Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system 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 females at the arrival at BSL: approx. 11-12 weeks old
- Age of the males at the start of pairing: approx. 11-12 weeks old
- Body weight at the allocation of the animals to the experimental groups:
males: 302 - 356 g (mean: 328.44 g, ± 20% = 262.75 – 394.13 g)
females: 187 - 236 g (mean: 207.71 g, ± 20% = 166.17 – 249.25 g)
- The animals were derived from a controlled full-barrier maintained breeding system (SPF).

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 (lot no. 1526)
- 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 (except during the pre-mating period when females were kept in groups of two animals and
mating period when two females were paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 290114)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Aqua ad injectionem
Details on exposure:
PREPARATION OF THE TEST ITEM FORMULATION

The test item was weighed into a tarred plastic vial on a precision balance.
The test item was dissolved in Aqua ad injectionem.
The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline.
The test item formulations were prepared freshly on each administration day before the administration procedure.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.
The vehicle was also used as control item.

CHARACTERISATION OF THE VEHICLE
- Name: Aqua ad injectionem
- Manufacturer: AlleMan Pharma
- Batch No.: 311651
- Physical State: liquid
- Storage Conditions: at room temperature
- Expiry Date: 10/2016
- 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 5 mL/kg bw/day.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Concentration in vehicle: 0, 20, 60, 200 mg/ml
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The assessment of homogeneity as well as a determination of the measured concentration of the test item in the vehicle was performed at
various intervals. Samples for analysis of the dose formulations of the test item in the vehicle (concentration) were taken in the first and last week of
the study for all doses. Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. Samples
were taken in the first and last week of the study. Samples for stability analysis were taken before in life initiation 0 hours after the preparation,
6 hours after the preparation (at room temperature) and another sample 10 days after the preparation (at room temperature) from high and low dose formulations. Each sample was retained twice (sample A, sample B, each of at least 50 mL). All formulation samples were stored at -15 to -35°C and
the A samples were shipped on dry ice after completion of the in-life phase of the toxicity study to:
CIP
Chemisches Institut Pforzheim GmbH
Schulberg 17
75175 Pforzheim
Germany
The determination of the test item concentration in the dosing formulations was performed by CIP GmbH, in accordance with GLP.
The procedures followed were described in a detailed analytical phase plan (phase study no. 14B07113-01RARW) that was attached to the study plan per amendment. The results are reported in an analytical phase report which is attached to this report.
The B samples were retained at BSL until the completion of the final study report and will be discarded thereafter.
Details on mating procedure:
PREPARATION OF THE ANIMALS
After the acclimatisation period of at least 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.
Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to regularise the number of
animals for terminal sacrifice on a particular day. At the subsequent mornings, the vaginal smear of each 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 the mean body weights were comparable to 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 test item was orally administered daily in graduated doses to several groups of pregnant females from the gestation day (GD) 5 to gestation day
(GD) 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.
No. of animals per sex per dose:
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 25 female Wistar rats in each the control, the LD group, the MD group and the HD
group, respectively. 156 animals (52 males and 104 females) were included in the study.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 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.
- 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:
CAGE SIDE OBSERVATIONS: Yes
- 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.


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the
treatment. 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 on the day of their arrival.


