Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-09 to 2017-08-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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)
Deviations:
yes
Remarks:
Before the first administration, due to irregularity of estrous cycle 4 already randomised females were replaced with 4 regularly cycling females taking their body weight into account. TSH-assessment by ELISA method was not validated according to GLP.
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4'-Ethyl-2,3-difluorbiphenyl-4-boronic acid
EC Number:
929-209-3
Cas Number:
1123312-95-7
Molecular formula:
C14 H13 B F2 O2
IUPAC Name:
4'-Ethyl-2,3-difluorbiphenyl-4-boronic acid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female;
the female animals were non-pregnant and nulliparous.
Age at the start
of the treatment period: approx. 14-15 weeks old
Body weight at the
allocation of the
animals to the
experimental groups: males: 316 - 369 g (mean: 346.68 g, ± 20 % = 277.34 - 416.01 g)
females: 204 - 246 g (mean: 224.50 g, ± 20 % = 179.60 - 269.40 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Nesting material was provided latest on GD 18 for all mated females
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (6 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. One female with milky opacity of the right eye was excluded from the study. A supplementary animal from the same delivery was provided in exchange.
Before initiation of dosing, all females were screened for two weeks for regular estrous cyclicity and females (10 females/ group) with regular estrous cycle (4-5 day cycle) were used in the study.
Before the first administration, all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively. Randomisation was performed with validated IDBS Workbook 10.1.2 software. After randomisation before the first administration, due to irregularity of estrous cycle 4 already randomised females were manually replaced with 4 regularly cycling females taking their body weight into account.
Each animal was marked with its identification number by individual ear tattoo and/or tail marking.


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.25 % aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)
Remarks:
hydroxypropyl methylcellulose, colorcon, Batch No.: 3B12012N02, Expiry Date/Retest Date: 11 February 2019 aqua ad injectionem, AlleManPharma, Batch No.: 511535 Expiry Date: October 2018 (each bottle was only used up to one week after opening)
Details on exposure:
The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.
The test item, as delivered, was weighed into a tared plastic vial on a suitable precision balance and coated with approx. half the target volume with vehicle. A glass rod was used to bray the test item in the container. The formulation was then dispersed by ultraturrax for approx.
2.5 minutes. Subsequently the formulation was shortly placed into an ultrasonic bath. The vehicle 0.25 % aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium) was added to give the appropriate final concentration. Then the formulation was dispersed by ultraturrax again for
1 minute until a homogenous suspension was achieved.
The test item formulation was prepared at least once every ten days based on available stability data (Eurofins Munich study no. 152933). The prepared formulation was stored at 2-8 °C and protected from light.
Formulates were kept under magnetic stirring during the daily administration.
The vehicle was also used as control item.

The following doses were evaluated:
Control: 0 mg/kg/d
Low Dose: 25 mg/kg/d
Medium Dose: 100 mg/kg/d
High Dose: 400 mg/kg/d


The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and a NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle using the same volume as used for the dose groups.

The test item and vehicle were administered daily as single doses to the animals by oral gavage. The application volume for all groups was 10 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.



Details on mating procedure:
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.
The cages were arranged in such a way that possible effects due to cage placement were minimized.
Due to reduced health condition of male no. 33 of the HD group, this mating partner of female no. 73 of the HD group was exchanged on mating day 9 with male no. 34 of the HD group.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
daily
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days, i.e. during 14 days of pre-mating and
maximum 14 days of mating in both males and females. Then in females treatment was done during the gestation period and up to post-natal day 12.
Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.
Frequency of treatment:
daily
Details on study schedule:
Arrival of the Test Item: 28 April 2016
Study Initiation Date: 09 August 2016
Amendment to Study Plan: 24 August 2016
2nd Amendment to Study Plan: 12 September 2016
Delivery of Animals: 04 August 2016
Acclimatisation Period: 04 August 2016 to 09 August 2016
Experimental Starting Date: 10 August 2016
Treatment Period: males: 24/25 August 2016 to 20/21 September 2016
females: 24/25 August 2016 up to 25 October 2016
Necropsies: males: 28 August 2016, 16 September 2016, 21 September 2016, 22 September 2016
females: 20 September 2016, 04 October 2016, 05 October 2016, 07 October 2016, 08 October 2016,
12 October 2016 to 17 October 2016, 26 October 2016
Experimental Completion Date: 26 October 2016
Completion Date of Delegated Phase (Histopathology): 09 August 2017
Completion Date of Delegated Phase (Formulation Analysis): 11 May 2017
Study Completion Date: 17 August 2017


Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
25 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
400 mg/kg bw/day
No. of animals per sex per dose:
100 animals (40 males and 60 females) were included in the study. All females were screened for regular estrous cycles for 14 days before the treatment
initiation and only 40 females (10 females/group) showing regular estrous cycles were continued in the study. The unstained samples were evaluated freshly
by light microscope after obtaining vaginal smears by lavage using saline.
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included check of spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Any changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded if present.

Functional Observations
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of lactation of the lactation period in 5 randomly selected females (only lactating females were evaluated except for non-pregnant female no. 79 of the HD group) of each group outside the home cage using a functional observational battery of tests 0:
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).


Body Weight and Food Consumption
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with the pups. All animals were weighed directly before termination. Any animals prematurely sacrificed were weighed prior to the sacrifice.
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.


Oestrous cyclicity (parental animals):
Estrous cycles were monitored before start of treatment to select for the study females with regular cyclicity (using vaginal smears). Further on, vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.
Litter observations:
The duration of gestation was recorded and calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring was recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / Cube root of pup weight). The number of nipples / areolae in male pups was counted on PND 12.

Postmortem examinations (parental animals):
Pathology
All surviving male animals were sacrificed after the completion of the mating period (after a total dosing period of 28 days) on study day 29, while female animals were sacrificed on the respective PND 13 by using anaesthesia (ketamine/xylazine, 2:1). Pups used for blood collection on PND 4 were sacrificed by bleeding after cardiac puncture under anesthesia. All surviving pups were killed by cervical dislocation/decapitation on
PND 13.
Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.
Dead pups and all surviving pups on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
Non-pregnant females (no. 68 of the MD group and no. 76 and 79 of the HD group) were sacrificed on day 26 from the day of mating or from the last day of mating period.
All adult animals were subjected to a detailed gross necropsy which includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. If appropriate and possible, the number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating and or on day 26 post-coitum due to non-delivery.

Organ Weights
The wet weight of the organs (testes (paired weight), epididymides (paired weight), prostate, seminal vesicles , and coagulating glands (complete weight), Cowper’s gland (paired weight), thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) - weighed after fixation (complete weight), liver, kidneys (paired weight), adrenal (paired weight), pituitary gland, levator ani and bulbocavernosus
muscle complex (complete weight), glans penis, uterus with cervix, ovaries (paired weight), thymus, spleen, brain, heart) of five randomly selected male and female animals (only lactating females were evaluated except for non-pregnant female no. 79 of the HD group) from each group was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded.
Reproductive organs were weighed in all animals.
Thyroid/parathyroid glands from 1 pup/sex/litter/group (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.


