Strontium chloride

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

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

Guideline:

other: ICH Harmonised Tripartite Guideline - Detection of toxicity to reporduction for medical products, Washington June 24, 1993; ICH Harmonized Tripartite Guideline, Addendum: Toxicity to male fertility, July 1996.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Strontium ranelate

IUPAC Name:

Strontium ranelate

Constituent 2

Reference substance name:

5459-90-4

IUPAC Name:

5459-90-4

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

Sex:

male/female

Details on test animals or test system 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 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. 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: lenght=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).

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)

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

Duration of treatment / exposure:

F0 animals:
Male fertility and parturition of F0 females and pre-/postnatal development of F1 animals:
- subgroup B (females): day six of gestation and continued until day twenty of lactation inclusive
- subgroup B (males): twenty-eight days prior to pairing with treated females from the same subgroup and continued throughout pairing up to, and including, the day before terminal sacrifice (six days after the treated females started littering)

Toxicokinetics of F0 males and F0 females during lactation and F1 pups:
- subgroup D (females): day six of gestation and continued up to and including day twelve of lactation.
- subgroup D (males): twenty-eight-day pre-pairing period and finished the first day of the pairing period

Frequency of treatment:

F0 animals: daily, seven days a week (each treatment was carried out during the morning)

Details on study schedule:

- The method of reproduction used for F1 animals was identical to that used for F0 animals (nocturnal, monogamous pairing).
- Selection of parents from F1 generation when pups were eleven to twelve weeks of age at the start of the pairing period which lasted up to twelve days.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 500, 750 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Subgroup B: 25 females/25 males each for the control group and the 3 dose levels
Subgroup D: in each treated group five supplementary animals of each sex made up subgroup D for toxicokinetic investigations (exclusively toxicokinetic)
(Groups A and C: see section 7.8.2 Developmental toxicity / teratogenicity)

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 show 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 show 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.

The pre-pairing treatment period for F0 males was fixed at twenty eight days, because the results of subchronic and chronic toxicity studies (Müller, W. and Semich, R., 1994; Nuttall, J. et al., 1996)* did not reveal either a treatment-related decrease in the weight of male genital organs, or histopathological modifications of the same organs.

*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.

Positive control:

none

Examinations

Parental animals: Observations and examinations:

F0 GENERATION:
CAGE SIDE OBSERVATIONS: Yes
Time schedule:
- treated animals intended for toxicokinetic investigations (subgroup D) and untreated males (subgroup D) were subjected to a daily mortality survey only.
- treated F0 males from subgroup B were observed once daily during acclimation to detect mortality. During treatment, clinical monitoring was carried out prior to and after treatment each day until the day before the start of terminal sacrifice to get equal numbers of recordings for all males. Then treated males were subjected to a daily mortality survey only until their terminal sacrifice.
- treated F0 females from subgroup B 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 from both subgroups 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. During lactation partial or total litter loss was recorded for F0 females from subgroup B. Clinical signs, recorded for non-pregnant females after effective mating, were excluded from evaluation.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
Time schedule for examinations:
1) Before pairing:
- treated males of subgroup B were weighed once weekly
2) After pairing:
- males of subgroup B were weighed once weekly for three weeks, as well as on the day of terminal sacrifice
- each female of subgroup B effectively mated was weighed daily during gestation, from day zero to day twenty-one in subgroup B
- females of subgroup B, which littered, were weighed daily during lactation, from day zero to day twenty-one inclusive

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 three weeks of gestation in subgroups B, and the two first weeks of lactation in subgroup B.
Five different periods were defined for females of subgroup B as follows:
- from day zero to day five of gestation inclusive,
- from day six to day thirteen of gestation inclusive,
- from day fourteen to the end gestation inclusive,
- from day zero to day six of lactation inclusive,
- from day seven to day thirteen of lactation inclusive

Oestrous cyclicity (parental animals):

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.

Sperm parameters (parental animals):

Parameters examined in F0 male parental generations:
The testes and epididymis from the treated F0 males of subgroup B were systematically sampled and fixed after recording their organ weights and after sperm analysis 8number and viability of spermatozoa) in the left cauda epididymis.

