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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2004
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Guideline on Detection of Toxicity to Reproduction for Medicinal Products, (ICH Harmonised Tripartite Guideline) endorsed by the ICH Steering Committee at Step 4 of the ICH Process, 24 June 1993
Deviations:
yes
Remarks:
, but the observed deviations were not considered to have compromised the validity or integrity of the study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: brownish gelatinous mixture
Details on test material:
Batch number : CD05-054/05/CD06-287
Expiry date : not specified
Storage conditions : at +4°C and under nitrogen atmosphere

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
- Test animals are Specific Pathogen Free (S.P.F.).
- Breeder: Charles River Laboratories France (Elevage Scientifique des Dombes, Châtillon-sur-Chalaronne, France).
- Age/Weight: at the beginning of the treatment period, the females were approximately 18-20 weeks old and had a mean body weight of 3418 g (range from 2965 g to 4480 g). The females were sexually mature and primigravid.
- Mating: females were mated at the breeder's facilities. The day of confirmed mating (visual assessment) was designated as day 0 post-coitum.
- Acclimation: animals were acclimated for a period of 5 days before the beginning of the treatment period (arrival of females on day 1 post-coitum).
- The animal room conditions are set as follows:
. temperature : 18 ± 3°C
. relative humidity : 50 ± 20%
. light/dark cycle : 8h dark/16h light (5:00 - 21:00)
. ventilation : about 8 to 10 cycles/hour of filtered, non-recycled air.
The animals were housed individually in stainless steel cages (64.0 cm x 48.0 cm x 41.0 cm).
The animals had free access to food and water.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.4% w/w xanthan gum aqueous solution in purified water
Details on exposure:
Each animal was given the dosage forms once a day (50, 150, 500 mg/kg b.w./day or vehicle), at approximately the same time, from day 6 to day 18 post-coitum inclusive. Day 1 corresponds to the first day of treatment period.
The dosage forms were administered by gavage using a plastic syringe fitted with a plastic gavage tube.
The quantity of the dosage form administered to each animal was adjusted according to the most recently recorded body weight.
A constant dosage-volume of 3 mL/kg/day was used. Control animals (group 1) received the vehicle alone.
The dosage forms were stirred continuously throughout the dosing procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
An aliquot of each dosage form was diluted and analyzed by High Performance Liquid Chromatography with Ultra-Violet detection at 302 nm. The concentration of the test item was determined from a calibration curve of peak area against concentration of PIAS in standard solutions (external standard calibration).
Details on mating procedure:
Females were mated at the breeder's facilities. The day of confirmed mating (visual assessment) was designated as day 0 post-coitum. Mating of females took place between 10 September and 24 September 2003.
Duration of treatment / exposure:
Each animal was given the dosage forms once a day, at approximately the same time, from day 6 to day 18 post-coitum inclusive.
Day 1 corresponds to the first day of treatment period.
Frequency of treatment:
Once a day.
Duration of test:
From day 6 to day 18 post-coitum inclusive.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg b.w./day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg b.w./day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg b.w./day
Basis:
actual ingested
No. of animals per sex per dose:
20 mated females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
On day 18 post-coitum, blood was sampled one hour after dosing from three females of each group. Approximately 2 mL blood was taken into a tube containing lithium heparinate, from a peripheral vein of the animals (without anesthesia). The blood samples were centrifuged and the plasma kept frozen at -20°C at CIT for a period of at least 6 months after finalization of the final report.

Examinations

Maternal examinations:
Morbidity and mortality:
Each animal was checked for mortality or signs of morbidity: at least twice a day during treatment period and at least once a day on other days.
Animals found dead or killed prematurely were subjected to a macroscopic post-mortem examination.

Clinical signs:
From arrival, the animals were observed at least once a day as part of routine examinations. From the start of the treatment period, each animal was observed at least once a day, at approximately the same time for the recording of clinical signs (including evidence of abortion/resorption).

Body weight:
It was recorded for each female on days 2, 4, 5, 6, 9, 12, 15, 19, 24 and 29 post-coitum. The net body weight was calculated (gross body weight minus gravid uterine weight), as well as the gross and net body weight changes.