The following tissues (adrenal glands, all gross lesions, aorta, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, eyes with optic nerve and Harderian gland, femur with knee joint, heart, ileum (including Peyer´s patches), jejunum,
kidneys, liver, lungs, lymph nodes (mandibular), lymph nodes (mesenteric and axillary), mammary gland area (male and female), oesophagus,
ovaries, oviducts, pancreas, pituitary, prostate and seminal vesicles with coagulating glands as a whole, rectum, salivary glands (sublingual, submandibular), sciatic nerve, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar segments), spleen, sternum (with bone marrow),
stomach, testes, thymus, thyroid/parathyroid glands, tongue, trachea, ureters, urinary bladder, uterus with cervix and vagina) from the five randomly selected male and female animals from each group were preserved in 4 % neutral-buffered formaldehyde except for testes and epididymides that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 70 % ethanol.
All animals found dead and/or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for histopathological examination.
Thyroid/parathyroid glands from 1 pup/sex/litter/group sacrificed on PND 13 and non-selected adult animals were preserved for potential histopathological examination.


Histopathology
A full histopathology was carried out on the preserved organs and tissues of the selected animals of the control and high dose groups which were sacrificed at the end of the treatment period. Thyroid/parathyroid glands from pups and from the remaining non-selected adult animals were not histopathologically examined as not considered necessary.
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study or which were euthanised due to morbidity.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and examined in control and HD animals and in all non-pregnant female animals (no. 68, 76 and 79). Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female animals (no. 28, 36 and 39, respectively).
These examinations were extended to five randomly selected animals from the LD and the MD group for treatment-related changes that were observed in the HD group in stomach, liver (males only), kidneys, adrenal glands, spleen, bone marrow (males only) and thymus.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
For the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
The histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). The histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The study phases from test site 1 and 2 were performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation was performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all specimen and raw data (including blocks, slides, remaining wet tissues, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided a histopathology phase report including histopathology results to the study director electronically (by e-mail) and sent the final histopathology phase report (1 pdf and paper version with all signatures) to the study director upon the completion of the study.

Postmortem examinations (offspring):
Dead pups and all surviving pups on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
Statistics:
A statistical assessment of the results of body weight, food consumption, parameters of haematology, blood coagulation, clinical biochemistry and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights were statistically analyzed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
see Details on results P0
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
see Details on results P0
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Isee Details on results P0
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
see Details on results P0
Urinalysis findings:
no effects observed
Description (incidence and severity):
see Details on results P0
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
see Details on results P0
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):
see Details on results P0
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
see Details on results P0
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
see Details on results P0
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
see Details on results P0

Details on results (P0)

Mortality
Four animals of the HD group died prematurely during the course of treatment. Male no. 33 was sacrificed in a moribund condition for animal welfare reasons on study day 24. Male no. 35 was found dead on day 5 of the premating period without any preceding signs of morbidity. Female no. 71 and 74 were both found dead on study day 28 (gestation day 11 and 13, respectively). Female no. 71 showed slight loss of body weight in the first week of gestation as well as piloerection on gestation day 7. No signs of reduced health condition were observed in female no. 74 before death.
All remaining animals survived their scheduled study period.

Clinical Observations
One male of the HD group (no. 33) was euthanised for animal welfare reasons in a moribund condition with severe piloerection, sunken flanks, severely reduced spontaneous activity, ataxia, slow movements and half eyelid closure. On the day before euthanasia the animal was observed with reduced health condition in terms of moderate piloerection, moderately reduced spontaneous activity and hypothermia.
The unspecific clinical sign of piloerection was noted in one more male of the HD group (no. 37) at the end of the treatment period as well as in 2/10 females of the HD group (no. 71 and 80). Furthermore, reduced spontaneous activity was observed in total in 6/10 males of the HD group at the end of the treatment period and in 1/10 females of the HD group for 2 days after delivery of pups (still birth). Hunched posture was noted in 1/10 males (no. 37) of the HD group at the end of the treatment period and the slight sign of red nasal discharge in 2/10 males of the HD group and 1/10 females of the LD group. Nasal discharge of the female of the LD group (no. 54) was only observed on one single day of treatment and might be a slight reaction to incidental swallowing a part of the oral gavage cannula few days before.
Moving the bedding was observed transiently in almost all males of the HD group and all females of the MD and the HD group. Furthermore, salivation was noted transiently and dose dependently in 8/10 males and 5/10 females of the HD group and in 2/10 males and 3/10 females of the MD group. The slight clinical signs of moving the bedding and salivation were mainly seen at the end of the treatment period in males and during gestation and lactation period in females. Both signs were observed in short timely relation to dose application or in anticipation thereof and thus were considered to be a sign of discomfort or a local reaction to the test item. These slight signs were not considered as adverse systemic effects.
Low incidences of slight clinical signs like alopecia or crust in few animals of the control or dose groups were seen without dose dependency and were considered as incidental in nature.
None of the females showed clinical signs of abortion or premature delivery.
During the weekly detailed clinical observation, on few occasions salivation was noted in some males of the MD and the HD group and some females of the HD group.

No further relevant differences between the groups were found. Slightly individually deviating values were noted mainly for females for fear and response to finger approach, for males for response to handling and for males and females for spontaneous activity and response to head touch. As these deviating values were observed throughout the study period in all groups including control without clear dose dependency and without consistency, they were considered as incidental in nature without toxicological relevance.

Functional Observations
In males and females, no relevant effects were observed in any of the parameters of the functional observation battery at the end of the treatment period.
There were no biologically or statistically significant differences in body temperature between the dose groups and the controls.

Ophthalmoscopic Examination
There were no ophthalmoscopic findings in any of the animals of this study.

Body Weight Development
In males, the mean body weight increased in the control, the LD and the MD group with the progress of the study. After initiation of treatment, males of the HD group showed a moderate, statistically significant loss of body weight (mean body weight change -10.67 g compared to +6.60 g of the control group, p < 0.001). Body weight change was equal to a loss of -2.9 % in the male HD group and a gain of +1.8 % in the male control group. In the second week of premating a gain of body weight was noted in the HD group which was comparable to controls. However, this was followed by a slight loss of mean body weight in the remaining study period which gained statistical significance in the first week of the mating/postmating period (p < 0.01). Thus, mean body weight of the male HD group was moderately and statistically significantly lower in the first and second week of the mating/postmating period (8 % and 10 % below controls, respectively, each p < 0.05). Considering the whole treatment period, there was a biologically and statistically significant mean loss of -7.25 g body weight (equal to -1.9 % under exclusion of decedents) in the male HD group compared to a gain of +35.70 g (equal to +9.6 %) in the respective controls (p < 0.001). At terminal sacrifice, mean body weight of the male HD group was moderately reduced (10 % below controls, p < 0.05).
A comparable test item-related effect was noted for the female HD group. A slight but statistically significant mean loss of body weight occurred in the first week of treatment in the female HD group (-1.50 g) when compared to a gain of +4.60 g in the control group (p < 0.05). Body weight change was equal to a loss of -0.6 % in the female HD group and a gain of +2.0 % in the female control group. Although body weight increased subsequently, mean weight gain remained slightly lower in the female HD group throughout the whole study period when compared to controls. Statistical significance was achieved in the 1st week of gestation (p < 0.001), the last week of gestation (p < 0.001) and when related to the whole gestation period (day 0-20) (body weight gain 56 % below controls, p < 0.001). Thus, mean body weight of the female HD group was moderately and statistically significantly reduced on day 7 of gestation (8 % below controls, p < 0.01) and day 20 of gestation (18 % below controls, p < 0.001).
A biologically and statistically significant effect on mean body weight was also noted during the lactation period with a moderately lower body weight on day 4 (12 % below controls, p < 0.01) and day 13 (13 % below controls, p < 0.001).
After initiation of treatment, a slight but statistically significant loss of body weight occurred also in the female MD group (-3.10 g, p < 0.01) (equal to -1.3%) when compared to controls. However, without a biologically and statistically significant effect on mean body weight, this slight and transient effect on body weight gain was not considered as adverse.
No effects of toxicological relevance were noted in the male and female LD and MD groups for mean body weight and body weight gain. There were no further statistically or biologically significant differences to controls.

