Registration Dossier

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-05-01 to 1998-08-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
gravid uteri including the cervix were not weighed
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guideline S5 - Detection of toxicity to reporduction for medical products to male fertility, Washington June 24, 1993; ICH Harmonized Tripartite Guideline, Addendum: Toxicity to male fertility, July 1996.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Strontium Ranelate
Cas Number:
135459-87-9
Molecular formula:
C12H10N2O8S.2Sr
IUPAC Name:
Strontium Ranelate
Specific details on test material used for the study:
- Name of test material (as cited in study report): S 12911-2
- INN: distrontium ranelate
- Chemical name: 5-[bis(Carboxymethyl)amino]-2-carboxy-4-cyano-3-thiopheneacetic acid, distrontium salt
- Molecular formula: C12H6N2O8SSr2
- Molecular weight: 513.5
- Physical state: powder
- Storage condition of test material: stored at room temperature, in a closed container
- Batch No: 44697

Test animals

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS - New Zealand White rabbits (SPF)
- Source: C.P.A., 351, rue Roquemaure, 45160 OLIVET, France
- Age on mating day: sixteen to nineteen weeks
- Weight on mating day: 2.9 to 3.9 kg
- Housing: the F0 females were housed in individual cages; cage measurements: length = 75 cm, depth = 54.5 cm, height = 40 cm
- Diet (ad libitum): U.A.R (Usine Alimentation Rationnelle, Epinay-sur-Orge, France) sterilized feed pellets
- Water (ad libitum): sterilized drinking water

ENVIRONMENTAL CONDITIONS
- Temperature: 19 ± 2°C
- Relative humidity: 55 ± 15%
- Air changes: 14 to 19 times per hour
- Photoperiod (hrs dark / hrs light): 12/12
- Dust level at filter outlet: less than 40 000 particles ≥ 0.5 µm per m^3 an no particle > 5 µm

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: hydroxyethylcellulose
Details on exposure:
VEHICLE
- Batch no.: EI922
- Physical state: powder
- Stability: until April 2000
- Dissolved in demineralized water at the concentration of 1% w/v.

PREPARATION OF DOSING SOLUTIONS:
S 12911-2 was suspended in the vehicle prepared as mentioned above.
The concentrations of the various preparations were calculated to allow administration at a constant dose volume of 10 mL/kg bw.

The dose volume, calculated on the basis of 10 mL/kg bw, was administered by gavage to each F0 female after weighing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Tests of stability and homogeneity, carried out before the start of the study (Ginot, Y.M., 1990)*, showed that preparations of S12911-2 in 1% hydroxyethylcellulose (w/v) were stable for twenty-six days when stored in stoppered bottles at room temperature, at concentrations spanning those administered.
Chemical analysis of the preparations administered during the study showed that measured concentrations were close to the intended values.
The pH of the test substance preparations was checked and values of the first preparation were found between 7.3 and 7.5.

