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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:
key study
Study period:
1997-09-23 to 1998-04-08
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): S 12911-2
- INN: Strontium 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

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Iffa Credo, Domaine des Oncins, 69210 Saint-Germain sur L' arbresle, France
- Age and weight at study initiation: at the beginning of treatment, during the pre-pairing period, the F0 males were nine weeks old and their body weight ranged from 262.5 to 331.3 g, the F0 females intended for Caesarian section were eleven weeks old and their body weight ranged from 217.9 to 261.2 g. The F0 females intended for delivery of their offspring were thirteen weeks old at the beginning of the pairing period and their body weight ranged from 215.1 to 305.1 g on Gestation Day zero.
- Housing: except within the pairing period, the treated F0 males of subgroups B and D, as well as all females, were housed in individual cages. The untreated males of subgroups A and C were regrouped up to three per cages. After delivery, F0 dams were kept with their F1 offspring in the same cage until weaning. After weaning, all F1 animals from the same litter, not selected as breeders, were housed together by sex until sexual maturation. The F1 breeders were regrouped up to three per cage for each sex from weaning to pairing. After successful mating in F1 males remained regrouped up to three per cage, while the F1 females were housed in individual cages.
- Cage measurements: length =45 cm, width = 30 cm, height = 20 cm
- Diet (ad libitum): U.A.R (Usine Alimentation Rationnelle, Epinay-sur-Orge, France) sterilized feed pellets
- Water (ad libitum): sterilized drinking water
- Acclimation period: about two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 1°C
- 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^2 an no particle > 5 µm

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: hydroxyethylcellulose
Details on exposure:
VEHICLE
- Batch no.: EI 922
- 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 was calculated on the basis of 10 mL/kg bw (body weight not recorded).
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 were stable for twenty-six days when stored in stoppered flasks 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 were found equal to 7.6 for the first set of preparations.

*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:
- M/F ratio per cage: 1:1 ratio (subgroup B and D: males were treated)
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation (GD0)
Duration of treatment / exposure:
F0 animals:
Female fertility and embryofetal development:
- subgroup A (females): fourteen days prior to pairing with untreated males from the same subgroup and continued throughout pairing and until day seventeen of gestation inclusive

Toxicokinetics during pre-mating and gestation periopds:
- subgroup C (females): fourteen days prior to pairing with untreated males from the same subgroup and continued throughout pairing and until day seventeen of gestation inclusive
Frequency of treatment:
F0 animals: daily, seven days a week (each treatment was carried out during the morning)
Duration of test:
Until gestation day 20
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 500, 750 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Subgroup A: 20 females each for the control group and the 3 dose levels (males were also included)
Subgroup C: in each treated group five supplementary females made up subgroup C for toxicokinetic investigations
(Groups B and D: see section 7.8.1 Toxicity to reproduction)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were chosen on the basis of the results from previous reproductive and general toxicity studies in the rat with S 12911-2.
A study on male fertility, carried out by a laboratory (Müller, W. and Semich, R., 1994)* showed that the reproductive performance were not altered when S 12911-2 was administered by gavage at 825 mg/kg/day for eighty days before pairing with untreated female rats. The test substance was well tolerated. No treatment-related mortality was observed.

In a preliminary reproductive toxicity study (Momburg, r. et al., 1992)* female rats were treated with S 12911-2 by gavage at 750 mg/kg/day for fourteen days prior to pairing with untreated male rats, throughout pairing and until Gestation Day twenty or until Lactation Day four. The study results showed that S 12911-2 did not interfere with the general condition of the females, their fertility, the implantation process and prenatal development of the offspring, as well as the parturition and the viability of the newborn F1 pups until Lactation Day four. Only mean foetal weight on Gestation Day twoenty was slightly decreased.

Subchronic and chronic toxicity studies showed that S 12911-2 was well tolerated at 750 mg/kg day when administered by gavage over thirteen weeks to Wistar rats or over twenty-six weeks to Sprague Dawley rats (Bazot, D. and Lupart, M., 1997; Nuttall, J. et al., 1996, respectively)*.