Litter observations:

F1 GENERATION
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number of stillbirths, live births, dead births and cannibalized pups (litter affected by maternal cannibalism were excluded from the calculation of sex-ratio)
- clinical signs and mortality: after day zero of lactation the F1 pups were observed once a day until weaning, and each dead pup, either spontaneously or by cannibalism, was recorded
- body weight: all live pups from each litter were weighed individually on day zero, four, seven, fourteen and twenty-one of lactation; only the F1 breeders (one F1 male and female pup per litter) were weighed once weekly from week four to week ten postpartum

Postnatal development: pre-weaning landmarks of physical development and reflex acquisition were observed for all live F1 pups as follows:
- physical tests (incisor eruption on Lactation Day eleven, eye opening on Lactation day fifteen, auditory meatus opening on Lactation Day twenty)
- reflex tests (surface righting on Lactation Day six, prehensile traction on Lactation Day thirteen, pupillary reflex on Lactation Day nineteen, auditory startle on Lactation Day twenty)

Post-weaning landmarks of development (sexual maturation) were observed for all live F1 pups, and behaviour was assessed for all F1 breeders (one male and one female per litter where possible) as follows:
- sexual maturation (cleavage of the balanopreputial gland from day forty postpartum (p.p.), vaginal opening from day thirty p.p.)
- behaviour (locomotor activity in an open field at the age of six to seven weeks, learning ability in a water maze at the age of nine to ten weeks, memorizing ability in a water maze at the age of ten to eleven weeks)

GROSS EXAMINATION OF DEAD PUPS: YES
- F1 pups found dead between day zero and day eight of lactation were fixed in alcohol for an examination of their skeleton
- F1 pups which died after eight days of age were subjected to a detailed autopsy

F2 GENERATION
- on the day of birth (day zero of lactation) the number of live newborn pups, dead newborn pups, stillborn pups and cannibalized pups was recorded.
- after day zero of lactation the F2 pups were observed once a day until terminal sacrifice, and each dead pup, either spontaneously or by cannibalism, was recorded. Dead pups were fixed in alcohol for an examination of their skeleton.
- F2 pups from each litter were weighed individually on day zero, and three of lactation.

Postmortem examinations (parental animals):

F0 GENERATION:
SACRIFICE
- males of subgroup D and females of subgroups D were killed by carbon dioxide inhalation after successful mating for males, on Lactation Day thirteen, after the last blood sampling for females of subgroup D. These animals were eliminated without necropsy
- F0 males of subgroup B were killed by ether inhalation for detailed autopsy, after littering of their corresponding F0 females from subgroup B or from Gestation Day twenty-seven if the corresponding females did not litter
- F0 females of subgroup B, which delivered a litter, were killed by ether inhalation from Lactation Day twenty-one for a detailed autopsy. Those from the same subgroup with a sperm positive vaginal smear, but without delivery, were killed by ether inhalation from twenty-five days later for verification of pregnancy and detailed autopsy. 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 pairing day and eliminated without necropsy.

GROSS NECROPSY (TREATED ANIMALS)
- a detailed autopsy was carried out on each treated F0 male and 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 complete male genital tract (testes, epididymides, prostate, seminal vesicles and deferent ducts), from treated F0 males of subgroup B which did not fertilize their corresponding female, was sampled for light microscopic examination

HISTOPATHOLOGY / ORGAN WEIGHTS
- no histopathological examination was carried out on organs with macroscopic anomalies, which were sampled from F0 and F1 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 or F1 males which did not fertilize their corresponding females
- ovaries and oviducts of F0 or F1 females which were not pregnant (no implantation site detected after uterus staining)
- right knee joint of ten F1 and F2 males and females
The other organs, including the macroscopic anomalies, were kept in fixative without any further investigation.

Postmortem examinations (offspring):

F1 GENERATION AND THEIR F2 LITTERS
SACRIFICE
- F1 males and females not selected for behaviour testing and breeding, were killed separately by ether inhalation after achievement of sexual maturation of all littermates from the same sex in a litter. They were subjected to a detailed autopsy. When a large majority of F1 females had already been killed, and also the F1 males of approximately half of the litters, it was decided to perform an X-ray photography before autopsy on all remaining F1 males from the treated groups and also on one F1 male from each of the remaining control litters, to examine the ribs, the scapulae, the clavicles, the humerus and the long bones of the forelimbs and hind limbs.
- F1 males selected for breeding were killed by ether inhalation for a detailed autopsy, after littering of their corresponding F1 females, should the occasion arise, or after terminal sacrifice of apparently non-pregnant F1 females for verification of pregnancy.
- F1 females, which delivered a litter and showed no total litter loss, were killed by ether inhalation between Lactation Day three and five for a detailed autopsy. Their F2 pups were subjected to a macroscopic external examination to detect any anomaly, before killing.
- F1 females with a sperm positive vaginal smear, but without delivery, were killed by ether inhalation from twenty-six days later for verification of pregnancy and a detailed autopsy.