Food Consumption
In males, in correlation to the body weight change, mean food consumption per animal in the HD group was moderately lower in the first week of the premating period when compared to controls (27 % below controls). No toxicologically relevant differences were noted in the second week of the premating period.
In females, a slight tendency towards lower food consumption was observed in the first week of the premating period. In accordance to the attenuated body weight gain during gestation, food consumption of the HD group was also noted to be moderately lower when compared to the control group (1st week of gestation 30 % below controls, 2nd week of gestation 26 % below controls and 3rd week of gestation 28 % below controls, p < 0.001 each). Food consumption was further reduced in the HD group during lactation with values being 38 % below controls (p < 0.01) in the first week and 54 % below controls (p < 0.001) in the second week of lactation.
Slightly lower food consumption was observed in the 2nd week of gestation in the MD group (15 % below controls, p < 0.01). This slight transient effect on food consumption without relevant effect on body weight was not considered to be within a toxicologically relevant range.
No effects of toxicological relevance were noted in the male and female LD and MD groups for food consumption. There were no further statistically or biologically significant differences to controls.

Estrous Cyclicity
The test item had no biologically significant effect on the estrous cyclicity analyzed during the 2 weeks premating period after the first administration. There were no considerable differences in the length or sequence of cycle stages between the dose groups and the control group. There were no statistically significant differences for the number of abnormal and normal cycles in the dose groups compared to the control group. Slight deviations to the normal sequence of cycle stages were rarely observed in single animals of the control group and the dose groups without a dose dependent pattern and were considered as biological variation not related to the treatment with the test item.

Precoital Interval and Duration of Gestation
Length of the precoital interval was within the normal range of variation for animals of this age and strain in the dose groups and the control group. There were no toxicologically relevant differences.
A biologically and statistically significant effect was noted on the duration of gestation in the HD group. The mean gestation period was found to be prolonged in the HD group (23.80 days) when compared to 22.30 days in the control group (p < 0.001). Two females of the HD group were noted with a gestation length of 24 days (no. 73 and 80) and one female with a gestation length of 25 days (no. 77). Two of these females (no. 80 and no. 77) had only stillbirths.

Pre- and Post-Natal Data
Post-implantation loss was markedly and statistically significantly higher in the HD group (76.81 %) when compared to the control group (5.07 %) (p < 0.001). This was mainly related to the increased number of still births and one dam which was not observed littering. No toxicologically relevant effect on post-implantation loss was noted for the LD and the MD group.
There were no biologically or statistically significant effects on the number of corpora lutea and number of implantation sites in the dose groups when compared to the control group. Slight differences between the dose groups and the control group were seen without dose dependency and were considered incidental. Percentage of pre-implantation loss was within the normal range of biological variation.

Reproductive Indices
The copulation index (no. of animals copulated/no. of pairs x 100) was 100 % in all groups including control.
The fertility index (number of pregnant females/number of copulated females x 100) was slightly reduced in the MD group (90 %) and the HD group (80 %) when compared to the control group (100 %). Histologically there was no morphological reason for the non mating of females no. 68 of the MD group and no. 76 and 79 of the HD group with their male pairing partners (no. 28, 36 and 39), respectively. Therefore, the slightly reduced fertility index in the MD and the HD group was considered as incidental in nature.
The delivery index (no. of dams with live pups born / no. of pregnant dams x 100) was markedly reduced in the HD group (50 %) when compared to the control group (100 %). The delivery index was 100 % in the LD and the MD group.
There was also a moderately reduced viability index during post-natal days 0-4 (no. of live offspring at day 4 / no. of live offspring at birth x 100) of 79.17 % in the HD group when compared to 99.23 % in the control group. Viability index during post-natal days 0-4 was slightly reduced in the MD group (94.80 %). No effect was noted in the LD group (100 %).

Thyroid Hormone Analysis
Serum analysis in adult males at terminal sacrifice revealed dose dependently and statistically significantly lower thyroxine (T4) hormone levels throughout all dose groups when compared to the control group (LD: 81.92 nmol/L (p < 0.05), MD: 55.97 nmol/L (p <0.001), HD: 37.91 nmol/L (p < 0.001) compared to 93.40 nmol/L in controls).
Analysis of TSH levels in serum samples of adult males showed no statistically significant differences between the dose groups and the control group. Furthermore, no correlation between TSH and T4-values was observed for the individual animals.

Haematology and Coagulation
In males, slightly and statistically significantly lower mean percentage of reticulocytes was noted in the HD group (0.92 %) when compared to the control group (1.68 %) (45 % below controls). Though not observed in females, a relationship with the treatment of the test item cannot be excluded for this biologically relevant observation.
Marginally but statistically significantly lower value for mean corpuscular volume (MCV) was noted for the male HD group (50.30) when compared to controls (53.72) (6 % below controls, p < 0.05). As this marginal change was within the normal range of variation, did not follow a dose-dependent pattern and without further changes to red blood cell parameters, this was not considered to be toxicologically relevant.
There were no further biologically or statistically relevant effects on haematology at the end of the treatment period. Mean values were within the normal range of variation and slight differences to controls were not assumed to be biologically relevant.
Blood coagulation was not affected by the treatment with the test item. Prothrombin time (PT) was noted to be marginally but statistically significantly higher in males but not females of the HD group (23.9 s) when compared to controls (20.15 s, p < 0.001). As values were within the normal range of variation, this marginal and isolated difference was not considered as adverse.

Clinical Biochemistry
No toxicologically relevant effects on clinical biochemistry parameters were found at the end of the treatment period of this study. Single deviating values in individual animals compared to controls were considered as incidental.
Statistically significantly lower mean value for alkaline phosphatase in the female HD group when compared to controls (p < 0.01) was considered as incidental.
Statistically significantly lower mean cholesterol (p < 0.05) and higher mean glucose (p < 0.05) were seen in the female HD group when compared to controls. Though achieving statistical significance, these changes were not considered as toxicological relevant but incidental as values were within the normal range of variation and no such changes were seen in males. Furthermore, no glucosuria was detected in any female at necropsy.

Urinalysis
All parameters of urinalysis of the dose groups at the end of the treatment period were not considerably different to the corresponding control group and were within the normal range of variation. Slightly deviating values for individual animals throughout all groups were considered as incidental, isolated findings without relation to the treatment with the test item.

