Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-03-23 to 2018-03-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
2 HD animals were dosed with a wrongly prepared (inadvertently prolonged sonication) formulation on 2 single days. Two females (C, LD) were inadvertently sacrificed on gestation day 21 instead of 20 and therefore excluded from calculation of means.
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST SYSTEM
Species/strain: healthy 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 of the animals at the arrival at BSL Munich: approx. 11-12 weeks old
Body weight before initiation of pairing: males: 307 - 352 g (mean: 328.50 g, ± 20 % = 262.80 - 394.20 g)
females: 190 - 235 g (mean: 209.12 g, ± 20 % = 167.29 - 250.94 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to 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)
- The animals were kept individually in IVC cages (type III H, polysulphone cages) on Altromin saw fibre bedding (except during the pre-mating period when animals were kept in groups of up to 5 animals / gender / cage in IVC double decker type GR1800 cages or during mating period when two females were paired with one male)
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (5 days)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF THE TEST ITEM FORMULATION
The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.
The test item formulation was prepared by weighing in the test item into a tared plastic vial on a suitable precision balance, adding the vehicle corn oil to give the appropriate final concentration of the test item formulation and further stirring it for 2-3 minutes. The solution was sonicated for 5-10 minutes. However, the first high dose formulation that was prepared for dosing in this study was inadvertently sonicated for 30 minutes. 2 HD animals (no. 76 and no. 77) were dosed with this wrongly prepared formulation on 2 single days (gestation day 5 and 6) after which the remaining aliquots were discarded.
Based on the results of stability testing (Eurofins Munich study no. 169064), the test item formulations were prepared at least once every 10 days. The prepared formulations were stored at room temperature.
Homogeneity of the test item in the vehicle was maintained by keeping formulates under magnetic stirring during the daily administration.
The vehicle was also used as control item.

CHARACTERISATION OF THE VEHICLE
Name: corn oil
Manufacturer: Sigma Aldrich
Batch No.: MKBZ9899V
Physical State: liquid
Storage Conditions: room temperature
Expiry Date: 27 May 2017
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.


ADMINISTRATION OF DOSES
The test item formulation or vehicle was administered at a single dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Dose levels: 0, 100, 300, 1000 mg/kg bw (concentration in vehicle: 0, 25, 75, 250 mg/ml)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich (Eurofins Munich study no. 169064).
Prepared low dose and high dose formulations were shown to be stable at all conditions tested (6 hours at RT, 10 days at RT, 10 days at 2-8 °C, 10 days at -15 to -35 °C).
Prestart homogeneity investigation included samples collected from various levels (top, middle and bottom) of a high dose and low dose formulation preparation.
As the test item formulations were shown to be homogenous after 60 min without stirring, during the study samples were not collected for the investigation of homogeneity but for determination of test item concentration in the first and the last week of the study from all groups (8 samples in total).
In study week 1, measured concentration of the prepared high dose formulation did not meet the acceptance criterion of 10 % with a recovery of 82.6 % due to wrong preparation of formulation. Therefore, after 2 days of dosing of two HD animals the remaining high dose aliquots were discarded and a new formulation prepared and analysed again for study week 1.
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich and until then stored under appropriate conditions based on available stability data (Eurofins Munich study no. 169064). The B-samples will be retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report.
The exact procedure and analytical results are reported in the appendix of this report (Eurofins Munich study phase no. 169065).
Details on mating procedure:
PREPARATION OF THE ANIMALS
After the acclimatisation period of 5 days, females were paired with males as per the ratio of 1:2 (male to female). Prior to the start of the mating a detailed clinical observation outside the home cage was made. No animal showed remarkable clinical signs before mating.
Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day. At the subsequent mornings, the vaginal smear of the female was checked to confirm the pregnancy. The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body weights were comparable with each other. Each animal was assigned a unique identification number. After getting 100 sperm positive females, the remaining females and males were discarded without any observations.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week from gestation day 5 until gestation day 19.
Frequency of treatment:
Once per day. 7 days per week
Duration of test:
On GD 20, i.e. the day prior to the expected day of delivery, the presumed pregnant females were subjected to a caesarean section.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Remarks:
25 mg/mL
Dose / conc.:
300 mg/kg bw/day
Remarks:
75 mg/mL
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
100 mg/mL
No. of animals per sex per dose:
156 animals (52 males and 104 females) were included in the study.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Doses were selected according to the results of the dose range finding study (BSL Munich study no. 169062, non-GxP) and in consultation with the sponsor. The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-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 treatment groups.

- Rationale for animal assignment (if not random): Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that the mean body weights were comparable to each other

Examinations

Maternal 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.
Clinical observations included check for 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. 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.

Body Weight and Food Consumption
All animals were weighed once before initiation of pairing to ensure that the body weights are within + 20 % variation.
The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.
Food consumption of sperm positive females was measured on gestations days 5, 8, 11, 14, 17 and 20.
Food consumption was not measured for males during the entire study or for both male and females during the mating period.

Post-Mortem Examination
On gestation day 20, sperm positive (presumed pregnant females) were subjected to a caesarean section after sacrificing the animals using an overdose of pentobarbital injected intraperitoneally at a dosage of approximately 8 mL/kg bw.
Two females (animal no. 4 of the control group and no. 29 of the LD group) were sacrificed on gestation day 21. Individual values of the dams and foetuses were excluded from calculation of mean as appropriate.
At the time of termination or death during the study, the dam (presumed pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Any macroscopic findings were preserved in 4% neutral-buffered formaldehyde.
Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed. Uteri that appear non-gravid were further examined by staining with 10 % ammonium sulphide solution to confirm the non-pregnant status.
Each gravid uterus with the cervix was weighed.
The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or foetal deaths as well as the number of viable foetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death of the conceptus. The position and number of foetuses in each uterine horn was also recorded.
Males were sacrificed without any observations at any time after the completion of the mating or were used for other studies.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

FOETAL EVALUATIONS
All foetuses from a particular dam were identified by using numbered plates and were weighed and sexed based on the anogenital distance. Each foetus was examined for external anomalies.
One half of each litter was examined for soft tissue anomalies by a microdissection technique.
The remaining foetuses were processed by Alizarin staining and the first 20 litters per group after caesarian section on gestation day 20 were examined for skeletal alterations.
Craniofacial examination of the heads of the foetuses used for the soft tissue examination of the first 20 litters per group (with caesarian section on gestation day 20) were performed for internal structure including the eyes, brain, nasal passage and tongue by razor blade serial sectioning technique.