GROSS NECROPSY
- a detailed autopsy was carried out on each F1 male and female at terminal sacrifice, and on all F1 animals found dead during the study if they were aged at least nine days
- organs with macroscopic anomalies were sampled and preserved for a possible histopathological evaluation; corresponding organs of sufficient controls were preserved for comparison
- the complete male genital tract (testes, epididymides, prostate, seminal vesicles and deferent ducts), from F1 males which did not fertilize their corresponding females, was sampled for light microscopic examination
- the ovaries and oviducts were sampled for a possible light microscopic examination from F1 females which were apparently not pregnant at terminal sacrifice
- the right knee joint (femoro-tibial joint) of F1 males and females from the first ten litters, with a least one viable F2 male and female pup, was sampled for light microscopic examination
- in each of these litters, one F2 male and female were selected at random for the same examination

Statistics:

Please refer to the field "Any other information on materials and methods incl. tables" below.

Reproductive indices:

- gestation index (number of females with a live litter / number of pregnant females)
- mean live birth index (number of live newborn pups / number of implantation sites)

Offspring viability indices:

- weaning index
- viability index
- survival index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

1) F0 GENERATION (F0 males from subgroup B (F0 male general condition and fertility))
CLINICAL SIGNS AND MORTALITY
- administration of S 12911-2 to F0 males from subgroup B did not provoke mortality or changes in behaviour; only discolouration of faeces for all animals of all dose groups was seen

BODY WEIGHT AND FOOD CONSUMPTION
- body weight of F0 males was not adversely affected by the test substance in any of the treated groups

REPRODUCTIVE FUNCTION: SPERM MEASURES
- determination of the mean number of spermatozoa per gram cauda epididymis (x10^6) revealed similar values for all groups (not statistically significant)
- evaluation of sperm viability (5 motile sperm) per cauda epididymis led to similar values for all groups (not statistically significant)

REPRODUCTIVE PERFORMANCE
- libido of F0 males was not altered by treatment with S 12911-2 in any dose group
- the effective mating rate was similar for all groups
- the mean time necessary for successful copulation, expressed in days, was also similar for all groups
- the fertility index was similar for F0 males from treated and control groups

ORGAN WEIGHTS
- reproductive organ weights: absolute mean and relative (% to body weight) mean weights of testes and epididymides were similar between treated and control groups

GROSS PATHOLOGY (PARENTAL ANIMALS)
- findings are not attributed to an adverse effect of the test substance

HISTOPATHOLOGY
- systemic histomorphological examination of the testes and epididymides in all F0 males from the high dose and control groups, as well as examination of the complete genital tract in those of the F0 males that did not fertilize their corresponding females, did not reveal any anomaly attributable to treatment with the test substance

2) F0 GENERATION (F0 females from subgroup B (F0 female parturition and general condition during gestation and lactation))
CLINICAL SIGNS AND MORTALITY
- administration of S 12911-2 t F0 females of subgroup B, from Gestation Day six to Lactation Day twenty, did not provoke mortality or changes in behaviour; only discolouration of faeces for all animals of all dose groups was seen

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

WATER CONSUMPTION
- treatment of F0 females from subgroup B with S 12911-2 did not interfere with their water consumption up to the highest dose administered

GROSS PATHOLOGY
- autopsy of the F0 females showed findings which are not attributed to an adverse effect of the test substance

GESTATION and PARTURITION
- no interference with normal progress of gestation and parturition
- no effects on mean duration of gestation or gestaion index
- mean live birth index not affected by treatment
- slight differences between treated groups were due to one female in each group mated during metoestrus (without biological relevance)

Effect levels (P0)

Dose descriptor:

NOAEL

Remarks:

(general toxicity, male fertility and reproductive performance)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Remarks on result:

other: Generation: F0 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Details on results (F1)

1) F1 pups

OTHER FINDINGS
- the sex ratio of F1 pups from treated females was close to 1.00, and therefore, was not modified by the test substance