Pathology
Macroscopic findings that could be attributed to the treatment with the test item consisted of grossly enlarged kidneys (in one male of the HD group along with abnormal color, spotted) (males: 1/10 of the MD, 5/10 of the HD group; females: 2/10 of the HD group) and enlarged adrenal glands (males: 3/10 of the HD group; females: 2/10 of the HD group). Furthermore, small spleen was noted in 4/10 males of the HD group and small thymus in 6/10 males of the HD group.
At premature death of female no. 71, blood was found in its uterus. At necropsy of the other female no. 74 which was found dead prematurely, jejunum and ileum were noted to be filled with blood.
All other gross lesions recorded at necropsy were considered to be within the range of normal background alterations which may be seen in rats of this strain, age and in this study type. The distributions among the groups were considered to be incidental, reflecting the usual individual variability.

Organ Weight
At the end of the treatment period, a toxicologically relevant effect was noted for absolute and relative (to body weight) spleen weight in the HD group. Spleen weight was moderately decreased in males of the HD group (absolute weight 42 % below controls, p < 0.01) and females of the HD group (absolute weight 35 % below controls, p < 0.01).
Furthermore, the relative kidney weight to body weight ratio was moderately and dose dependently higher in males of the MD and the HD group (16 % and 28 % above controls, respectively, p < 0.05 each) and in females of the HD group (15 above controls, p < 0.05) when compared to the respective controls.
In males of the HD group, the absolute thymus weight was markedly reduced (45 % below controls, p < 0.05). No such effect was noted in females.
Slightly but statistically significantly lower absolute but not relative weight of levator ani and bulbocavernosus muscle complex was seen in the HD group when compared to the control group (17 % below controls, p < 0.05). Without dose dependency this slight and isolated finding was not assumed to be toxicologically relevant but incidental or secondary to the lower body weight in the HD group.
Other slight but statistically significant changes in absolute weight of the heart of the male and female HD group and in absolute and relative (to body weight) weight of prostate with seminal vesicles and coagulating glands in the male HD group did not correlate with microscopic findings. They were considered incidental or secondary to body weight changes.
There were no further statistically or biologically significant differences between the dose groups and the control group for the remaining organ weight data.

Histopathology
Test item-related changes were detected in the stomach, liver (males only), kidneys, adrenal glands, spleen, bone marrow (males only) and thymus in the HD group. Therefore examinations of these organs were extended to five randomly selected animals from the LD and the MD group.
In the kidneys, minimal to moderate degrees of tubular degeneration/necrosis along with tubular regeneration and tubular dilation were observerd in proximal tubules, distal tubules and/or collecting ducts of animals of the HD group. The tubular degeneration/necrosis was mainly characterized by vacuolar degeneration of cortical tubular epithelial cells and/or cell swelling, nuclear pyknosis and cellular sloughing at the corticomedullary junction and/or in the renal papilla. This finding was sometimes along with foci of mineralization (in the cortex) or inflammatory cell infiltrates (in the papilla). In the HD group, these findings were sometimes accompanied by slight cortical tubular vacuolation, slightly increased mononuclear cell infiltrates and minor degrees of urothelial hyperplasia. Minimal or slight degrees of papillary necrosis were also recorded in two animals. Some animals of the LD and MD group showed also minimal and/or slight cortical tubular vacuolation. In the MD group, there was also minimal tubular degeneration/necrosis, tubular regeneration and tubular dilation in one male.
Minimal to moderate epithelial hyperplasia along with parakeratosis and minimal submucosal inflammation was recorded in the forestomach of males and females of the HD group.
A minimal centrilobular hepatocellular hypertrophy was recorded in the liver of males of the HD group. Minimal hypertrophy recorded in few control and high dose females was considered incidental.
Males and females of the HD group showed minimal to slight diffuse hypertrophy in the Zona fascilulata of the adrenal cortex. In addition, a moderate to marked cortical diffuse hemorrhage was present in both males that died prematurely. Minimal hemorrhage was also present in another high dose male.
Slight to marked lymphoid atrophy was recorded in the spleen of males and females of the HD group. A further finding consisted of a slight increase of hemosiderin deposits in these animals.
Some males of the HD group showed minimal to slight focal hypocellularity in the sternal bone marrow.
An increased severity grade of atrophy was noted in males of the HD group. In females of the HD group, the severity grade for this finding was increased only in one female that was found dead.
Besides those lesions, there were further microscopic findings in the prematurely sacrificed male no. 33 that were considered to be related to its morbidity. They consisted of moderate multifocal mucosal mineralization in the glandular stomach, moderate multifocal hepatocellular necrosis in the liver, marked hemorrhagic necrosis in the prostate gland and minor degrees of lymphoid atrophy in the axillary lymph nodes and alveolar edema in the lung. Further findings related to the premature death of male no. 35 and female no. 71 and 74 consisted of minor degrees of alveolar edema in the lung, of lymphoid atrophy in the mesenteric lymph nodes (no. 35) and lymphocyte apoptosis in the Peyer’s Patches (no. 74).

There were no microscopic findings in the male and female reproductive organs that could be attributed to the treatment with the test item. The cause of non pregnancy of females no. 68, 76 and 79 could not be established from the tissues and organs examined from these animals and from males no. 28, 36 and 39 which were paired with these females. The treatment with the test item did not reveal effects on the completeness of sperm stages or cell populations. There was no indication for maturation arrest, reabsorption of sperm or any other degenerative type. All findings recorded are within the range of normal background alterations.
All other findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age and in this study
type.

Dose Formulation Analysis
Homogeneity and mean concentration of the test item in formulation samples was determined in study weeks 1, 3, 5 and in the last week for all dose groups.
The mean recoveries observed from all study weeks for low dose (LD), medium dose (MD) and high dose (HD) groups were 98.2 %, 103.7 % and 102.2 % of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations for each week were within acceptance criterion of 15 %. All samples were homogenous, as COV was below or equal 15 %.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Some of the pups which did not survive until terminal sacrifice were seen without indication of suckling after birth

see Details on results F1
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The markedly and statistically significantly increased mortality of pups in the HD group with a total mortality (post-natal day 0 to 4) of 20.83 % was based on 3 missing pups attributed to cannibalism by the dam) from in total 2 litters on post-natal day 1. Observed mortality in the MD group was related to pups missing (on post-natal day 1 (3 pups), day 3 (1 pups) and day 4 (1 pup) from in total 3 litters. Though not statistically significant, this dose dependently and biologically significantly increased mortality in the MD group was considered as toxicologically relevant. All live pups on post-natal day 4 in the dose groups and the control group survived until terminal sacrifice on post-natal day 13.

see Details on results F1
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly lower mean weight of pups on PND 0 was noted in the HD group (5.06 g) when compared to the control group (6.05 g). Though only three litters with live pups were delivered in the HD group, this biologically relevant 15 % difference of mean pup weight compared to controls was considered as an effect of the treatment with the test item. Mean pup weight remained slightly (but not statistically significantly) below controls on post-natal day 4 and day 13. Markedly and statistically significantly lower total litter weight was noted on post-natal day 0, 4 and 13 for the HD group which was mainly related to the markedly lower number of live pups.