EXTERNAL EXAMINATION
Lip and palate were examined for cleft lip and palate by gently opening the mouth with forceps. The head, eyes, ears, jaw and snout was examined for the shape and size. The trunk was examined for any external abnormalities. Limbs were examined for shape, size, position and digits for number and depth of digital furrows. The tail was examined for presence, size, shape and position.

SKELETAL EXAMINATION
Foetuses scheduled for the skeletal examination were eviscerated and the entire litter was transferred into plastic bottles containing 95 % ethanol. These foetuses were processed using the Alizarin red staining technique. After fixation in 95 % ethanol, the foetuses were macerated with a 1 % aqueous potassium hydroxide solution for 1 day and transferred to an Alizarin red solution (0.0025% in 1 % aqueous potassium hydroxide) for 1 day. After that the foetuses were again transferred to 1 % KOH. Alizarin stained foetuses were then cleared and dehydrated in a solution containing 2 parts of 70 % ethanol, 2 parts of glycerin and one part of benzyl alcohol for 1 day and finally preserved in a 1:1 solution of 70 % ethanol and glycerin.
The stained foetuses were examined under the stereomicroscope, the skull was examined for size, shape and degree of ossification of nasal, parietal, interparietal, supraoccipital, exoccipital, lacrimal, zygomatic (malar), squamosal (temporal), premaxillary, maxillary, basisphenoid, hyoid and tympanic ring (annulus). Similarly, the vertebral centres, ribs and sterna centres were also examined for size, shape and counted for the number of ossification centers. The cervical, thoracic, lumbar, sacral, caudal vertebrae were observed for the ossification of centers and arches. Pelvic girdles, fore limbs and hind limbs were examined for the development of the bones. Any deviation from the normal development was recorded for each foetus.

VISCERAL EXAMINATION
Foetuses scheduled for the visceral examination were pinned to a paraffin covered petri dish with the ventral side up under a stereo microscope. The abdominal and thoracic cavities of all foetuses were dissected and examined for visceral anomalies.
The intestine, stomach, spleen and pancreas were examined for size and position. The liver was examined for size, shape, colour and number of lobes. The kidney and adrenal glands were observed for size, position and colour. The kidneys were further observed for the presence of clear fluid-filled cysts, cortical cysts, pitting or granular appearance and then sectioned with a sharp scalpel blade to examine the pelvis for distention or the presence of calculi or white granular material.
The left kidney was sectioned with one longitudinal slice just off center and the right kidney was sectioned with one transverse slice directly through the papilla. The capsule, cortex, medulla, renal papilla, and renal pelvis were checked for the presence and the pelvis for distension with fluid.
The reproductive organs were exposed by raising the intestine and the attached viscera from the dorsal wall and examined for any developmental defect.
The rib cage was cut from the side of the sternebrae to examine the thoracic organs. The lung was observed for size, colour and number of lobes. The thymus gland was checked for size and position. The trachea and oesophagus were exposed by removing the thymus gland and examined for fusion or tracheaoesophageal fistula.
The position, size, colour and shape of the heart was recorded. The pericardial sac was opened and the heart was fully exposed and examined for the presence or absence of major blood vessels like aortic arch, pulmonary artery and ductus arteriosus. For an examination of the internal anatomy of the heart, the heart was then repositioned and two cuts through the right ventricle were made using micro-dissecting scissors. The first cut was taken starting from the right of the ventral midline surface at the apex to the base of the pulmonary artery and the second cut was made through the midline surface at the apex extending to the left ventricle in to the ascending aorta. Incisions were opened with fine forceps for the examination of interventricular and auriculo ventricular septum.
After the completion of the visceral examination by the microdissection technique, foetuses were transferred to plastic bottles containing formalin-aceto-alcohol for later craniofacial examination by the razor-blade-serial-section technique.

CRANIOFACIAL EXAMINATION
Before initiating the serial sectioning with a razor blade, foetuses were transferred to the beaker containing tap water for deformalisation. After deformalisation, the foetus was decapitated and the head of the foetus was subjected to 5-7 sections in order to observe the internal structures of the head including the symmetry of the external nares, nasal conche, nasal septum, palate, the development of the cerebellum and brain stem. Transverse sections of the cephalic region were observed under the stereomicroscope and any anomalies were recorded.
Statistics:
A statistical assessment of the results of the body weight, food consumption and litter data was performed by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were analysed using Fisher’s exact test. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).
Historical control data:
Please refer to "Attached background material"