*Reference:
Ginot, Y.M.
Technologie Servier.
Stability Study no.: PA.R. TOX.G04.R02.12911.01, 1990.
Homogeneity Study No.: PA.R. TOX.G02.R02.12911.01, 1990
Details on mating procedure:
Impregnation procedure: female New Zealand white rabbits, aged at least sixteen weeks, were paired at the breeder's facilities on three or four consecutive days during four weeks (one delivery per week to the testing laboratory Biologie Servier). During the first three weeks fifty-five female rabbits were mated on the basis of five females per day. During the fourth week twenty-four females were mated on the basis of six females per day. Each female was paired successively with two different untreated mature male rabbits from the same strain. Effective mating was checked visually and the mating day was considered as day zero of gestation.
Duration of treatment / exposure:
Day six of gestation to day eighteen of gestation inclusive
Frequency of treatment:
Once daily and seven days a week
Duration of test:
ca. 30 days
No. of animals per sex per dose:
Treatment groups: 19 mated female rabbits
Control group: 18 mated female rabbits
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale:
The doses were chosen on the basis of the results from the preliminary reproductive toxicity study with New Zealand White rabbits, in which pregnant females were treated at doses of 100, 300, 1000 or 1500 mg/kg, expressed as anhydrous product, once daily by gavage, from day six to day eighteen of gestation.
The results showed that S 12911-2 was well tolerated and did not provoke maternotoxic effects up to the highest dose of 1500 mg/kg. Embryotoxic effects, occurred at 1500 mg/kg and were characterized by an increase in the number of early resorptions and, as a consequence, in the post-implantation loss rate. However, the number of live foetuses was not affected. No drug-related foetotoxic or teratogenic effect was observed, whatever the dose used.
Systemic exposure, expressed as AUC24, increased proportionally with dose between 100 and 1500 mg/kg, reaching a maximum value of 984 µg x h/mL at the highest dose. After repeated administration, the compound did not accumulate in animal plasma or very slightly (0.82 to 2.1-fold), except for one female treated at 1000 mg/kg 84 times, 1456 µg x h/mL).
The dose of 1500 mg/kg represents the limit of feasibility in terms of homogenous ad stable suspension in relationship with a dose volume tolerable by pregnant does. This dose was used again as highest dose. Two lower doses were determined by a geometric progression using a factor of approximately three. Hence these doses were 150 and 500 mg/kg.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during gestation the F0 females were observed at least once daily and during the treatment period clinical monitoring was carried out prior to and after treatment. Any sign of abortion was recorded during gestation.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: each female was weighed daily from the day three of gestation to day thirty of gestation inclusive.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
The parameter was determined daily from day three to day twenty-nine of gestation inclusive, only for the pregnant females alive on Gestation Day thirty.
Mean feed consumption per group, expressed in g/day, were calculated for the following four periods:
- from day three to day five of gestation inclusive
- from day six to day twelve of gestation inclusive
- from day thirteen to day eighteen of gestation inclusive
- from day nineteen to day twenty-nine of gestation inclusive

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: the parameter was determined daily from day three to day twenty-nine of gestation inclusive, only for the pregnant females alive on Gestation Day thirty.
Mean water consumption per group, expressed in g/day, were calculated for the following four periods:
- from day three to day five of gestation inclusive
- from day six to day twelve of gestation inclusive
- from day thirteen to day eighteen of gestation inclusive
- from day nineteen to day twenty-nine of gestation inclusive

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 30
- Organs examined: uterine contents, as well as a detailed autopsy on each F0 female.
- Organs with macroscopic anomalies were sampled and preserved for a possible histopathological evaluation. Corresponding organs of sufficient controls were preserved for comparison.
Ovaries and uterine content:
The uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Number of foetuses: Yes
An external macroscopic examination was performed on the placentas which were then weighed individually.
Fetal examinations:
- External examinations: Yes, an external macroscopic examination was performed on the foetuses which were then weighed individually.
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
- The sex of each foetus was determined during organ inspection
Statistics:
1) F0 females during gestation
- one way analysis of variance (group): global body weight gain between gestation days 6 and 19
- two-way analysis of variance (group, time) with repeated measures on time for: body weight during gestation, every day between days 3 and 5, 6 and 19, 20 and 30; daily mean body weight gain during gestation between days 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30; daily mean feed and water consumption during different periods ( gestation days 3 - 5, 6 - 12, 13 - 18, 19 - 29)

2) Implantation - Embryonic and foetal development
- Kruskal-Wallis test on the following data: number of corpora lutea, number of implantation sites, number of live foetuses, number of dead foetuses, number of early resorptions, number of late resorptions, loss rate before implantation and loss rate after implantation

For each analysis the critical significance level is 5%.