see Details on results F1
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
effects observed, treatment-related
Description (incidence and severity):
Despite the lower pup weight in the HD group, the mean absolute anogenital distance of male pups was marginally but statistically significantly higher in the HD group (2.69 mm) when compared to the control group (2.36 mm) (14 % above controls). Statistical significance was also achieved for the relative (to cube root of pup weight) anogenital distance of male pups. Values for anogenital distance showed high variability throughout the groups and differences in the HD group to controls was mainly based on one single pup of the HD group with a comparably high value (absolute anogenital distance 3.36 mm). Due to the high variability and the small sample size (7 live male pups) it remains unclear if the observed effect was incidental or induced by the test item. No statistically significant differences were observed for anogenital distance of female pups.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No effect of toxicological relevance was observed for nipple retention in the pups of any group.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
see Details on results F1
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
see Details on results F1

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Litter Data
Litters were delivered by 10 dams of the control, 10 of the LD, 9 of the MD and 5 of the HD group.
The mean number of total pups delivered (live and dead) was markedly and statistically significantly reduced in the HD group (4.67 compared to 11.90 pups in the control group, p < 0.001) due to an adverse effect of the test item. Accordingly, the mean number of male pups (2.00) (p < 0.05) and female pups (2.33) (p < 0.01) was also markedly lower when compared to controls (5.20 and 6.70, respectively). Furthermore, the number of live pups delivered in the HD group (2.83) was markedly and statistically significantly lower when compared to the control group (11.90, p < 0.001). These markedly and statistically significantly differences for number of pups between the HD group and the control group remained throughout the remaining study period (post-natal day 4 and 13).
The number of still births was biologically and statistically significantly higher in the HD group when compared to the control group (1.83 compared to 0.00, p < 0.01) based on two fully stillborn litters (female no. 77 and 80), two stillbirths in female no. 72 and two stillbirths in female no. 75. No littering was observed in female no. 78.
Only one litter of the HD group (female no. 73) was observed without any still births. Marginally but not statistically significantly higher number of still births was observed in the LD group (0.40) and the MD group (0.22). There were two still births in female no. 51, one still birth in female no. 55 and one still birth in female no. 56 of the LD group. One still birth was noted in female no. 65 and also one still birth in female no. 70 of the MD group. Without dose dependency, this marginally higher number of still births in the LD and the MD group was not considered to be toxicologically relevant.
There was no biologically or statistically significant effect on the sex ratio of pups on post-natal day 0, 4 or 13. Slight differences to the control group were considered as incidental. Male and female pups were comparably affected by death at birth or in the post-natal period.
There were no runts in any of the dose groups or control group.

Litter Weight Data
Slightly lower mean weight of pups on PND 0 was noted in the HD group (5.06 g) when compared to the control group (6.05 g). Mean pup weight remained slightly (but not statistically significantly by taking litter as unit) below controls on post-natal day 4 and day 13.
When considering all individual pup weights per group irrespective of the litter, mean male pup weight as well as mean female pup weight on post-natal day 0 were slightly but statistically significantly below the respective controls (males 13 % below controls, p < 0.05; females 13 % below controls, p < 0.01). However, the sample size of the HD group was very low with only 7 live male pups and 10 live female pups born.
Markedly and statistically significantly lower total litter weight was noted on post-natal day 0 and 4 (p < 0.01) and day 13 (p < 0.001) for the HD group when compared to the control group.
There were no biologically or statistically significant effects for litter weight data of the LD and the MD group.

Pup Survival Data
One single pup of the control group was found dead on post-natal day 1 leading to a total mortality for post-natal day 0 to 4 of 0.77 %. All live pups of the LD group survived from post-natal day 0 to 4. A slightly but not statistically significantly higher mortality was observed in the MD group with a total mortality (post-natal day 0 to 4) of 5.20 %. This was related to pups missing (attributed to cannibalism by the dam) on post-natal day 1 (3 pups), day 3 (1 pups) and day 4 (1 pup) from in total 3 litters. A markedly and statistically significantly increased mortality was noted in the HD group with a total mortality (post-natal day 0 to 4) of 20.83 % (p < 0.01). This was based on 3 missing pups from in total 2 litters on post-natal day 1.
All live pups on post-natal day 4 in the dose groups and the control group survived until terminal sacrifice on post-natal day 13.

Anogenital Distance and Nipple Retention
The mean absolute anogenital distance of male pups was marginally but statistically significantly higher in the HD group (2.69 mm) when compared to the control group (2.36 mm) (14 % above controls, p < 0.05). Statistical significance was also achieved for the relative (to cube root of pup weight) anogenital distance of male pups. Values for anogenital distance showed high variability throughout the groups and differences in the HD group to controls was mainly based on one single pup of the HD group with a comparably high value (absolute anogenital distance 3.36 mm). No statistically significant differences were observed for anogenital distance of female pups.
No effect of toxicological relevance was observed for nipple retention in the pups of any group.

Thyroid Hormone Analysis
There were no statistically or biologically significant differences for T4 level of pups measured on post-natal day 13 between the dose groups and controls. However, only pups from 2 dams of the HD group were analyzed with one single pup showing comparably low T4 value (88.6 nmol/L compared to the other examined HD litter with 2 pups with a mean of 149 nmol/L and controls with a mean of 145 nmol/L). As only one pup was affected, this might be considered as outlier and incidental.
There was no effect of toxicological relevance on mean pup thyroid weight on post-natal day 13.

Pup External Findings
Two of the three pups of the HD group which did not survive the post-natal phase were seen pale and without indication of suckling after birth. One pup of the MD group was also seen without indication of suckling after birth and was missing (attributed to cannibalism by the dam) afterwards. No indication of suckling was also observed in one pup of the control group which, however, survived until terminal sacrifice. The only pup of the control group which did not survive until terminal sacrifice was observed to be pale with haematoma at the neck and a blue colored abdomen after birth.
There were few other gross external abnormalities in few pups of the dose groups and/or the control group such as haematoma on the skin which were considered as spontaneous and not related to the treatment with the test item. All pups in two litters of the LD group were noted with their hair coat partly missing at terminal sacrifice which was assumed to be related to barbering by the respective dam. Without dose-dependency this was not considered to be test item-related. There were no remarkable, toxicological relevant external abnormalities.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes