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Moving the bedding was noted dose-dependently in 2/25 females of the MD group on one day of treatment and in 4/25 females of the HD group on few days of treatment. This slight clinical sign was seen directly after dose administration, it was considered as slight clinical sign elicited by local effects of the test item formulation and/or attributed to discomfort of the animals due to oral administration, but not systemic toxicity.
Female no. 91 of the HD group was noted with slight piloerection and sticky fur at its abdomen on gestation day 17. At time of caesarian section, implantation sites but no foetuses were observed in the uterus. As this female showed body weight loss at the end of the gestation (day 14-17: 27 g, day 17-20: 23 g), abortion of foetuses cannot be excluded. Female no. 6 of the control group was also seen with implantation sites but without foetuses at caesarian section but without related clinical signs or body weight loss at the end of the gestation. As only one dam of the HD group and one dam of the control group were affected, this was assumed to be incidental.
No further female showed signs of abortion or premature delivery prior to the scheduled sacrifice.
Piloerection as general sign of discomfort or slightly reduced health condition was observed in further 3/25 females of the HD group on few days at the end of the gestation phase. As only two animals were shortly affected without any further indication of morbidity in terms of clinical signs and body weight development, this was not considered as toxicologically relevant.
Local alopecia on various body parts was noted in few females of the control and the dose groups and was considered incidental in nature.
One female of the control group was seen with ataxia from gestation day 11 until necropsy. This was assumed to be related to the clinical observation of a local bruise which was also apparent from gestation day 11 onwards and might be based on a local injury by accident.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred in the control or any of the dose groups during the treatment period of this study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight increased in all groups before initiation of treatment and further increased with the progress of the study comparably in the control, the LD and the MD group.
After initiation of treatment, mean body weight gain was moderately and statistically significantly lower during gestation days 5-8 in the HD group when compared to controls (0.52 g when compared to 5.23 g). Few individual HD females were seen with a slight loss of body weight.
Throughout the remaining treatment period, mean body weight gain remained moderately to slightly lower in the HD group when compared to the control group with the greatest effect and statistical significance noted during gestation days 8-11 (40 % of controls) and 11-14 (50 % of controls).
When related to the entire study period (gestation day 0-20), moderately and statistically significantly lower body weight gain was observed in the HD group (70 % of controls).
Differences in body weight gain compared to controls resulted in slightly to moderately and statistically significantly reduced mean body weight of the HD group from gestation day 8 (96 % of controls) onwards with the greatest effect on gestation day 20 (90 % of controls). This moderate effect on body weight development in HD group was considered as test item related.
Statistically significantly lower mean body weight gain was noted for gestation day 8-11 in the MD group (67 % of controls). Body weight development was comparable to controls throughout the remaining treatment period. This transient difference to controls without relevant impact on mean body weight was not considered as toxicologically relevant.
Body weight development in the LD group was comparable to controls.

see Tables under Section Any other relevant information including tables
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In correlation to the lower body weight gain after initiation of treatment, mean food consumption in the HD group was moderately and statistically significantly lower when compared to controls throughout the whole treatment period. The greatest effect was noted during gestation day 8-11 (77 % of controls). When related to the entire study period (gestation day 0-20) mean food consumption was 90 % below controls in the HD group with achieving statistical significance. This moderate effect on food consumption of the HD group was considered as an adverse effect of the treatment with the test item
Food consumption of the LD and the MD group was comparable to controls throughout the study period without biologically or statistically significant difference.

see Tables under Section Any other relevant information including tables
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross pathological changes were observed during the macroscopic examination of females of any of the groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Female no. 91 of the HD group was noted with slight piloerection and sticky fur at its abdomen on gestation day 17. At time of caesarian section, implantation sites but no foetuses were observed in the uterus. As this female showed body weight loss at the end of the gestation (day 14-17: 27 g, day 17-20: 23 g), abortion of foetuses cannot be excluded.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on maternal toxic effects
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on maternal toxic effects
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
see Details on maternal toxic effects
Dead fetuses:
no effects observed
Description (incidence and severity):
see Details on maternal toxic effects
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No female showed signs of premature delivery prior to the scheduled sacrifice on gestation day 20.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Successful mating resulted in 22/25 pregnancies in the LD group, 21/25 in the MD group and 24/25 in the HD group when compared to 22/25 pregnancies in the control group.
Details on maternal toxic effects:
Details on maternal toxic effects:

PRENATAL DATA
Successful mating resulted in 22/25 pregnancies in the LD group, 21/25 in the MD group and 24/25 in the HD group when compared to 22/25 pregnancies in the control group.
Test item-related, moderately and statistically significantly lower mean terminal body weight was observed in the HD group (90 % of controls) when compared to controls. As mean uterus weight was comparable to controls, also moderately and statistically significantly lower mean adjusted maternal weight (maternal weight minus gravid uterus weight) was observed in the HD group (87 % of controls).
Mean terminal body weight of the LD and the MD group on gestation day 20 was comparable to controls (99 % and 97 % of controls, respectively). Slightly and not dose-dependently higher mean uterus weight was observed in the LD (114 % of controls) and the MD group (108 % of controls) without achieving statistical significance. This was considered as incidental and mainly related to single dams of the control group with particularly low uterus weight due to no or low number of live foetuses (female no. 6, 23 and 24). Mean differences in uterus weight led to slightly lower mean adjusted maternal weight in the LD and the MD group (94 and 95 % of controls, respectively). This achieved statistical significance but was not considered as toxicologically relevant.

No test item-related effects of toxicological relevance were noted for number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, male and female foetuses, sex ratio and percent pre- and post-implantation loss. There were no statistically significant differences between the dose groups and controls.
100 % post-implantation loss was observed in one female of the HD group (no. 91) and one female of the control group (no. 6) with implantation sites but without foetuses at caesarian section. As no further dam was affected with 100 % post-implantation loss and as mean post-implantation was comparable between the dose groups and controls with values in the normal range of variation, this was not considered as toxicologically relevant. No dead foetuses were observed in any of the groups.


see Tables under Section Any other relevant information including tables

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Mean foetal weight and litter weight of the dose groups was unaffected by the test item administration. Values were comparable to controls without dose-dependent differences. Statistical analysis showed no significant differences to the control group.

see Tables under Section Any other relevant information including tables
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for number of live foetuses. There were no statistically significant differences between the dose groups and controls.

see Tables under Section Any other relevant information including tables
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for number of male and female foetuses and sex ratio. There were no statistically significant differences between the dose groups and controls.

see Tables under Section Any other relevant information including tables
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for litter size and weight.

see Tables under Section Any other relevant information including tables
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical analysis showed no significant differences for external abnormalities between the dose groups and the control group. However, the external malformation of mandibular micrognathia with absent tongue was observed in one foetus of the MD group and a slightly misshapen snout in one foetus of another litter of the MD group. As these findings were observed in single foetuses in an intermediate dose group without dose-dependency, they were not considered as toxicologically relevant. The external finding of mandibular micrognathia correlated with malformations of skull bones at the time of skeletal examination.
Low incidences of haematoma on various body parts were noted in more foetuses of the control group (in total three foetuses) than in the dose groups (in total one foetus of the MD group) and were considered to be spontaneous in nature without relation to the treatment with the test item. Furthermore, the observation of small size in one foetus of the LD group was also considered to be incidental.