FOOD CONSUMPTION: Yes
- Food consumption of pregnant females was measured on gestations days 5, 8, 11, 14, 17 and 20.
- Food consumption was measured neither for males during the entire study nor for both males and females during the mating period.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes
- On gestation day 20, sperm positive females no. 1-11 (control group), no. 26-36 (LD group), no. 51-62 (MD group) and 76-87 (HD group) were subjected to a caesarean section after sacrificing the animals using an overdose of pentobarbital injected intraperitoneally (Narcoren®, Merial; lot no.: 236014; expiry date: 30.01.2017) at a dosage of approximately 8 mL/kg bw. The remaining sperm positive females were subjected to a caesarean
section under ketamine/xylazine-anaesthesia (ketamine/xylazine, 2:1, Pharmanovo, lot no: 24863, expiry date: 10/2015 and Serumwerk,
lot no: 01213, expiry date: 10/2015 and Serumwerk, lot no: 00513, expiry date: 05/2015). After removing the uterus, blood from the abdominal aorta was collected in serum separator tubes and the females were exsanguinated. After completion of the experimental phase, serum samples were sent
to the sponsor for analysis. Results of the analysis will be reported separately by the sponsor and will not be part of this study.
- At the time of termination, the dam (presumably pregnant female) was examined macroscopically for any structural abnormalities or pathological
changes which may have influenced the pregnancy.
- After removing the uterus, 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 any time after the mating.
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 strings with 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 processed by Alizarin red staining and examined for skeletal alterations. The remaining litter was examined for soft tissue anomalies by a microdissection technique.
Craniofacial examination of the heads of the foetuses used for the soft tissue examination was 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 centers, ribs and sterna centers 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 and xyphisternum (6th sternebra) 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 were 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, a single foetus was decapitated and the head of the fetus 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, prenatal parameters and litter weight 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 a Fisher’s exact test. Litter incidence was the primary unit for the statistical analysis and
interpretation. The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
MORTALITIY
No mortality occurred in the control or any of the dose groups during the treatment period of this study.

CLINICAL OBSERVATIONS
There were no clinical signs of toxicological relevance in the dose groups when compared to the control group.
Low incidences of slight clinical signs like alopecia on various body parts, chromodacryorrhea and moving the bedding were noted in isolated females of the dose groups and/or the control group. As these findings were seen irrespective of the groups in isolated animals, they are considered to be incidental.
None of the females showed signs of abortion prior to the scheduled sacrifice.
After littering on study day 15 (8 pups), female no. 59 of the MD group was discarded without further observations, as apparently this animal had
already been pregnant before the first detection of sperm in the vaginal smear.

BODY WEIGHT DEVELOPMENT
The mean body weight increased with the progress of the study in the control, the LD, the MD and the HD group.
There were no test item-related effects of toxicological relevance noted for body weight and body weight gain in the females. Throughout the
treatment period, body weights were within the normal range of variation for this strain.

FOOD CONSUMPTION
In correlation to the body weight and body weight change, the food consumption increased with the progress of the study in the control, the LD, the MD and the HD group.
There were no test item-related effects of toxicological relevance on food consumption during the treatment period.

PRENATAL DATA
Prenatal parameters like group mean terminal body weight, gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantation
sites, number of live and dead foetuses, number of late resorptions, number of male and female foetuses, sex ratio and postimplantation loss
remained unaffected in the dose groups when compared to the control group. However, the preimplantation loss was marginally lower in the HD
group (10%) and the LD group (9%) when compared to the control group (16%) and the MD group (16%). Due to lack of dose dependency and without achieving statistical significance, this is considered to be incidental and not related to the test item.
Successful mating resulted in 23/25 pregnancies in the LD group, 19/25 in the MD group and 23/25 in the HD group, compared to 24/25
pregnancies in the control group. In the MD group a moderately lower pregnancy rate (number of pregnancies / number of females mated or
sperm positive x 100) was observed compared to the control group (96% in control, 92% in the LD, 76% in the MD and 92% in the HD group).
This is considered to be incidental in nature.

PATHOLOGY
No gross pathological changes were observed during the macroscopic examination of the females of any group.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Remarks on result:
other: 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:
Remarks:
No effects of the test item on females and foetuses were found at dose levels of 1000 mg/kg body weight/day. Under the condition of the study, 1000 mg/kg body weight/day is considered as no observed adverse effect level (NOAEL) for both maternal and embryo-fetal toxicity of the test item.

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
LITTER DATA

There were no test item-related effects of toxicological relevance noted for the total number of foetuses, number of male and female foetuses,
mean foetus weight, total litter weight and male and female litter weight. Slight differences in the parameters were within the normal range of
variation for this strain.