On the basis of these results and taking into account the duration of treatment (eight to ten weeks for male rats and five to eight weeks for female rats), the high dose was set at 1000 mg/kg/day. Two lower doses were defined by an arithmetical progression, using a factor of 250. Hence, the doses were 500 and 750 mg/kg/day.

*References:
- Momburg, R., Legrain, B. and Sterz, H.. Etude d'information du S 12911-02 par voie orale chez le rat Wistar. Biologie Servier Internal Report No.: 2349, 1992.
- Müller, W. and Semich, R. S12911 - Oral (Gavage) Fertility Study in the Male Rat. Hazleton -18-RFA Project No.: 303-093, 1994
- Bazot, D. and Lupart, M. S 12911-2 Toxicity Study by Repeated Oral Administration for 13 Weeks in Wistar Rats. biologie Servier Internal Report No.: 2123,1997.
- Nuttall, J. Kelly, J. Barton, C. and Brown, P. S 12911 - 26 Weeks Oral (Gavage) Chronic Toxicity Study in the Rat. Hazleton-25-UK Project No.: 303-88, 1996.

Examinations

Maternal examinations:
F0 GENERATION:
CAGE SIDE OBSERVATIONS: Yes
Time schedule:
- treated animals intended for toxicokinetic investigations (subgroup C) and untreated males (subgroups A) were subjected to a daily mortality survey only.
- treated F0 females from subgroup A were observed daily during acclimation to detect mortality. During treatment periods, clinical monitoring was carried out prior to and after treatment, except during pairing. From the start of the pairing period to effective mating, females were subjected to a daily mortality survey only. After each pairing the supposed date of fertilization of the F0 females was determined by examination of the vaginal smears. During gestation phases without treatment, F0 females were observed at least once daily, and any sign of abortion was recorded.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
Time schedule for examinations:
1) Before pairing:
- treated females of subgroup A were weighed once weekly.
2) After pairing:
- each female of subgroups A effectively mated was weighed daily during gestation, from day zero to day twenty in subgroup A.

FOOD CONSUMPTION AND WATER CONSUMPTION: YES (females only)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
These parameters were calculated for the two weeks of the premating period in subgroup A, the three weeks of gestation in subgroups A.
Six different periods were defined for females of subgroup A as follows:
- from day fourteen to day eight inclusive prior to the start of animal pairing,
- from day seven to day two inclusive prior to the start of animal pairing,
- from day zero to day five of gestation inclusive,
- from day six to day eleven of gestation inclusive,
- from day twelve to day seventeen of gestation inclusive,
- from day eighteen to day nineteen of gestation inclusive
Five different periods were defined for females of subgroup B as follows:

OESTRUS CYCLICITY
F0 GENERATION:
For each female, vaginal smears were recorded every day from the first day of pairing until effective mating to determine the stage of oestrous cycle, and to detect potential adverse effects on the normal cycle.

F0 GENERATION:
SACRIFICE
- females of subgroup C were killed by carbon dioxide inhalation, after successful mating for males, on Gestation Day twenty for females of subgroup C. These animals were eliminated without necropsy.
- untreated F0 males of subgroups A and C were killed by carbon dioxide inhalation after successful mating or at the end of the pairing period and eliminated without necropsy.
- F0 animals of subgroup A with a sperm positive vaginal smear were killed by ether inhalation on Gestation Day twenty. Detailed autopsy including an examination of their uterine contents was performed. Those from the same subgroup with a sperm negative vaginal smear at the end of the pairing period were killed by ether inhalation ten days after the last mating day for verification of pregnancy and detailed autopsy.

GROSS NECROPSY (TREATED ANIMALS)
- a detailed autopsy was carried out on each treated F0 female, except those intended exclusively for toxicokinetic investigations. Organs with macroscopic anomalies were sampled and preserved for a possible histopathological evaluation. Corresponding organs of sufficient controls were preserved for comparison.
- the ovaries and oviducts were sampled for a possible light microscopic examination from F0 females of subgroup A which were apparently not pregnant at terminal sacrifice. These organs were discarded if any implantation site was detected after uterus staining.