see Tables under Section Any other relevant information including tables
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Skeletal examination of the Alizarin red stained foetuses revealed a range of findings in all groups including control.
There was no general trend towards dose-dependently reduced ossification. As usual for foetuses at this stage of gestation (day 20) incomplete ossification was seen in several bones of several litters in all groups including control. Mostly bones of the skull, sternum, paws and vertebra were affected by slight variations in the status of expected ossification in terms of incomplete ossification, misaligned ossification, irregular ossification, unossification or increased ossification throughout all groups. Values were well within the normal range of variation and historical control data and/or showed no dose-dependent pattern. Litter incidences for ossification status were statistically insignificant between the dose groups and controls except for the finding of increased ossification of the calcaneus (10.00 % in the HD group compared to 45 % in controls) which was considered as variation.
Additionally, humeri of litters (1 HD, 1 MD, each one foetus) were affected with incomplete ossification. Incomplete ossification was also noted for pubis in 2 foetuses of 2 litters of the HD group and 3 foetuses of 2 litters of the MD group (litter incidence 10.00 %). In the HD group only foetuses with particularly low body weight were affected. (below 3.0 g compared to a group mean weight of 3.55 g). Incomplete ossification of humerus was not seen before in historical control data and incomplete ossification of pubis was seen at a slightly less maximum litter incidence of 4.55 %. Though long bones of the limbs and pelvic bones should normally ossify early during gestation, with only few foetuses affected without clear dose-dependency this was not considered as an adverse effect.
Misshapen humerus was noted in total in 6 foetuses of 3 litters of the HD group (litter incidence 15.00 %, foetal incidence 5.22 %) with moderately misshapen (bilateral) in two cases, moderately misshapen (right side) in two cases, slightly misshapen (bilateral) in one case and slightly misshapen (left side) in two cases. Misshapen humerus was also observed in total in 3 foetuses of 3 litters of the MD group (litter incidence 15.00 %, foetal incidence 2.65 %) with moderately misshapen (bilateral) in one case and moderately misshapen (right side) in two cases. Moderately misshapen humerus was seen short and thick which was classified as malformation as this finding was assumed to be permanent and possibly affecting health. Slightly misshapen humerus was only seen thick which could be considered as variation. As the malformation of moderately misshapen humerus was only observed at the higher tested dose levels and slightly beyond historical control data (humerus short litter incidence 8.70 %, foetal incidence 1.52 %) an effect of the treatment with the test item was assumed for the MD and the HD group.
Fusion of zygomatic arch(es) was seen dose-dependently in total in 2 litters (3 foetuses) of the LD, 4 litters (4 foetuses) of the MD and 6 litters (11 foetuses) of the HD group compared to none in the concurrent control group. Litter incidences of 20.00 % and 30.00 % in the MD and the HD group, respectively, were moderately beyond historical control data with a maximum historical litter incidence of 14.29 %. Moderate fusion was noted in 1 litter of the LD group (bilateral in 2 foetuses, right side in 1 foetus), in 1 litter of the MD group (right side in 1 foetus) and in 2 litters of the HD group (left side in 2 foetuses). Furthermore, marked fusion was noted in 2 litters of the HD group (bilateral in 1 foetus, right side in 1 foetus). The remaining affected foetuses were only seen with slight fusion.
One foetus of the MD group was noted with several malformations of skull bones (misshapen mandibles, tympanic rings and pterygoid processes of the basisphenoid) which were seen as mandibular micrognathia at external examination. As only one foetus was affected, this was considered as incidental.
2 foetuses from different litters of the HD group were seen with the finding of split thoracic centrum (5th or 12th). Due to the separation of the precursor for the bone in two parts, this change was considered as permanent and as such as malformation.
Markedly and statistically significantly higher litter incidence of wavy ribs was noted in the HD group (75.00 %) when compared to the concurrent control group (35.00 %). Incidence of the HD group was also marginally above maximum litter incidence of historical control data (72.73 %). Furthermore, a dose-dependent pattern was observed with a litter incidence of 60 % in the LD group and 70 % in the MD group. Wavy ribs are common findings in rodent studies and are classified as variations and are considered to be postnatally reversible.
Bent scapula and/or scapular spine were observed at low incidences in all groups including control. Though incidence of scapula bent was highest in the HD group with 3 foetuses of 3 different litters affected (right side), severity grading was lowest (slight) compared to moderately bent scapula in the control group (left side) and MD group (right side). The finding of bent scapula appears to be transient and completely repaired post-natally. As this finding should be classified as variation and, moreover, was seen without clear consistency throughout the groups, the finding of bent scapula was not considered as an adverse effect of the test item.
Supernumerary (14th) thoracolumbal ribs (bilateral, right side, left side and rudimentary or full) were noted in several foetuses throughout all groups including control without statistically significant difference to controls. Values were within the normal range of historical control data and/or showed no consistent, dose-dependent trend. Supernumerary full ribs (bilateral) in the control group (35 %), full rib (right side) in the MD and the HD group (20 % each) and rudimentary rib (right side) in the HD group (65 %) were noted at marginally higher litter incidences when compared to maximum historical control data values (31.58 %, 18.18 % and 58.33 %, respectively). Without consistency, this was considered as incidental.
Rudimentary cervical ribs were noted in few foetuses throughout all groups including control without statistically significant difference and without a dose-dependent trend. Cervical ribs are skeletal alterations that can occasionally be seen in animals of this strain and age. Rudimentary/short ribs are considered as transient abnormalities. Full/long cervical rib which is considered as permanent was only seen in one foetus of the LD group (left side) which was considered as incidental.
Pelvic girdle caudal shift (bilateral / left side / right side) was noted in few foetuses throughout all groups including control without statistically significant difference to controls and without a clear dose-dependent trend. Changes of the position of the pelvic girdle relative to the number of pre-pelvic vertebrae can occasionally be seen in animals of this strain. Though litter incidences were partly marginally above historical control data (bilateral: concurrent controls and MD group 35.00 % compared to maximum historical data of 30.00 %; right side: HD group 15.00 % compared to maximum historical data of 11.00 %), without dose-dependency, this was considered as incidental.
Fusion of centrum with arch of sacral and caudal vertebrae was noted in some foetuses throughout all groups including control without dose-dependency and was considered as incidental.