References:
- Winer, B.J. Statistical Principles in Experimental Design. Mc Graw-Hill - New York, 160 -167, 518 -539, 1971. Second Edition.
- Miller, G. Simultaneous Statistical Inference. Springer-Verlag, 76 -81, 1981.
- Conover, J. Practical Nonparametric Statistics, Wiley, 229 -237, 1980.
- Snedecor, G.W. & Cochran, W.G. In: Statistical Methods. Iowa State University Press, IA 7th Edition, 250 -251, 1980.
- SAS/STAT User's Guide. Version 6, fourth edition, 1990.
- IMSL STAT/LIBRARY, 1989.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Administration of S 12911-2 to pregnant female New Zealand White rabbits did not provoke any changes in behaviour.
One control female found dead on gestation Day 13, had blood in the thoracic cavity and congestive left pulmonary lobe. One female of the 1500 mg/kg dose level group, found dead on Gestation Day 16, had perforation of the oesophagus and haemorrhagic lungs. Another female of the 1500 mg/kg dose level group, found dead on Gestation Day 15, showed mainly yellowish contents (probably test compound) in the thoracic cavity and partially haemorrhagic left pulmonary lobe. An intubation error is likely to be the origin of these three deaths.
Mortality:
no mortality observed
Description (incidence):
Administration of S 12911-2 to pregnant female New Zealand White rabbits did not provoke test substance-related mortality.
One control female found dead on gestation Day 13, had blood in the thoracic cavity and congestive left pulmonary lobe. One female of the 1500 mg/kg dose level group, found dead on Gestation Day 16, had perforation of the oesophagus and haemorrhagic lungs. Another female of the 1500 mg/kg dose level group, found dead on Gestation Day 15, showed mainly yellowish contents (probably test compound) in the thoracic cavity and partially haemorrhagic left pulmonary lobe. An intubation error is likely to be the origin of these three deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Global mean body weight gain of F0 females during treatment, from Gestation Day 6 through Gestation Day 19, was reduced at 1500 mg/kg when compared with that of control females (+270, +249, +257 and +164 g at 0, 150, 500 and 1500 mg/kg respectively). The difference from the control group was statistically significant at 1500 mg/kg (p < 0.01). After the withdrawal of treatment on Gestation Day 19, the mean body weight gain until terminal sacrifice on Gestation Day 30 was similar for all groups (+253, +238, +237 and +241 g at 0, 150, 500 and 1500 mg/kg respectively).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean feed consumption of females treated at 150 and 500 mg/kg, was similar to that of controls during the entire treatment period, whereas that of females treated at 1500 mg/kg was lower than that of controls during the first and second week of treatment (150, 154, 158, and 122 g/day at 0, 150, 500, and 1500 mg/kg respectively for the second week). The difference from the control group were statistically significant at 1500 mg/kg (p < 0.01). After the withdrawal of treatment, mean feed consumption showed a slight rebound effect at 1500 mg/kg and was similar for all groups (147, 156, 155 and 163 g/day at 0, 150, 500 and 1500 mg/kg respectively).
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Mean water consumption of F0 females was similar for all groups during the whole gestation period. The differences between groups were not statistically significant.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Administration of S 12911-2 to pregnant female New Zealand White rabbits did not provoke any changes in behaviour.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
One female of the 150 mg/kg dose level group killed prematurely on Gestation Day 4 in the lowest dose group had a sacro-lumbar desmorrhexia and also several localized losses of substance on the mucosa of the stomach fundus.
One aborted female of the 500 mg/kg dose level group, showed aerocoly, as well as dark spots on the lungs and white areas on the liver at autopsy.
Autopsy at termianl sacrifice on Gestation Day 30 of all remaining F0 females revealed congestive ovarian follicles in one non-pregnant female of the 500 mg/kg dose group, accentuated hepatic lobular pattern in one pregnant female of the same dose group, one localized loss of substance on the fundic mucosa in another pregnant female of the same dose group, and in two pregnant females of the 1500 mg/kg dose group one depressed, greyish area or congestive areas on the fundic mucosa respectively. As these macroscopic findings can be seen spontaneously, they were not attributed to an adverse effect of the test substance, and therefore not subjected to a histopathological examination.
Other effects:
no effects observed
Description (incidence and severity):
- Uterine content: treatment of pregnant females during organogenesis with S 12911-2 up to the top dose of 1500 mg/kg, had no impact on the uterine contents.
- Mean foetal and placental weights and sex ratio were also similar between all group.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
No impact on uterine contents, mean foetal and placental weights.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
A non dose- and treatment-related higher mean rate of pre-implanatation loss in all treated groups (0.13, 0.22, 0.25, 0.18 at 0, 150, 500 and 1500 mg/kg, respectively) resulting slightly lower mean no. of implantations and mean no. of live foetuses.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No impact on sex ratio.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
A non dose- and treatment-related higher mean rate of pre-implanatation loss in all treated groups (0.13, 0.22, 0.25, 0.18 at 0, 150, 500 and 1500 mg/kg, respectively) resulting slightly lower mean no. of implantations and mean no. of live foetuses.
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- articular stiffness in 0.6 , 0.7 2.4 and 6.2% of foetuses at 0, 150, 500 and 1500 mg/kg, respectively (spontaneous finding, not test item-related)
- one live foetus of the 500 mg/kg dose group was small sized, with shortened limbs, head deformities and a protruding tongue. This foetus was examined for the skeleton by a double staining method instead of X-ray radiography used for all other live foetuses.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
An increase in the frequency of delays in the ossification of several bones, mainly at 1500 mg/kg:
- skeletal anaomalies >/= 3 (retarded ossification/live foetuses): 3/17, 2/16, 3/15, 10/18 at 0, 150, 500 and 1500 mg/kg, respectively
- hyoid bone (8.3, 9.7, 8.9 and 22.8% at 0, 150, 500 and 1500 mg/kg respectively, historical control mean and maximum 6.4 and 17.2%, respectively)
- pubic bone (1.9, 0.0, 1.6 and 5.5% at 0, 150, 500, and 1500 mg/kg respectively, historical control mean and maximum 3.8 and 15.8%, respectively)
- talus (3.8, 3.0, 3.2 and 7.6% at 0, 150, 500, and 1500 mg/kg respectively, historical control mean and maximum 7.2 and 23.2%, respectively)
- phalanges (0.0, 0.0, 12.1 and 18.6% at 0, 150, 500 and 1500 mg/kg respectively, historical control mean and maximum 5.5 and 15.8%, respectively)