HISTOPATHOLOGY / ORGAN WEIGHTS
- no histopathological examination was carried out on organs with macroscopic anomalies, which were sampled from F0 animals at terminal sacrifice.
Microscopic examination was performed on:
- testes and epididymides of high dose and control F0 males
- complete genital tract of any F0 males which did not fertilize their corresponding females
- ovaries and oviducts of F0 females which were not pregnant (no implantation site detected after uterus staining)
The other organs, including the macroscopic anomalies, were kept in fixative without any further investigation.
Ovaries and uterine content:
EXAMINATION OF UTERINE CONTENTS (F0 FEMALES OF SUBGROUP A)
- after hysterectomy, the corpora lutea, the implantation sites, the live foetuses and the resorptions were counted
- an external macroscopic examination was performed on the foetuses and the placentas, which were then weighed individually
- the sex of each foetus was determined either during the organ inspection or during the evisceration of those intended for the skeletal examination
Fetal examinations:
Examination of foetuses of F0 females of subgroup A)
Foetuses were killed and half of the foetuses from each litter were allocated to skeletal examination, and half of them were allocated to soft tissue inspection
- skeletal examination was performed on foetuses of all groups
- organ examination included only foetuses of the high dose group and control group because no biologically significant difference was observed in the findings between these two groups.
- the sex of each foetus was determined either during the organ inspection or during the evisceration of those intended for the skeletal examination.
Statistics:
Please refer to the field "Any other information on materials and methods incl. tables" below.
Indices:
Fertility Index

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
F0 females from subgroup A (F0 female general condition and fertility)
CLINICAL SIGNS AND MORTALITY
- administration of S 12911-2 to F0 females of subgroup A, from fourteen days prior to pairing until Gestation Day seventeen, did not provoke mortality or changes in behaviour, but only discolouration of faeces for all animals of all dose groups

BODY WEIGHT AND FOOD CONSUMPTION
- body weight of F0 females was not adversely affected by the test substance in any of the treated groups
- treatment of F0 females from subgroup A with S 12911-2 did not interfere with feed consumption up to the highest dose administered

REPRODUCTIVE PERFORMANCE
- the number of effectively mated F0 females (sperm positive vaginal smear) was similar for all groups
- the effective mating rate of the treated F0 females was not affected by the test substance up to the high dose
- the mean time necessary for successful copulation, expressed in days, was similar for all groups
- the fertility index was similar for F0 females from treated and control groups
- evaluation of the uterine contents in the F0 females on day twenty of gestation revealed a slight and not statistically significant increase in the pre-implantation loss rate at 1000 mg/kg/day (0.24 versus 0.14 in controls). Consequently, the mean numbers of implantation sites and live foetuses were slightly decreased at 1000 mg/kg/day (11.1 versus 12.1 in controls for the former, and 10.1 versus 11.4 in controls for the latter). These differences were also not statistically significant. The post-implantation loss rate was also increased at 1000 mg/kg/day (0.17 against 0.05 in controls, not statistically significant), but without increase in early and late resorptions. All these parameters showed generally better values for females treated at 500 and 750 mg/kg/day than for controls.

GROSS PATHOLOGY
- autopsy revealed no macroscopic organ anomaly

HISTOPATHOLOGY
- systematic histopathological examination of the ovaries and the oviducts in the non-pregnant females did not reveal any abnormal finding

WATER CONSUMPTION
Mean water consumption of F0 females from subgroup A was slightly higher in the treated groups than in the control group during the pre-pairing period. However, there was no dose-response relationship, and the differences between treated and control groups were not statistically significant (26.6, 28.8, 31.3, 28.8 g/day at 0, 500, 750 and 1000 mg/kg/day respectively during the second week). During gestation mean water consumption remained higher in the treated than control groups (35.0, 37.9, 41.0, 40.1 g/day on the average at 0, 500, 750 and 1000 mg/kg/day respectively). The differences in group mean values for the entire gestation period, between treated and control groups, were statistically significant at 750 and 1000 mg/kg/day (p < 0.01 and p< 0.05 respectively).