Applicant's summary and conclusion

Conclusions:
On the basis of this combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test with the test item in male and female Wistar rats with dose levels of 25, 100 and 400 mg/kg body weight/day the following conclusions can be made:
At a dose level of 400 mg/kg body weight/day toxicologically relevant, unspecific clinical signs of reduced health condition (piloerection, reduced spontaneous activity, hunched posture and red nasal discharge) were noted. Two males and two females dosed with 400 mg/kg body weight/day died prematurely during the course of treatment. One male was found dead on study day 5 and the other male was sacrificed in a moribund condition on study day 24. Both decedent females were found dead on study day 28.
Significantly reduced body weight and food consumption were noted in adult males and females at 400 mg/kg body weight/day.
Test item-related, adverse effects were also observed for litter data at 400 mg/kg body weight/day. In relation to an increased number of stillbirths, gestation period was noted to be prolonged. The delivery index (no. of dams with live pups born / no. of pregnant dams x 100) was shown to be markedly reduced (50 %) when compared to 100 % in the control group. The mean number of total pups delivered (live and dead) was markedly reduced (4.67 compared to 11.90 in controls) and there was also an adverse effect on mean number of live pups born (2.83 compared to 11.90 in controls). Due to still births, non-littering and/or resorptions, post-implantation loss at 400 mg/kg body weight/day was markedly higher (76.81 %) compared to the control group (5.07 %). Furthermore, slightly lower mean weight of pups on PND 0 (15 % below controls) was also considered as biologically relevant. Total litter weight was markedly reduced at the same dose level due to the markedly lower number of live pups. No such effects on litter data were observed at the lower dose levels of 100 and 25 mg/kg body weight/day.
Test item-related, adverse effects were also noted for pup survival. At 400 mg/kg body weight/day a reduced viability index of 79.17 % was noted during post-natal days 0-4 and of 94.80 % at 100 mg/kg body weight/day when compared to 99.23 % in the control group. The dose dependently and biologically significantly increased mortality of pups at 100 and 400 mg/kg body weight/day was considered as an adverse effect of the treatment with the test item.
The toxicological relevance of slightly higher mean absolute anogenital distance in male but not female pups at 400 mg/kg body weight/day was unclear due to high variability and small sample size (7 live male pups).
Slightly lower mean percentage of reticulocytes was noted at 400 mg/kg body weight/day in adult males but not females (45 % below controls) which could be related to the microscopic finding of slight focal hypocellularity in the sternal bone marrow.
Under the conditions of this study, treatment with the test item induced a tubulopathy in the kidneys, characterized by minimal to moderate degrees of tubular degeneration/necrosis, tubular regeneration as well as tubular dilation in proximal tubules, distal tubules and/or collecting ducts at 400 mg/kg body weight/day. These findings were sometimes accompanied by slight cortical tubular vacuolation, slightly increased mononuclear cell infiltrates and minor degrees of papillary necrosis or urothelial hyperplasia. Minimal and/or slight cortical tubular vacuolation was also recorded in the kidneys of the lower dose groups, and minimal tubulopathy was present in one male at 100 mg/kg body weight/day.

The tubulopathy correlated with increased kidneys weights and grossly visible enlarged kidneys and was considered to be of adverse nature. Predominantly, morbidity and mortality of the adult males and females in this study was considered to be related to the observed tubulopathy.
No adverse effects of the test item were noted at a dose level of 25 mg/kg body weight/day. Thus, the NOAEL for general systemic toxicity as well as for developmental toxicity could be established at 25 mg/kg body weight/day.
Executive summary:

The aim of this study was to assess the possible effects of the test item on male and female fertility and development after repeated dose administration in Wistar rats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of a maximum of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed. Animals of an additional control group were handled identically as the dose groups but received 0.25 % aqueous hydroxypropyl methylcellulose, the vehicle used in this study. The 4 groups comprised 10 male and 10 femaleWistarrats each.

Before initiation of dosing, all females were screened for two weeks for regular estrous cyclicity and animals (10 females / group) with regular estrous cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

Control (C):                   0         mg/kg body weight/day

Low Dose (LD):             25       mg/kg body weight/day

Medium Dose (MD)    100     mg/kg body weight/day

High Dose (HD):         400     mg/kg body weight/day

The test item formulation was prepared with the vehicle, 0.25 % aqueous hydroxypropyl methylcellulose, at least once every ten days based on available stability data. The prepared formulation was stored at 2-8°C and protected from light.Formulates were kept under magnetic stirring during the daily administration.The vehicle was also used as control item. Dose volumes were adjusted individually based on the body weight most recently measured. Theadministration volume was 10 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died or were euthanised for animal welfare reasons were examined macroscopically and at the conclusion of the test all surviving animals were sacrificed and observed macroscopically.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behavior observations were performed before the treatment period and in the last week of treatment infive randomly selected males and femalesof each group.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not performed during the mating period in males and females and the post-mating period in males. During pregnancy, females were weighed on gestation days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with the pups.

After 14 days of treatment of males and females, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum.

The anogenital distance of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).

Males were sacrificed after completion of the mating period on day 29 and females along with their pups were sacrificed on post-natal day 13. Non-pregnant females were sacrificed on day 26. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on post-natal day 4 for interim sacrifice for blood sampling and those found dead were carefully examined for gross external abnormalities.

At the conclusion of the test, all surviving adult animals were sacrificed and observed macroscopically. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved.

Haematological and clinical biochemistry evaluations and coagulation measurements were performedon blood samples collected at terminal sacrifice fromfive randomly selected males and females from each group. Urinalysis wasperformedon samples collected at terminal sacrifice fromfive randomly selected males and females from each group.

A full histopathological evaluation of preserved organs/tissues was performed in 5 randomly selected males and females of the control and HD group and in all animals that died prematurely during the course of treatment. Due to test item-related morphologic changes detected in stomach, liver (males only), kidneys, adrenal glands, spleen, bone marrow (males only) and thymus of high dose animals, these organs were also examined from five randomly selected animals from the low and medium dose groups.Testes, epididymides, ovaries, uterus with cervix, vagina and accessory sex organs were examined in all control and HD animals and in non-pregnant females of the LD and MD group and their male mating partners. Any gross lesions macroscopically identified were examined microscopically in all animals.

Summary Results

Four animals of the HD group died prematurely during the course of treatment. Male no. 33 was sacrificed in a moribund condition for animal welfare reasons on study day 24. Microscopic findings related to its morbidity consisted ofmultifocal mucosal mineralization in the glandular stomach, multifocal hepatocellular necrosis in the liver, hemorrhagic necrosis in the prostate gland, minor degrees of lymphoid atrophy in the axillary lymph nodes, alveolar edema in the lung and tubulopathy of the kidneys.Male no. 35 (high dose) was found dead on day 5 of the premating period without any preceding signs of morbidity. Female no. 71 and 74 (high dose) were both found dead on study day 28. Before female no. 71 showed slight loss of body weight and piloerection. No signs of reduced health condition were observed in female no. 74 before death. At necropsy, blood was found in the uterus of female no. 71 and in the jejunum and ileum of female no. 74.Tubulopathy of the kidneys was considered to be the main cause of morbidity of male no. 33 and, along with papillary necrosis, to be the cause of death (renal failure) of female no. 71. Further microscopic findings related to the premature death of male no. 35 and female no. 71 and 74 consisted of minor degrees of alveolar edema in the lung, of lymphoid atrophy in the mesenteric lymph nodes (no. 35) and lymphocyte apoptosis in the Peyer’s Patches (no. 74). The exact cause of death of the male no. 35 and female no. 74 could not be established from the organs and tissues examined. No mortality of adult animals occurred in the control, the LD and the MD group during the treatment period of this study.

Various unspecific clinical signs of reduced health condition were noted in some males and females of the HD group mainly at the end of the treatment period: piloerection, reduced spontaneous activity, hunched posture and red nasal discharge. These signs in the HD group were considered as toxicologically relevant effects of the treatment with the test item.