see Tables under Section Any other relevant information including tables
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
Visceral Examination
Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including control.
Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls.
Litter incidences were statistically insignificant except for the finding of transposed umbilical artery. Litter incidences were dose-dependently higher in the dose groups (LD 52.38 %, MD 55.00 % and HD 65.22 %) when compared to the control group (15.00 %). Similarly, foetal incidences were also dose-dependently higher in the dose groups (LD 11.02 %, MD 18.18 % and HD 19.35 %) when compared to the control group (2.88 %). However, values were all within historical control data (maximum litter incidence 85.71 %, maximum foetal incidence 25.42 %). Thus, differences to controls for this variation were not considered as an adverse effect of the treatment with the test item.

Thick heart ventricle wall was seen in 5 foetuses from 4 litters of the HD group (litter incidence 17.39 %, foetal incidence 4.03 %). This finding was not seen in concurrent control animals or historical control data and was considered as malformation as it was considered likely to affect adversely survival or health. Furthermore, one single foetus of the LD group was affected. However, without clear dose-dependcy and complete absence of effects in the MD group this single observation in the LD group was considered as incidental.
The isolated finding of dilated aortic arch was observed in one foetus of the MD group and one foetus of the HD group. As this finding might result in functional consequences possibly influencing survival or health, it was considered as malformation.
Absent innominate artery was observed in two foetuses from different litters of the HD group. This finding is considered to reflect only a small change in the arrangement of arteries arising from the aortic arch in terms of a carotic branching variation and should as such be classified as a variation. However, as this finding was neither observed in concurrent controls nor documented in historical control data, it cannot be excluded that this abnormality was induced by exposure to the test item but was not considered as an adverse effect.
Furthermore, the finding of a short innominate artery was observed in one foetus of the LD group but not at higher dose levels. This was within the range of historical control data and was considered as incidental.
Discolouration of the liver (dark / red, complete / separate lobes), spleen (white, complete), urinary bladder (red, spotted) or thymus (red, focus) was seen in few foetuses of the dose groups and/or control group without following a dose-dependent pattern. Discolourations of organs were considered likely to reflect the consequence of a functional disorder and thus not strictly as developmental anomalies. Based on the low incidences without dose-dependency these findings were not considered as toxicologically relevant.
As the remaining observed findings (ureter convoluted, ureter dilated, renal pelvis dilated, abdominal cavity filled with red fluid) were either minor variations and/or due to a lack of dose-dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature.
The reproductive tract observation during fetal examination revealed no test item realted visceral findings in treatment groups andcontrol group.

Craniofacial Examination
Craniofacial examination by razor blade serial sectioning technique revealed few findings (dilated third ventricle, dilated lateral ventricle, retinal fold and small pituitary gland) at low frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls without any dose-dependent trend. Statistical analysis of the data revealed no significant effect. Values were within the normal range of historical control data. Thus, these findings were considered to be spontaneous in nature and not related to the treatment with the test item.
The finding of a subcutaneous haematoma was seen in one foetus of the MD group and three foetuses of the control group. Furthermore, subcutaneous oedema was also observed in one foetus of the MD group and subdural oedema in one foetus of another litter of the MD group. Slightly dilated nasal cavity was noted in one single foetus of the HD group. These findings were considered as incidental based on the low incidence without dose-dependency.

see Tables under Section Any other relevant information including tables

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
visceral malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: see Description
Description (incidence and severity):
Mandibular micrognathia, tongue absent, forelimb humerus(i) misshapen, skull zygomatic arch (B) fused, skull zygomatic arch (L) fused, skull zygomatic arch (R) fused, vertebra thoracic centrum(-tra) split, aortic arch dilated, ventricle wall thick.

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects in the absence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Dose Formulation Analysis

Formulation analysis for concentration verification was performed on collected samples of all groups in the first and the last week of the study.Mean recoveries calculated from all samples in the LD, MD and HD groups were 93.1%, 96.9%, and 92.4% of nominal concentration, respectively.In the first week of the study, recovery in the first sample from the HD group was 82.6% and acceptance criterion of 10 % was not met. This was related to inadvertent mistake at formulation preparation (extended sonication time). Therefore, remaining HD aliquots were discarded and a new formulation prepared and again analysed within study week 1 which met the acceptance criterion (100.8 % recovery). As only two animals of the HD group (female no. 76 and 77) were dosed on two single days (gestation day 5 and 6) with the wrongly prepared formulation showing slightly lower analytical recovery, it was not considered to have relevant impact on the study outcome.

Deviations from the Study Plan

There were the following deviations from the study plan including amendments:

Concerning:

Preparation of the Test Item Formulation

Before:

The test item formulation will be prepared by weighing in the test item into a tared plastic vial on a suitable precision balance, adding the vehicle corn oil to give the appropriate final concentration of the test item formulation and further stirring it for 2-3 minutes. The solution will be sonicated for 5-10 minutes.

New:

The test item formulation was prepared by weighing in the test item into a tared plastic vial on a suitable precision balance, adding the vehicle corn oil to give the appropriate final concentration of the test item formulation and further stirring it for 2-3 minutes. The solution was sonicated for 5-10 minutes. However, the first high dose formulation that was prepared for dosing in this study was inadvertently sonicated for 30 minutes. 2 HD animals (no. 76 and no. 77) were dosed with this wrongly prepared formulation on 2 single days (gestation day 5 and 6) after which the remaining aliquots were discarded.