Food consumption:
The quantity of food consumed by each female was recorded for the following intervals: 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-19, 19-24 and 24-29 post-coitum. Any obvious spillage of food was documented.
Ovaries and uterine content:
Hysterectomies:
On day 29 post-coitum, all the females were killed by an intravenous injection of thiopental sodium. The weight of the gravid uterus was recorded for each pregnant female (with at least one live fetus) at hysterectomy. The ovaries and uterus of the females were examined to determine number of corpora lutea, number and distribution of dead and live fetuses, number and distribution of early and late resorptions and number and distribution of implantation sites (or uterine scars).
Early resorptions refer to evidence of implantation without recognizable embryo or fetus; late resorptions refer to dead embryo or fetus with external degenerative changes; scars refer to evidence of implantation without recognizable structure (embryo, fetus or placenta). Then the fetuses were killed by subcutaneous injection of thiopental sodium.
For the litters 17 to 20, sex determination (internal gonad determination) and recording of fetal body weight was performed on all fetuses at the time of hysterectomy.

Macroscopic post-mortem examination:
After hysterectomy, the females were subjected to a macroscopic post-mortem examination of the principal thoracic and abdominal organs.
A gross evaluation of placentas was also performed. The number of corpora lutea and implantation sites were recorded whenever possible for the females that died, aborted, or were killed prematurely.

Microscopic examination:
No microscopic examination was deemed necessary.
Fetal examinations:
These examinations were carried out only for the first 16 litters (females with at least one live fetus). The other litters were discarded without further investigations.

Body weight:
The body weight of each live fetus was recorded.

External examination:
Each fetus was submitted to a detailed external examination, which included the observation of all visible structures, surfaces or orifices. Dead fetuses were then discarded.

Soft tissue examination:
All live fetuses were subjected to a detailed fresh dissection of the soft tissue, which included the observation of all the organs and structures of the head, neck, thorax and abdomen. The brain of each fetus was sampled and fixed in Bouin's fluid. It was then horizontally sectioned and examined.

Skeletal examination:
The carcasses of the fetuses were fixed with ethyl alcohol. A detailed examination of the skeleton was performed after staining with alizarin red S which included the observation of all the bone structures of the head, spine, rib cage, pelvis and limbs.

Sex of fetuses:
The sex of each live fetus was determined at the time of the fresh dissection. The sex of each dead fetus was determined at the time of the hysterectomy.
Statistics:
Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.
Indices:
The following calculations were made:
Pre-implantation loss = (Number of corpora lutea - Number of implantation sites)*100/Number of corpora lutea
Post-implantation loss = (Number of implantation sites - Number of live fetuses)*100/Number of implantation sites
Fetal or litter incidence = Total number of fetuses or litters with a particular finding*100/Total number of fetuses or litters examined
Mean proportion of affected fetuses = Sum of proportion of fetuses affected in each litter*100/Total number of litters examined
Historical control data:
CIT historical control data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
see below (in "Any other information on results incl. tables")

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
= 150 mg/kg b.w./day
Based on:
test mat.
Remarks:
PIAS
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Remarks:
= 150 mg/kg b.w./day
Based on:
test mat.
Remarks:
PIAS
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
see below (in "Any other information on results incl. tables")

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Maternal toxic effects

Mortality and abortion: no treatment-related deaths or abortions occurred during the study.

Clinical signs: at 50 mg/kg/day, loud breathing was noted in one female from day 14 to day 18 post-coitum. At 500 mg/kg/day, absence of feces was noted in three females during a few days of the dosing period. This sign was attributed to the markedly lower food consumption of these females.

Body weight: the net body weight change was significantly lower than controls at 500 mg/kg b.w./day, but not at 50 and 150 mg/kg b.w./day.

Food consumption: the food consumption was significantly lower than controls at 500 mg/kg b.w./day, but not at 50 and 150 mg/kg b.w./day.

Necropsy findings: the few findings noted at autopsy were not considered to be related to treatment.

Uterus weight: it was unaffected by the treatment at 50 and 150 mg/kg b.w./day while it was significantly lower at 500 mg/kg b.w./day.

Litter data

No treatment-related effect was noted on the pre-implantation loss, fetal weight or sex ratio at any dose-level.

At 500 mg/kg b.w./day, there was a significantly higher incidence of early resorption and post-implantation loss and a significantly lower number of live fetuses per female.

Embryotoxic/teratogenic effects

Fetal external malformation: at 50 and 150 mg/kg b.w./day, no external malformation was recorded. At 500 mg/kg b.w./day, the significantly higher incidence of abdominal wall malformations (omphalocele and gastroschisis) when compared to controls were considered to be most probably related to the treatment.

Fetal external variation: malrotated limbs were recorded in a single dead fetus at 500 mg/kg b.w./day and malrotated paws were observed in few animals of the control and treated groups, without indication of a clear relationship to treatment with the test item.

Fetal soft tissue examination: at 50 and 150 mg/kg b.w./day, the described malformations were low in incidence and/or neither dose-related nor statistically significant, they were consequently not considered to be related to the treatment with the test item but to be of spontaneous occurrence.