Moving the bedding and salivation were observedtransiently and dose dependently in animals of the HD and the MD group. These slight clinical signs were mainly seen at the end of the treatment period in males and during gestation and lactation period in females. Both signs were observed in short timely relation to dose application or in anticipation thereof and thus were considered to be a sign of discomfort or a local reaction to the test item applied by gavage. These signs were not considered as adverse systemic effects.

None of the females showed signs of abortion or premature delivery.

During the weekly detailed clinical observation, no relevant differences between the groups were found except rarely salivation in some animals of the MD and HD group.

In males and females, no relevant effects were observed in any of the parameters of the functional observation battery at the end of the treatment period. There were no biologically or statistically significant differences in body temperature between the dose groups and the controls. There were no ophthalmoscopic findings in any of the animals of this study.

Test item-related, adverse effects were noted for body weight, body weight development and food consumption in the HD group:

After initiation of treatment, males of the HD group showed a moderate and statistically significant loss of body weight compared to a slight gain of body weight in controls. Except for the second week of the study, mean body weight further decreased slightly until the end of the study. Considering the whole treatment period, there was a biologically and statistically significant mean loss of -7.25 g body weight (equal to -1.9 % under exclusion of decedents) in the male HD group compared to a gain of +35.70 g (equal to +9.6 %) in the respective controls. Thus, mean body weight of the male HD group was moderately and statistically significantly lower throughout the mating/postmating period and at terminal sacrifice (up to 10 % below controls). A comparable test item-related effect was noted for the female HD group. A slight but statistically significant mean loss of body weight occurred in the first week of treatment in the female HD group. Although body weight increased subsequently, mean weight gain remained slightly lower in the female HD group throughout the whole study period when compared to controls. Statistical significance was achieved in the 1stweek of gestation, the last week of gestation and when related to the whole gestation period (day 0-20) (body weight gain 56 % below controls). Thus, mean body weight of the female HD group was moderately and statistically significantly reduced on day 7 and day 20 of gestation and day 4 and day 13 of lactation (up to 18 % below controls).

In males, in correlation to the body weight loss, mean food consumption per animal in the HD group was moderately lower in the first week of the premating period when compared to controls (27 % below controls).In females, a slight tendency towards lower food consumption was also observed in the first week of the premating period. In accordance to the attenuated body weight gain during gestation, food consumption of the HD group was also noted to be moderately and statistically significantly lower when compared to the control group during the whole gestation period (up to 30 % below controls). Food consumption was further reduced in the HD group during lactation with values being 38 % below controls in the first week and 54 % below controls in the second week of lactation.

No effects of toxicological relevance were noted in the male and female LD and MD groups for mean body weight, body weight gain and food consumption.

The test item had no biologically significant effect on the estrous cyclicity analyzed during the 2 weeks premating period after the first administration.

The copulation index was 100 % in all groups. Length of the precoital interval was within the normal range of variation for animals of this age and strain in the dose groups and the control group. However, a biologically and statistically significant effect was noted on the duration of gestation in the HD group (mean 23.80 days compared to 22.30 days in controls) with two females showing a gestation length of 24 days and one female 25 days. The observed prolonged gestation period in the HD group was considered to be related to the increased number of stillbirths.

The fertility index (number of pregnant females/number of copulated females x 100) was slightly reduced in the MD group (90 %) and the HD group (80 %) when compared to the control group (100 %): As histologically no morphological reason for non-mating of those females and their male mating partners was seen, this slightly reduced fertility index was considered as incidental in nature.

There were no biologically or statistically significant effects on the number of corpora lutea and number of implantation sites in the dose groups when compared to the control group.

Test item-related, adverse effects were observed for litter data.

Litters were delivered by 10 dams of the control, 10 of the LD, 9 of the MD and 5 of the HD group.The delivery index(no. of dams with live pups born / no. of pregnant dams x 100)was markedly reducedin the HD group (50 %) when compared to the control group (100 %). The delivery index was 100 % in the LD and the MD group. The mean number of total pups delivered (live and dead) was markedly and statistically significantly reduced in the HD group (4.67 compared to 11.90 pups in the control group). The number of live pups delivered in the HD group (2.83) was also markedly and statistically significantly lower when compared to the control group (11.90). These significant differences for number of pups between the HD group and the control group remained until the end of the study period (post-natal day 4 and 13). The number of still births was biologically and statistically significantly higher in the HD group when compared to the control group (1.83 compared to 0.00) based on two fully stillborn litters and two more stillborn pups in two HD females each. Only one litter of the HD group was observed without any still births. Marginally but not statistically significantly higher number of still births was observed in the LD group (0.40) and the MD group (0.22). Without dose dependency, this marginally higher number of still births in the LD and the MD group was not considered as an adverse effect of the test item. Due to still births, non-littering and/or resorptions, post-implantation loss in the HD group (76.81 %) was markedly and statistically significantly higher compared to the control group (5.07 %). No toxicologically relevant effect on post-implantation loss and litter data was noted for the LD and the MD group.Percentage of pre-implantation loss was within the normal range of biological variation for all groups.

There was no biologically or statistically significant effect on the sex ratio of pups on post-natal day 0, 4 or 13.

Slightly lower mean weight of pups on PND 0 was noted in the HD group (5.06 g) when compared to the control group (6.05 g). Though only three litters with live pups were delivered in the HD group, this biologically relevant 15 % difference of mean pup weight compared to controls was considered as an effect of the treatment with the test item. Mean pup weight remained slightly (but not statistically significantly) below controls on post-natal day 4 and day 13. Markedly and statistically significantly lower total litter weight was noted on post-natal day 0, 4 and 13 for the HD group which was mainly related to the markedly lower number of live pups.

Test item-related, adverse effects were also noted for pup survival.There was a moderately reduced viability index during post-natal days 0-4 (no. of live offspring at day 4 / no. of live offspring at birth x 100) of 79.17 % in the HD group when compared to 99.23 % in the control group. Viability index during post-natal days 0-4 was slightly reduced in the MD group (94.80 %). No effect was noted in the LD group (100 %).

The markedly and statistically significantly increased mortality of pups in the HD group with a total mortality (post-natal day 0 to 4) of 20.83 % was based on 3 missing pups attributed to cannibalism by the dam) from in total 2 litters on post-natal day 1. Observed mortality in the MD group was related to pups missing (on post-natal day 1 (3 pups), day 3 (1 pups) and day 4 (1 pup) from in total 3 litters. Though not statistically significant, this dose dependently and biologically significantly increased mortality in the MD group was considered as toxicologically relevant. All live pups on post-natal day 4 in the dose groups and the control group survived until terminal sacrifice on post-natal day 13.

Occasionally some of the pups of the MD and the HD group which did not survive until terminal sacrifice were seen without indication of suckling after birth. There were no remarkable, toxicologically relevant external abnormalities in the pups of any group.