FOETAL EVALUATION
External examination
There were no external abnormalities considered to be of toxicological relevance noted in any of the dose groups. The statistical analysis showed no statistically significant changes.
One foetus of the HD group was observed with a red snout. As this finding was noted only in one single foetus, it is considered to be incidental in
nature and unrelated to the treatment with the test item.
Visceral examination
Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including
control.
Extra tissue at the median hepatic lobe was noted with a statistically significantly lower litter incidence in the LD group compared to the control
group and supernumerary liver lobe was seen at a statistically higher frequency in the MD group compared to the control group.
Without dose-dependency, these findings are considered to be incidental.
The remaining 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 the controls and were statistically insignificant. As observed findings are either minor variations and/or due to a lack of dose dependency and consistency, no serious toxicological significance can be attributed to these findings and they are considered to be
spontaneous in nature.
Craniofacial examination
Craniofacial examination by a razor blade serial sectioning technique revealed few abnormalities in all groups including controls.
Statistical analysis of the data revealed no significant effect in any of the findings.
Retinal folds and slightly dilated third and lateral ventricles were observed at low frequencies comparable to the control group and are considered spontaneous in nature.
Anophthalmia was noted in one foetus of the HD group. Without achieving statistical significance and with just one single foetus being affected,
this finding is considered incidental and not related to the treatment with the test item.
Skeletal examination
Skeletal examination of the Alizarin red stained foetuses revealed a range of findings which occurred at an incidence comparable to or lower in the dose groups when compared to the control group.
A statistically significantly higher litter incidence of an extra ossification site at the 4th left sacral vertebral arch in the LD group compared to the
control group is considered to be incidental as there was no dose-dependent pattern.
There was no indication of a test item-related trend in the type and incidences of the skeletal findings and they were therefore considered to be
spontaneous in nature.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: fetotoxicity
Remarks on result:
other: Under the condition of the study, 1000 mg/kg body weight/day is considered as no observed adverse effect level (NOAEL) for both maternal and embryo-fetal toxicity of the test item.
Remarks:
No effects of the test item on females and foetuses were found at dose levels of 1000 mg/kg body weight/day.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

DOSE FORMULATION ANALYSIS

Formulation analysis was performed on samples of all dose groups collected at various intervals during the study.

Concentration verification of samples of all dose groups was investigated in the first and the last week of the study. The recoveries were 99.8% and 101.5% in the LD group, 97.3% and 99.9% in the MD group and 97.2 and 98.7% in the HD group, respectively.

Stability of formulation samples was investigated for concentration of the LD and the HD group before in life initiation. After 6 hours storage at room temperature, recoveries were 96.3% for the HD group and 98.4% for the LD group. After 10 days storage at room temperature, recoveries were 95.1% for the HD group and 99.0% for the LD group.

Homogeneity of formulation samples was determined in the first and the last week of the study for the LD and the HD group. Recoveries for the different sampling locations (top, middle, bottom) were between 98.8% and 101.8% in the LD group and between 95.7% and 98.2% in the HD group. DEVIATIONS FROM THE STUDY PLAN There were the following deviations from the study plan:

Before:

A statistical assessment of the results of the body weight, food consumption will be 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 will be analysed using a Chi-square test.The statistics will be performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

New:

A statistical assessment of the results of the body weight, food consumption, prenatal parameters and litter weight 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 a Fisher’s exact test. Litter incidence was the primary unit for the statistical analysis and interpretation.The statistics were performed with GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Reason:

It was decided to use Fisher’s Exact test instead of Chi-square test in order to calculate an exact p-value for more meaningful evaluation.


Concerning:

Foetal Evaluations,study plan, p. 15

Before:

All foetuses from a particular dam will be identified by using different colour strings and will be weighed and sexed based on the anogenital distance. Each foetus will be examined for external anomalies.

New:

All foetuses from a particular dam were identified by using strings with numbered plates and were weighed and sexed based on the anogenital distance. Each foetus was examined for external anomalies.

Reason:

New foetal identification method with actual number written on plate has been introduced for quick identification of the foetuses.