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
(F0 female)
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:
Dose descriptor:
NOAEL
Effect level:
< 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
F0 females from subgroup A (embryo-foetal development)
External examination:
- macroscopic external examination of foetuses revealed no findings related to the test substance

Visceral examination:
- soft tissue inspection included only foetuses of females treated at the high does (1000 mg/kg/day) and control foetuses
- all findings belonged to the spontaneous anomalies of the strain, and no biologically significant difference was observed in the findings between these two groups.

Skeletal examination:
- an increase in the frequency of delays in the ossification of two sternebrae at all doses (1.9, 11.9, 19.2 and 13.2% at 0, 500, 750 and 1000 mg/kg/day respectively), and caudal vertebrae at all doses (6.7, 9.2, 14.4 and 8.8% at 0, 500, 750 and 1000 mg/kg/day respectively)
- an increase in the frequency of variations, which were characterized by wavy ribs, bent bones (scapula, radius, ulna, femur, tibia, fibula), shortened and thickened, humerus, and misshapen clavicle. the percentage of foetuses and litters affected by wavy ribs in each group was 18.3 and 47.4% for controls, 70.6 and 94.7% at 500 mg/kg7day, 75.0 and 100% at 750 mg/kg/day, and 89.0 and 88.9% at 1000 mg/kg/day
- the percentage of foetuses and litters affected by at least one skeletal variation of the limbs (clavicle included) in each group was 1.0 and 5.3% for controls, 21.1 and 42.1% at 500 mg/kg/day, 22.1 and 41.2% at 750 mg/kg/day, and 61.5 and 77.8% at 1000 mg/kg/day
- one stunted foetus at 0 and 1000 mg/kg/day
- one foetus at 750 mg/kg/day presenting one thoracic vertebra limited to a left hemivertebra and a right vertebral arch, and misshapen vertebrae (one thoracic and one lumber)
- one foetus at 1000 mg/kg/day presenting one thoracic vertebra limited to the left transverse process, and two fused ribs.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 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

Any other information on results incl. tables

TOXICOKINETIC EVALUATION

- after administration of S 12911 -2 to to F0 females from subgroups C, strontium and ranelate were detected in the plasma of all animals. This result proves that the compound was absorbed at each dose level.

- mean accumulation ratios were close to unity, however, it was unclear whether steady state was reached.

- AUC24 -values of strontium in females increased proportionally to dose on MD-1, but significantly less than proportionally to dose on Gestation Day 6. No correlation was found on Gestation Day 17.

- AUC24 -values of ranelate in females increased proportionally to dose on MD-1, Gestation Day 6 and gestation Day 17. No correlation was found on Lactation Day 12.

- Cmax-values of strontium and ranelate increased significantly less than proportionally to dose on MD-1 and proportionally to dose on Gestation Day 6. No correlation was found on Gestation Day 17.

Applicant's summary and conclusion

Conclusions:
No observed adverse effect levels (NOAELs) of S 12911-2 in the rat:
- The NOAEL for F0 male and female general toxicity and fertility is 1000 mg/kg/day, corresponding on the last day of the pre-pairing period to AUC24-values of 376 and 276 µg.h/mL of strontium, respectively, and 16.5 and 13.8 µg.h/mL of ranelate, respectively.
- The NOAEL for embryo-foetal development is less than 500 mg/kg/day, mainly with regard to the skeletal variations, which were however, reversible during postnatal development. The maternal systemic exposure (AUC24) at 500 mg/kg/day corresponded to 192 µg.h/mL of strontium and to 7.5 µg.h/mL of ranelate on Gestation Day 17.
- The NOAEL for teratogenicity is 1000 mg/kg/ day, corresponding to a maternal AUC24 value on Gestation Day 17 of 302 µg.h/mL for strontium and of 16.7 µg.h/mL of ranelate.