11 Methacrylate a clear increase in the incidence and severity of single cell (hepatocyte) necrosis and accompanying inflammatory cell infiltrates were observed in the centrilobular areas. In females treated with 360 and 1200 mg/kg bw/d C9-11 Methacrylate, the increase in severity of single cell necrosis and inflammatory cell infiltrates showed a dose-relationship. The increase incidence and severity of single cell necrosis is considered an adverse finding. In the forestomach of male and female animals in the group treated with 120, 360 and 1200 mg/kg bw/d C9-11 Methacrylate clear signs of irritation to the mucosa were present and included: erosion/ulceration found in one female treated with 120 mg/kg bw/d (minimal focal erosion) and one male treated with 1200 mg/kg bw/d (slight focal erosion) and in a single male treated with 360 mg/kg bw/d (slight focal ulceration), hyperplasia, squamous cell and/or hyperkeratosis noted in two males and two females treated with 1200 mg/kg bw/d, and submucosal edema that was found to be present in several males and females treated with 1200 mg/kg bw/d C9-11 Methacrylate. In two animals the submucosal edema was grossly visible as focal thickening of the wall. A typical focal vacuolar degeneration of the keratinocytes within the stratum granulosum was observed in a single male treated with 1200 mg/kg bw/d. Basophilic material was noted within the keratin of the stratum corneum in a single male and female of the 360 mg/kg bw/d treated group and in 3 out of 10 males and a single female treated with 1200 mg/kg bw/d C9-11 Methacrylate. In few cases this keratin layer showed a wide-mesh appearance. All these changes in the forestomach are considered local adverse effects and are believed to be caused by the chemical properties of the test-item by the principle investigator of the pathological assessment. The study director agreed with this evaluation for the test group 2 and 3 (360 and 1200 mg/kg bw/d). In dose group 1 (120 mg/kg bw/d) no macroscopical finding but a minimal focal erosion in one animal (female) was observed. Since it was the only finding on the forestomach at this dose group and it`s low severity, this finding was assessed as non adverse by the study director. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item. The spermatogenic staging profiles were normal for all males examined.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Body weight data
No significant changes in mean body weights and mean body weight change values were observed for male and female animals of test groups 1-3 (120, 360 and 1200 mg/kg bw/d) when compared to the control group.

Detailed clinical observations (DCO)
In the detailed clinical observations on study days 0, 7, 14, 21 and 28 in parental males and females and additionally, on study days 35, 42, 49, 56 and 63 in parental female animals no adverse findings were observed.

Functional observational battery (FOB)
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental. The following examinations were performed during FOB and are assessed individually:
Home cage observations
No test substance-related effects were observed.
Open field observations
No test substance-related effects were observed.
Sensorimotor tests/reflexes
No test substance-related effects were observed.
Quantitative parameters
No test substance-related effects were observed.

Motor activity measurement (MA)
Regarding the overall motor activity, no test substance-related deviations were noted for male and female animals. Comparing the single intervals with the control groups, one significantly increased value was measured for female animals of test group 1 (120 mg/kg bw/d) at interval 1. The change was regarded to be incidental and not related to treatment as neither other single intervals nor the overall motor activity was affected as well as there was no dosedependency given. No deviations to control values were observed for male animals in test groups 1 to 3 (120, 360 and 1200 mg/kg bw/d) when compared to the control group.

Clinical Pathology
Hematology
No treatment-related changes among hematological parameters were observed.

Clinical chemistry
No treatment-related changes among clinical chemistry parameters were observed.
In males of test groups 1, 2 and 3 (120, 360 and 1200 mg/kg bw/d) sodium and chloride levels were significantly higher compared to controls. However, all values were within historical control ranges (males, sodium 140.4-145.8 mmol/L; chloride 97.9-104.8 mmol/L). In females of test group 3 (1200 mg/kg bw/d) creatinine levels were significantly increased. Again, this isolated changed value was within the historical control range (females, creatinine 27.4-35.4 µmol/L). Therefore, all mentioned alterations were regarded as incidental and not treatment-related.

Thyroid hormones
In parental males (test groups 1, 2 and 3; 120, 360 and 1200 mg/kg bw/d) and in male and female pups at PND13 (test groups 11, 12 and 13; 120, 360 and 1200 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Estrous cycle
Estrous cycle data revealed regular cycles in the rearing F1 females of all test groups including the control. The mean estrous cycle duration in the different test groups (0-3) ranged from 3.8 to 3.9 days.

Male reproduction data
Male mating index
The male mating index calculated after the mating period to produce F1 litter was 100% in all test groups.
Male fertility index
Fertility was proven for all F0 parental males within the scheduled mating interval to
produce F1 litter. Thus, the male fertility index was 100% in all test groups 0-3.

Female reproduction and delivery data
Female mating index
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) was 2.3 days for test group 0, 2.2 days for test group 1, 2.9 days for test group 2 and 2.2 days for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
Female fertility index
All sperm positive rats delivered pups. Thus, the female fertility index was 100% in all test groups 0-3. The mean duration of gestation was 21.8 days (control group 0), 22.1 days (test group 1), 22.2 days (test group 2) and 22.1 days (test group 3).
These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Gestation index
The gestation index was 100% in all test groups.

Live birth indices
The rate live birth indices were 100% in all test groups.

Postimplantation loss
The postimplantation loss was 4.3% in control group 0, 5.7% in test groups 1 (120 mg/kg bw/d), 9.7% in test group 2 (360 mg/kg bw/d) and 6.2% in test group 3 (1200 mg/kg bw/d). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. A treatment-related increase in postimplantation loss was not observed in any test group.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproductive performance and fertility
Effect level:
1 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Effect level:
1 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
120 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
general systemic toxicity
Effect level:
360 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
local toxicity
Effect level:
120 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: irritation in the forestomach

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One male pup of test group 1 (120 mg/kg bw/d) showed reddish discolored testis. One female pup of the control group and one female pup of test group 2 (360 mg/kg bw/d) showed empty stomach. This finding occurred without any relation to dosing and can be found in the historical control data at comparable or even higher incidences. All other F1 pups of any test groups (0-3) did not show adverse findings during necropsy.

Details on results (F1)

F1 generation litter/pups
Litter data
Pup number and status at delivery
The mean number of delivered F1 pups per dam was equally distributed among test groups 0, 1, 2 and 3. No significant deviations occurred.

Pup viability index/mortality
The viability index indicating pup mortality between PND 0 and 4 was 100.0% in all test groups.
The survival index indicating pup mortality during PND 4-13 was 100.0% in all test groups.

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups. The sex distribution and sex ratios of test group 1 (120 mg/kg bw/d) showed significant differences towards the control group as the ratio of male pups was decreased (35.5%*) and the ratio of females was increased (64.5%*) (see Tab. 4.2.2.1.3.1.). Without a dosedependency this isolated finding, was assessed as incidental and not related to treatment.

Pup clinical observations
All F1 pups of any test group (0-3) did not show adverse clinical signs up to scheduled sacrifice on PND 4 or PND 13.

Pup body weight data
Mean pup body weights/pup body weight changes of all pups in all test groups were
comparable to the control group. No runts were seen in all test groups.

Anogenital distance/ anogenital index
Anogenital distance and anogenital distance index of male and female pups was not
influenced in all test groups.

Nipple/areola anlagen
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.


Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL for reproductive performance and fertility was set to 1200 mg/kg bw/d for male and female Wistar rats. This, the NOAEL for developmental toxicity was 1200 mg/kg bw/d.