Furthermore, the nature and fetal incidence of the soft tissue variations were randomly distributed and commonly observed in rabbit fetuses of this strain, e.g.: dilatation of renal pelvis or ureter, absence of brachiocephalic trunk, short innominate artery, dilated aorta and pulmonary trunk.

At 500 mg/kg b.w./day, the slightly higher incidence of fetal malformation and variations of the eyes e.g. small eye bulge recorded in three fetuses from two different litters and microphthalmia in one fetus from another litter were associated for most of these fetuses with hemorrhage of the eye and soft lens. These findings were considered to be most probably related to the treatment.

Fetal skeletal observation: there was a quite low incidence in both the control and all treated groups.

Applicant's summary and conclusion

Conclusions:
The test item, PIAS, batch No. CD05-054/05/CD06-287, was administered daily to pregnant female KBL New Zealand White rabbits, from day 6 to day 18 post-coitum, at 50, 150 or 500 mg/kg b.w./day.
At 500 mg/kg b.w./day, marked maternotoxicity was substantiated by significant body weight loss and severe decreased of food consumption during the dosing period. Higher incidence of early resorption, lower number of live fetuses and slightly higher incidence of omphalocele, gastroschisis and small eyes were also noted at 500 mg/kg b.w./day.
Consequently, under the same experimental condition of this study, the NOEL (No Observed Effect Level) for maternotoxicity and for embryofetal development is 150 mg/kg b.w./day.
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item, PIAS, on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rabbits during the period of organogenesis (from implantation to

closure of the hard palate: day 6 to day 18 post-coitum inclusive).

Methods

Four groups of 20 pregnant females KBL New Zealand White rabbits received the test item at 50, 150, 500 mg/kg b.w./day or the vehicle (0.4% w/v xanthan gum aqueous solution), by daily oral administration, from days 6 to 18 post-coitum (p.c.) inclusive and at a constant volume of

3 mL/kg.

Clinical signs, mortality, body weight and food consumption were recorded at designated intervals.

On day 29 p.c., the dams were sacrificed and subjected to a macroscopic post-mortem examination. The gravid uterus was weighed to allow calculation of the net body weight gain. At hysterectomy, the litter parameters were recorded. The fetuses from all dams were weighed (live only); and sexed and the fetuses from the first 16 dams were submitted to external, visceral and skeletal examination.

Results

Maternal data

- Mortality and abortion: no treatment related deaths or abortions were recorded in any group.

- Clinical signs: with the exception of absence of feces recorded in few animals (3/20 females) at 500 mg/kg b.w./day, no clinical sign ascribed to the treatment was noted in any group.

- Body weight and food consumption: no treatment-related effect on body weight and food consumption was noted at 50 and 150 mg/kg b.w./day.

At 500 mg/kg b.w./day, marked body weight loss, significantly lower body weight gain, and net body weight gain were noted during the dosing period. The food consumption was severely affected during the treatment.

- Uterus weight: the uterus weight was significantly lower at 500 mg/kg b.w./day.

- Macroscopic post-mortem examination: no macroscopic findings in any group were ascribed to the treatment with the test item.

Litter data

No treatment-related effect was noted on the pre-implantation loss, fetal weight or sex ratio at any dose-level.

At 500 mg/kg b.w./day, there was a significantly higher incidence of early resorption and post-implantation loss and a significantly lower number of live fetuses per female.

Fetal examinations

No treatment related malformation or variations (external, visceral or skeletal) were noted at 50 and 150 mg/kg b.w./day.

At 500 mg/kg b.w./day, the slightly higher incidence of malformations of the anterior abdominal wall represented by omphalocele and gastroschisis, and the presence of fetuses with malformed eyes (microphthalmia, small eye bulge) were considered to be most probably related to the treatment.

Conclusion

The test item, PIAS, batch No. CD05-054/05/CD06-287, was administered daily to pregnant female KBL New Zealand White rabbits, from day 6 to day 18 post-coitum, at 50, 150 or 500 mg/kg b.w./day.

At 500 mg/kg b.w./day, marked maternotoxicity was substantiated by significant body weight loss and severe decreased of food consumption during the dosing period. Higher incidence of early resorption, lower number of live fetuses and slightly higher incidence of omphalocele, gastroschisis and small eyes were also noted at 500 mg/kg b.w./day.

Consequently, under the same experimental condition of this study, the NOEL (No Observed Effect Level) for maternotoxicity and for embryofetal development is 150 mg/kg b.w./day.