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 effects of the test item on females and foetuses were found at dose levels of 1000 mg/kg body weight/day. Under the condition of the study,
1000 mg/kg body weight/day is considered as no observed adverse effect level (NOAEL) for both maternal and embryo-fetal toxicity of the test item.
Executive summary:
This study was performed in order to be compliant with Chinese regulations for notification. The aim of this study was to assess possible adverse effects on pregnant females and embryo-foetal development which could arise from repeated exposure of the test item 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 25 female Wistar rats in each the control, the LD group, the MD group and the HD group, respectively.

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 freshly on each day of administration. The test item was dissolved in Aqua ad injectionem and administered daily during gestation days 5 to 19 to the female animals. Dose volumes were adjusted individually based on the most recently measured body weight.

Animals of the control group were handled identically as the dose groups, but received Aqua ad injectionem, the vehicle used in this study.

During the period of administration, the animals were observed precisely each day for signs of toxicity and mortality. All female animals were sacrificed on the respective gestation day 20. 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 the early embryonic deaths.

Foetuses were identified by strings with numbered plates, sexed and weighed. All foetuses were observed for external abnormalities, half of the foetuses for visceral and craniofacial abnormalities and the remaining half of the litter was observed for skeletal abnormalities.

Body weight and food consumption were measured on gestation days 0, 5, 8, 11, 14, 17 and 20.

Summary Results

Maternal Findings

No mortality occurred in the control group or any of the dose groups during the treatment period of this study.

There were no clinical signs of toxicological relevance in the dose groups when compared to the control group. None of the females showed signs of abortion prior to the scheduled sacrifice.

The mean body weight increased with the progress of the study in the control, the low dose (LD), the medium dose (MD) and the high dose (HD) group. There were no test item-related effects of toxicological relevance noted for body weight and body weight gain in the females.

In correlation to the body weight and body weight change, the food consumption increased with the progress of the study in the control, the LD, the MD and the HD group. There were no test item-related effects of toxicological relevance on food consumption during the treatment period.

Prenatal parameters like mean gravid uterus weight, adjusted maternal weight, number of corpora lutea, implantation sites, number of live and dead foetuses, number of late resorptions, number of male and female foetuses, sex ratio and post- and pre-implantation loss remained unaffected in the dose groups when compared to the control group.

No gross pathological changes were observed during the macroscopic examination of the females of any group.

Foetal Findings

There were no test item-related effects of toxicological relevance noted for the total number of foetuses, number of male and female foetuses, mean foetus weight, total litter weight and male and female litter weight.

There were no external abnormalities considered to be of toxicological relevance noted in any of the dose groups. The statistical analysis showed no statistically significant changes.

Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including control. As observed findings are either minor variations and/or due to lack of dose dependency and consistency, no serious toxicological significance can be attributed to these findings and they are considered to be spontaneous in nature.

Craniofacial examination by a razor blade serial sectioning technique revealed few findings in all groups including control. Statistical analysis of the data revealed no significant effect in any of these findings.

Skeletal examination of the Alizarin red stained foetuses revealed a range of findings which occurred at an incidence comparable to

or lower in dose groups when compared to the control group.There was no indication of a test item-related trend in the type and incidences of the skeletal findings and they were therefore considered to be spontaneous in nature.

Dose Formulation Analysis

Concentration verification of samples of all dose groups was investigated in the first and the last week of the study. The recoveries were 99.8% and 101.5% in the LD group, 97.3% and 99.9% in the MD group and 97.2 and 98.7% in the HD group, respectively.

Stability of formulation samples was investigated for concentration of the LD and the HD group before in life initiation. After 6 hours storage at room temperature, recoveries were 96.3% for the HD group and 98.4% for the LD group. After 10 days storage at room temperature, recoveries were 95.1% for the HD group and 99.0% for the LD group.

Homogeneity of formulation samples was determined in the first and the last week of the study for the LD and the HD group. Recoveries for the different sampling locations (top, middle and bottom) were between 98.8% and 101.8% in the LD group and between 95.7% and 98.2% in the HD group.

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 effects of the test item on females and foetuses were found at dose levels of 1000 mg/kg body weight/day. Under the condition of the study, 1000 mg/kg body weight/day is considered as no observed adverse effect level (NOAEL) for both maternal and embryo-fetal toxicity of the test item.