Reason:

Inadvertent mistake at formulation preparation

Concerning:

Dose Formulation Analysis

Before:

As the test item formulations were shown to be homogenous after 60 min without stirring, during the study samples will not be collected for the investigation of homogeneity but for determination of test item concentration in the first and the last week of the study from all groups (8 samples in total).

New:

As the test item formulations were shown to be homogenous after 60 min without stirring, during the study samples were not collected for the investigation of homogeneity but for determination of test item concentration in the first and the last week of the study from all groups (8 samples in total).

In study week 1, measured concentration of the prepared high dose formulation did not meet the acceptance criterion of 10 % with a recovery of 82.6 % due to wrong preparation of formulation. Therefore, after 2 days of dosing of two HD animals the remaining high dose aliquots were discarded and a new formulation prepared and analysed again for study week 1.

Reason:

Inadvertent mistake at formulation preparation

Concerning:

Preparation of the Test Item Formulation

Before:

On gestation day 20, sperm positive (presumed pregnant females) will be subjected to a caesarean section after sacrificing the animals using an overdose of sodium pentobarbital.

New:

On gestation day 20, sperm positive (presumed pregnant females) were subjected to a caesarean section after sacrificing the animals using an overdose of pentobarbital injected intraperitoneallyat a dosage of approximately 8 mL/kg bw.

Two females (animal no. 4 of the control group and no. 29 of the LD group) were sacrificed on gestation day 21. Individual values of the dams and foetuses were excluded from calculation of mean as appropriate.

Reason:

Inadvertent mistake in the lab

These deviations did not influence the quality or integrity of the present study.


 


 

Applicant's summary and conclusion

Conclusions:
On the basis of this prenatal developmental toxicity study in pregnant female Wistar rats with the test item at dose levels of 100, 300 and 1000 mg/kg body weight/day, administered on gestation days 5 to 19, the following conclusions can be made:
No clinical signs of systemic toxicity or mortality were observed in this study. However, at a dose level of 1000 mg/kg body weight/day moderately lower body weight gain and food intake were observed resulting in a moderately reduced terminal body weight. Neither uterus, foetus and litter weight nor prenatal and litter data were affected.
A tendency towards dose-dependently higher incidences of visceral and skeletal foetal abnormalities (variations as well as malformations) was noted:
The rare malformation of cardiac hypertrophy occurred in five foetuses (four litters) at 1000 mg/kg body weight/day and dilatation of aortic arch in one foetus at 300 mg/kg body weight/day and in one foetus at 1000 mg/kg body weight/day. Both findings were only observed in male foetuses. As these findings were neither seen in concurrent control animals nor in historical control data, they were assumed to be test item-related. A further rare abnormality affecting the greater vessels was the finding of absent innominate artery in two foetues at 1000 mg/kg body weight/day. Though this is regarded only as a slight carotic branching variation, it might also have been induced by exposure to the test item.
At skeletal examination, the malformation of moderately misshapen (short and thick) humerus was seen in three foetuses (three litters) at 300 mg/kg body weight/day and in four foetuses (two litters) at 1000 mg/kg body weight/day. Furthermore, the malformation of fused zygomatic arch(es) was noted at increased severity and incidence at the highest dose level above historical control data which was considered as an effect of the treatment with the test item. Two foetuses of different litters at 1000 mg/kg body weight/day were seen with split single thoracic centrum. As this finding was marginally beyond historical control data, a relation to the treatment with the test item cannot be excluded. Next to these malformations, there were markedly and dose-dependently higher incidences of wavy ribs which were marginally beyond historical data at 1000 mg/kg body weight/day. Though wavy ribs are common variations which are considered to be postnatally reversible, they are reported to occur more often in the presence of maternal and/or foetal toxicity, secondary to generalized toxicity or stress.
Thus, the NOAEL for maternal toxicity in this study is considered to be 300 mg/kg body weight/day and for foetal toxicity 100 mg/kg body weight/day.

Executive summary:

The aim of this study was to assesspossible adverse effects on pregnant females and embryo-foetal developmentwhich could arise from repeated exposure of the test item via oral administration (gavage) to female rats during gestation days 5 to 19. Nulliparous and non-pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on the day of sperm positive vaginal smears (GD 0).The 4 groups comprised each 25 femaleWistar rats in the control (C) group, the low dose (LD) group, the medium dose (MD) group and the high dose (HD) group.

The following doses were evaluated:

Control:                        0         mg/kg body weight/day

Low Dose:                    100     mg/kg body weight/day

Medium Dose:              300     mg/kg body weight/day

High Dose:                   1000   mg/kg body weight/day

The test item formulation was prepared with the vehicle corn oil. The solution was sonicated for 5-10 minutes. Based on the results of stability testing (Eurofins Munich study no. 169064), the test item formulations were prepared at least once every 10 days and the prepared formulations were stored at room temperature.Formulates were kept under magnetic stirring during the daily administration.Dose volumes were adjusted individually based on the body weight most recently measured. Animals of the control group were handled identically as the dose groups, but received corn oil, the vehicle used in this study.

Sperm positive females were weighed on gestation days 0, 5, 8, 11, 14, 17 and 20.

Food consumption of sperm positive females was measured on gestation days 5, 8, 11, 14, 17 and 20.

During the period of administration, the animals were observed precisely each day for signs of toxicity and mortality. Females were sacrificed on the respective gestation day 20 except for one female of the control and one of the LD group which were sacrificed on gestation day 21. Following the gross necropsy, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late), live and dead foetuses. The uteri of the non-pregnant females were processed with 10 % ammonium sulphide solution and checked for early embryonic deaths.

Foetuses were identified by using strings with numbered plates, sexed and weighed. One half of each litter was examined for soft tissue anomalies by a microdissection technique. The remaining foetuses were processed by Alizarin red staining and the first 20 litters per group after caesarian section on gestation day 20 were examined for skeletal alterations. Craniofacial examination of the heads of the foetuses used for the soft tissue examination of 20 litters per group was performed for evaluation of the internal structure including the eyes, brain, nasal passage and tongue by razor blade serial sectioning technique.