Despite the lower pup weight in the HD group, the mean absolute anogenital distance of male pups was marginally but statistically significantly higher in the HD group (2.69 mm) when compared to the control group (2.36 mm) (14 % above controls). Statistical significance was also achieved for the relative (to cube root of pup weight) anogenital distance of male pups. Values for anogenital distance showed high variability throughout the groups and differences in the HD group to controls was mainly based on one single pup of the HD group with a comparably high value (absolute anogenital distance 3.36 mm). Due to the high variability and the small sample size (7 live male pups) it remains unclear if the observed effect was incidental or induced by the test item. No statistically significant differences were observed for anogenital distance of female pups.

No effect of toxicological relevance was observed for nipple retention in the pups of any group.

Serum analysis in adult males at terminal sacrifice revealed dose dependently and statistically significantly lower thyroxine (T4) hormone levels throughout all dose groups when compared to the control group (LD: 81.92 nmol/L (p < 0.05), MD: 55.97 nmol/L (p <0.001), HD: 37.91 nmol/L (p < 0.001) compared to 93.40 nmol/L in controls). However, there were no statistically or biologically significant weight changes in pituitary or thyroid/parathyroid glands which could have been further indicative of disturbances of the normal function of the hypothalamic-pituitary-thyroid axis (HPT axis). There were also no corresponding microscopic findings in the histopathologically examined thyroid/parathyroid glands of the male and female HD group such as increased cell proliferation. Thyroid-stimulating hormone (TSH) levels are regulated by negative feedback of thyroid hormones. In general, when thyroid hormone serum levels are decreased, thyroid-releasing hormone (TRH) and TSH release are stimulated. However, in this study, analysis of circulating TSH levels in serum samples of adult males showed no dose dependency and no statistically significant difference between the dose groups and the control group. No correlation between TSH- and T4-values was observed for the individual animals. Furthermore, there were no alterations to certain endpoints such as sperm maturation in males or estrous cycles in females which could be influenced by changed thyroid hormone levels though increased number of stillbirths was noted in the HD group. The reason for the statistically significantly lower T4 levels in the dose groups remains unclear. However, in the absence of changes to thyroid weight, histopathological findings, clinical manifestation and changes of TSH levels, the observed isolated finding of statistically significant changes to thyroxine levels was not considered as adverse.

There were no statistically or biologically significant differences for T4 level of pups measured on post-natal day 13 between the dose groups and controls. There was no effect of toxicological relevance on mean pup thyroid/parathyroid weight on post-natal day 13.

In adult males but not females at necropsy,slightly lower mean percentage of reticulocytes was noted in the HD group (0.92 %) when compared to the control group (1.68 %) (45 % below controls). A relationship with the treatment of the test item cannot be excluded for this biologically relevant observation and could be related to the microscopic finding of minimal to slight focal hypocellularity in the sternal bone marrow in the male HD group. There were no further toxicologically relevant effects on haematology, blood coagulation and clinical biochemistry in any group. Slight differences to controls were not assumed to be biologically relevant.Single deviating values in individual animals compared to controls were considered as incidental.

There were no toxicologically relevant effects on parameters of urinalysis in any group at the end of the treatment period.

Macroscopic findings that could be attributed to the treatment with the test item consisted of grossly enlarged kidneys (males: 1/10 of the MD, 5/10 of the HD group; females: 2/10 of the HD group) and enlarged adrenal glands (males: 3/10 of the HD group; females: 2/10 of the HD group). Kidneys of 1/10 males of the HD group were also seen with abnormal color (spotted). Furthermore, small spleen was noted in 4/10 males of the HD group and small thymus in 6/10 males of the HD group.

Statistically significant changes in organ weights correlating to macroscopic and microscopic findings were recorded in spleen, thymus and kidneys.

At the end of the treatment period, a toxicologically relevant effect was noted for absolute and relative (to body weight) spleen weight in the HD group. Spleen weight was moderately decreased in males of the HD group (absolute weight 42 % below controls) and females of the HD group (absolute weight 35 % below controls). Furthermore, the relative kidney weight to body weight ratio was moderately and dose dependently higher in males of the MD and the HD group (16 % and 28 % above controls) and in females of the HD group (15 % above controls) when compared to the respective controls. In males of the HD group, the absolute thymus weight was markedly reduced (45 % below controls), whereas no such effect was noted in females. Other slight but statistically significant changes in organ weights were considered incidental or secondary to body weight changes and did not correlate with microscopic findings.

Under the conditions of this study, treatment with the test item induced a tubulopathy in the kidneys, characterized by minimal to moderate degrees of tubular degeneration/necrosis, tubular regeneration as well as tubular dilation in proximal tubules, distal tubules and/or collecting ducts of animals of the HD group. These findings were sometimes accompanied by slight cortical tubular vacuolation, slightly increased mononuclear cell infiltrates and minor degrees of papillary necrosis or urothelial hyperplasia. Minimal and/or slight cortical tubular vacuolation was also recorded in the kidneys of the LD and MD group, and minimal tubulopathy was present in one male of the MD group. The tubulopathy correlated with increased kidneys weights and grossly visible enlarged kidneys (and in one case along with abnormal color, spotted kidneys), and was considered to be of adverse nature.

Further treatment-related microscopic findings were observed in the stomach,liver (males only), adrenal glands, spleen, thymus and bone marrow (males only) in the HD group.

In the stomach, they consisted of minimal to moderate epithelial hyperplasia along with parakeratosis and minimal submucosal inflammation. These findings were considered to be due to a local irritant effect of the test item when administered orally and therefore non-adverse.

In the liver, minimal centrilobular hepatocellular hypertrophy was recorded in males of the HD group. Since there were no indicators of liver injury (Kupffer cell proliferation, necrosis, apoptosis, fibrosis, other degenerative changes, etc.), this lesion was considered to be of adaptive nature, reversible and therefore non-adverse.

Macroscopically enlarged adrenal glands correlated microscopically with minimal to slight diffuse hypertrophy in the Zona fasciculata of the adrenal glands in animals of both sexes treated with 400 mg/kg/day. In addition, a moderate to marked cortical diffuse hemorrhage was present in both males that died prematurely. Minimal hemorrhage was also present in another high dose male. The reason for this change remains unclear. In the spleen, a slight to marked lymphoid atrophy, correlating with decreased organs weights and small spleens (males only) was recorded in animals of both sexes of the HD group. A further finding consisted of slight increased hemosiderin deposits in the spleen of these animals. Decreased thymus weights and small thymuses correlated microscopically with an increased severity grade of atrophy in high dose males. In high dose females, the severity grade for this finding was increased only in one female that was found dead. In the bone marrow, some male animals treated with 400 mg/kg/day showed minimal to slight focal hypocellularity. All findings recorded in adrenal glands, spleen, thymus and bone marrow were considered to be indirect effects secondary to body weight loss and/or stress, and as such, were considered to be not systemic effects of oral treatment with the test item and, therefore, non-adverse.

There were no microscopic findings in the male and female reproductive organs that could be attributed tothetreatment with the test item. The treatment with the test item did not reveal effects on the completeness ofspermstages or cell populations. There was no indication for maturation arrest, reabsorption of sperm or any other degenerative type.

Based on microscopic findings noted in the kidneys, a histopathological NOAEL could be established at 25 mg/kg/day; a histopathological NOEL could not be established.

Nominal concentrations of the test item were confirmed in analyzed dose formulations of all dose groups in study weeks 1, 3, 5 and the last week. All samples were homogenous.