An increase in the incidence of skeletal variants, mainly at 1500 mg/kg:
- skeletal variations >/= 3 (live and dead foetuses): 6/17, 6/16, 7/15, 11/18 at 0, 150, 500 and 1500 mg/kg, respectively
- frequency of supernumerary ribs, mainly from 500 mg/kg (34.6, 36.6, 47.6 and 57.2% at 0, 150, 500, and 1500 mg/kg respectively, historical control mean and maximum 22.1 and 45.5% respectively)
- wavy ribs only at 1500 mg/kg (8.3%, unknown in this rabbit strain)
- two foetuses (1.4%) with one or two bent femurs at 1500 mg/kg (unknown in this rabbit strain)
- five cases of malformed foetuses: two in the control , one at 500 mg/kg and two at 1500 mg/kg

In sum, the substance interfered with the ossification process of the foetuses by inducing delays in the ossification of some bones, as well as skeletal variations in form of wavy ribs and bent femurs. The ossification process continues after birth and recovers the prenatal delay. The clinical relevance for the rib and femur anomalies appears slight, as the ossification status at birth is quite more advanced in man than in the rabbit.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- shortened innominate artery in 5.2, 5.2, 8.1 and 13.8% of foetuses at 0, 150, 500 and 1500 mg/kg, respectively (historical control mean and maximum 12.7 and 37.3%, respectively)
- absence of truncus brachiocephalicus in 10.3, 16.4, 16.1 and 17.2% of feotuses at 0, 150, 500 and 1500 mg/kg, respectively (historical control mean and maximum 16.4 and 44.4%, respectively)

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEL
Effect level:
1 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
1 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The susbtance did not provoke any teratogenic effect.
Under the conditions of this study, the NOAELs were 500 mg/kg for materno- and foetotoxicity, and 1500 mg/kg for embryotoxicity and teratogenicity.