Summary Results

Maternal Findings

No mortality or toxicologically relevant signs of morbidity occurred in any of the groups during the treatment period of this study.

Suspected abortion in one single female of the HD group was assumed to be incidental.

Moving the bedding was noted dose-dependently in single females of the MD and the HD group on few days of treatment. As this was observed directly after dose administration, it was considered as slight clinical sign elicited by local effects of the test item formulation and/or attributed to discomfort of the animals due to oral administration, but not systemic toxicity.

There was a moderate effect on body weight development for the HD group after initiation of treatment. Statistical significance on body weight gain was achieved on gestation days 5-8 (10 % of controls), 8-11 (40 % of controls) and 11-14 (50 % of controls). Overall, body weight gain of the HD group was 70 % of controls.Differences in body weight gain compared to controls resulted in slightly to moderately and statistically significantly reduced mean body weight of the HD group from gestation day 8 (96 % of controls) onwards with the greatest effect at the end of the study on gestation day 20 (90 % of controls). The LD and MD groups were not affected.

In correlation to the lower body weight gain after initiation of treatment, mean food consumption was moderately and statistically significantly lower when compared to controls throughout the whole treatment period. Overall, mean food consumption of the HD group was 90 % of controls.

This moderate effect on body weight development and food consumption of the HD group was considered as an adverse effect of the treatment with the test item. No toxicologically relevant effects were observed for the LD and the MD group.

There were no gross pathological findings at necropsy of adult females of any of the groups.

In correlation to the moderately lower mean terminal body weight of the HD group (90 % of controls), also moderately and statistically significantly lower mean adjusted maternal weight (maternal weight minus gravid uterus weight) was observed in the HD when compared to controls (87 % of controls) with no effect on mean uterus weight.

Marginal and statistically significant but not dose-dependent differences for mean uterus weight and adjusted maternal weight in the LD and the MD group compared to controls were not considered as biologically relevant and were assumed to be incidental.

Successful mating resulted in 22/25 pregnancies in the LD group, 21/25 in the MD group and 24/25 in the HD group when compared to 22/25 pregnancies in the control group.

No test item-related effects of toxicological relevance were noted for number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, male and female foetuses, sex ratio and percent pre- and post-implantation loss. No dead foetuses were observed in any of the groups.

Foetal Findings

No test item-related effects were noted for foetal weight and litter weight data.

There were no statistically significant foetal external and craniofacial abnormalities in any of the dose groups.

Overall a slight tendency towards accumulation of foetal visceral and skeletal abnormalities (variations as well as malformations) was observed . Because these were found dose-dependently and predominantly in the HD group they were assumed to be test item related.

Litter incidences for visceral findings were statistically significant for the finding of transposed umbilical artery.These were dose-dependently increased (LD 52.38 %, MD 55.00 % and HD 65.22 %) when compared to the control group (15.00 %). Foetal incidences were also dose-dependently increased up to 19.35 % in the HD group. The finding of a transposed (left-sided) umbilical artery is reported as one of the most common visceral variations in rat foetuses. Incidences are generally high in Wistar rats, especially in the strain used in this study (Crl:WI(Han)). Though values in the dose groups of this study were markedly higher compared to concurrent controls, they were all within historical control data (maximum litter incidence 85.71 %, maximum foetal incidence 25.42 %). Thus, differences to controls for this variation were not considered as an adverse effect of the treatment with the test item.

Rare abnormalities of the heart or greater vessels were seen in few foetuses of this study affecting mostly the HD group.

Thick heart ventricle wall (hypertrophy) was seen in 5 foetuses from 4 litters of the HD group (litter incidence 17.39 %, foetal incidence 4.03 %). This finding was not seen in concurrent control animals or historical control data and was considered as malformation as it was considered likely to affect adversely survival or health. Furthermore, one single foetus of the LD group was affected. However, without clear dose-dependcy and complete absence of effects in the MD group this single observation in the LD group was considered as incidental.

The isolated finding of dilated aortic arch was observed in one foetus of the MD group and one foetus of the HD group. As this finding might result in functional consequences possibly influencing survival or health, it was considered as malformation. None of these foetuses were affected with cardiac hypertrophy, however, the foetus with dilated aortic arch of the HD group was also noted with a variation of carotic branching. As dilation of the aortic arch was not documented before in historical control data, a relationship to the treatment with the test item cannot be fully excluded though only two foetuses were affected without clear dose-dependency.

Remarkably, the findings of cardiac hypertrophy and dilated aortic arch were only observed in male foetuses.

Absent innominate artery was observed in two foetuses from different litters of the HD group. This finding is considered to reflect only a small change in the arrangement of arteries arising from the aortic arch in terms of a carotic branching variation and should as such be classified as a variation. However, as this finding was neither observed in concurrent controls nor documented in historical control data, it cannot be excluded that this abnormality was induced by exposure to the test item but was not considered as an adverse effect.

The reproductive tract observation during fetal examination revealed no test item realted visceral findings in treatment groups andcontrol group.

Skeletal examination of the Alizarin red stained foetuses revealed statistically significant litter incidences for the finding of increased ossification of the calcaneus (10.00 % in the HD group compared to 45 % in controls). This is no deviation from the normal ossification sequence as the calcaneus is known to begin to ossify only during late gestation. As such variation in ossification status for this bone can be high. With values being within historical control data, this single statistical significant change in ossification was not considered as an adverse effect of the treatment with the test item. There was no general trend towards dose-dependently reduced ossification.


 

Additionally, humeri of litters (1 HD, 1 MD, each one foetus) were affected with incomplete ossification. Incomplete ossification was also noted for pubis in 2 foetuses of 2 litters of the HD group and 3 foetuses of 2 litters of the MD group (litter incidence 10.00 %). In the HD group only foetuses with particularly low body weight were affected. Incomplete ossification of humerus was not seen before in historical control data and incomplete ossification of pubis was seen at a slightly less maximum litter incidence of 4.55 %. Though long bones of the limbs and pelvic bones should normally ossify early during gestation, with only few foetuses affected without clear dose-dependency this was not considered as an adverse effect.

Misshapen humerus was noted in total in 6 foetuses of 3 litters of the HD group (litter incidence 15.00 %, foetal incidence 5.22 %) with moderately misshapen (bilateral) in two cases, moderately misshapen (right side) in two cases, slightly misshapen (bilateral) in one case and slightly misshapen (left side) in two cases. Misshapen humerus was also observed in total in 3 foetuses of 3 litters of the MD group (litter incidence 15.00 %, foetal incidence 2.65 %) with moderately misshapen (bilateral) in one case and moderately misshapen (right side) in two cases. Moderately misshapen humerus was seen short and thick which was classified as malformation as this finding was assumed to be permanent and possibly affecting health. Slightly misshapen humerus was only seen thick which could be considered as variation. As the malformation of moderately misshapen humerus was only observed at the higher tested dose levels and slightly beyond historical control data (humerus short litter incidence 8.70 %, foetal incidence 1.52 %) an effect of the treatment with the test item was assumed for the MD and the HD group.

Fusion of zygomatic arch(es) was seen dose-dependently in few foetuses in the dose groups (in total 3 foetuses of 2 litters of the LD group, 4 foetuses of 4 litters of the MD group and 11 foetuses of 6 litters of the HD group) compared to none in the concurrent control group. Litter incidences of 20.00 % and 30.00 % in the MD and the HD group, respectively, were moderately beyond historical control data with a maximum historical litter incidence of 14.29 %. Post-natal consequences of fusion of skull bones at this time-point of development are unknown. However, based on severity grading, especially moderate and marked fusion of zygomatic arch(es) are suspected to possibly affect health and were considered as malformation. Slight fusion was considered as variation. Moderate fusion was noted in 1 litter of the LD group (bilateral in 2 foetuses, right side in 1 foetus), in 1 litter of the MD group (right side in 1 foetus) and in 2 litters of the HD group (left side in 2 foetuses). Furthermore, marked fusion was noted in 2 litters of the HD group (bilateral in 1 foetus, right side in 1 foetus). Without clear consistency in terms of incidence and severity in the LD and MD group, findings of fusion of zygomatic arch(es) in these groups were not considered as toxicologically relevant whereas it was considered as an adverse effect of the treatment with the test item in the HD group based on the increased severity and incidence distinctly above historical control data.

2 foetuses from different litters of the HD group were seen with the finding of split single thoracic centrum. Due to the separation of the precursor for the bone in two parts, this change was considered as permanent and as such as malformation. As this finding was only seen at the highest dose and as litter incidence (10.00 %) and foetal incidence (1.74 %) was marginally beyond historical data (maximum litter incidence 8.70 %, maximum foetal incidence 1.55 %), a relation to the treatment with the test item cannot be excluded.

Dose-dependently higher litter incidence of wavy ribs was noted in the LD (60.00 %), the MD (70.00 %) and the HD group (75.00 %) when compared to the concurrent control group (35.00 %). Litter incidence of the HD group was marginally beyond the maximum historical litter incidence of 72.23 %.Wavy ribs are common findings in rodent studies and are considered to be postnatally reversible. However, they are reported to occur more often in the presence of maternal and/or foetal toxicity, secondary to generalized toxicity or stress.

Thus, significantly higher incidence of wavy ribs in the HD group of this study might be related to maternal toxicity with the observed reduced body weight and food consumption of dams of the HD group. Wavy ribs are classified as variations and as such were not considered as an adverse effect of the treatment with the test item.

Dose Formulation Analysis

Formulation analysis for concentration verification was performed on collected samples in the first and last week of the study. Mean recoveriescalculated from all samples in the LD, MD and HD groups were 93.1%, 96.9%, and 92.4% of nominal concentration, respectively.

Conclusion

On the basis of this prenatal developmentaltoxicity study in pregnant female Wistar rats with the test item at dose levels of 100, 300 and 1000 mg/kg body weight/day, administered on gestation days 5 to 19, the following conclusions can be made:

No clinical signs of systemic toxicity or mortality were observed in this study. However, at a dose level of 1000 mg/kg body weight/day moderately lower body weight gain and food intake were observed resulting in a moderately reduced terminal body weight. Neither uterus, foetus and litter weight nor prenatal and litter data were affected.

A tendency towards dose-dependently higher incidences of visceral and skeletal foetal abnormalities (variations as well as malformations) was noted:

The rare malformation of cardiac hypertrophy occurred in five foetuses (four litters) at 1000 mg/kg body weight/day and dilatation of aortic arch in one foetus at 300 mg/kg body weight/day and in one foetus at 1000 mg/kg body weight/day. Both findings were only observed in male foetuses. As these findings were neither seen in concurrent control animals nor in historical control data, they were assumed to be test item-related. A further rare abnormality affecting the greater vessels was the finding of absent innominate artery in two foetues at 1000 mg/kg body weight/day. Though this is regarded only as a slight carotic branching variation, it might also have been induced by exposure to the test item.

At skeletal examination, the malformation of moderately misshapen (short and thick) humerus was seen in three foetuses (three litters) at 300 mg/kg body weight/day and in four foetuses (two litters) at 1000 mg/kg body weight/day. Furthermore, the malformation of fused zygomatic arch(es) was noted at increased severity and incidence at the highest dose level above historical control data which was considered as an effect of the treatment with the test item. Two foetuses of different litters at 1000 mg/kg body weight/day were seen with split single thoracic centrum. As this finding was marginally beyond historical control data, a relation to the treatment with the test item cannot be excluded. Next to these malformations, there were markedly and dose-dependently higher incidences of wavy ribs which were marginally beyond historical data at 1000 mg/kg body weight/day. Though wavy ribs are common variations which are considered to be postnatally reversible, they are reported to occur more often in the presence of maternal and/or foetal toxicity, secondary to generalized toxicity or stress.

Thus, the NOAEL for maternal toxicity in this study is considered to be 300 mg/kg body weight/day and for foetal toxicity 100 mg/kg body weight/day.