VIABILITY
- S 12911-2 had no impact on F1 pup viability after birth
- mortality of F1 pups from birth to weaning was similar in all groups
- no litter loss was observed in any of the groups, consequently, the weaning index was similar in all groups

CLINICAL SIGNS (OFFSPRING)
- no treatment-related findings were made

BODY WEIGHT
- mean body weight of F1 pups, as well as changes in mean body weight of F1 pups, from birth to weaning, were similar for treated and control groups

SEXUAL MATURATION
- sexual maturation of F1 animals was not altered by treatment of the animals from the parental F0 generation

GROSS PATHOLOGY
- the external macroscopic examination of F1 pups at birth did not reveal any abnormality
Skeletal examination of F1 pups found dead before lactation day nine revealed the following findings:
- one control pup with one rudimentary rib and a thoracic vertebra limited to the right transverse process
- one pup at 750 mg/kg/day with one rudimentary rib and wavy ribs
- three pups at 1000 mg/kg/day with wavy ribs
- one pup at 1000 mg/kg/day with wavy ribs, one rudimentary rib, a bent scapula and a misshapen clavicle
- one pup at 1000 mg/kg/day with a delay in the ossification of two sternebrae and two caudal vertebrae, as well as a misshapen clavicle.

- autopsy at terminal sacrifice of the F1 animals not selected for breeding revealed no adverse effect of the test substance
- X- ray radiography of F1 males of approximately half of the litters, did not detect any bent, or shortened and thickened, or misshapen bone among the ribs, the scapulae, the clavicles, the humerus and the long bones of the forelimbs and hind limbs

OTHER FINDINGS
Physical development:
- F1 pups from treated groups showed a percentage of success equal to or higher than that of control pups for eye opening on Lactation Day fifteen (LD 15) and for the auditory meatus opening on LD 20
- the incisor eruption on LD 11 appeared to be delayed for a higher percentage of pups from the treated groups than from the control group, slightly at 500 and 750 mg/kg/day, moderately at 1000 mg/kg/day (3.7, 7.3, 10.6, 27.1% at 0, 500, 750 and 1000 mg/kg/day respectively). The difference from the control group was statistically significant at 750 and 1000 mg/kg/day (p<0.01), but had no consequential negative effect on animal growth
Reflex acquisition:
- similar or identical percentages of successful F1 pups in treated and control groups for, surface righting on LD 6, prehensile traction on LD 13, pupillary reflex on LD 19 and auditory startle on LD 20
- difference between treated and control groups were not statistically significant, except for surface righting at 500 mg/kg/day (73.2% against 63.7% for controls, p<0.05)

2) F1 BREEDERS
CLINICAL SIGNS
- treatment of parental F0 generation had no adverse effect on F1 male and female locomotor activity in an open field whatever the parameter evaluated
- treatment of parental F0 generation had also no adverse effect on learning and memorizing abilities of F1 males and females in a water maze; treated animals performed generally better than controls

BODY WEIGHT
- mean body weight gain of F1 males and females, randomly selected at weaning (three weeks old) as future breeders, was similar between the third and tenth week pp. for treated and control groups
- mean body weights of F1 males and females at the age of ten weeks were not statistically different between treated and control groups


REPRODUCTIVE PERFORMANCE
Libido:
- all F1 males mated their corresponding F1 female
- the effective mating rate was 1.00 for all groups
- the mean time necessary for successful copulation, expressed in days, was similar for all groups
Fertility index and reproductive performances:
- the fertility index was not adversely affected
- the delivery data for the F1 females on Lactation Day zero revealed no statistically or biologically significant differences between treated and control groups for any of the parameters evaluated
Gestation and Parturition:
- treatment of females from the parental F0 generation from implantation, through gestation and parturition until weaning, had no impact on the normal progress of gestation of the F1 females
- the mean duration of gestation was almost identical for all groups.
- the gestation index was slightly higher in all treated than control groups (0.94, 1.00, 0.95, 1.00, at 0, 500, 750 and 1000 mg/kg/day respectively)
- the mean live birth index was similar between groups (0.89, 0.88, 0.87, 0.88 at 0, 500, 750 and 1000 mg/kg/day respectively)

GROSS PATHOLOGY
- autopsy of F1 breeders at terminal sacrifice revealed no findings which were attributed to an adverse effect of the test substance
- the systemic histological examination of the femoro-tibial joints of F1 breeders from control and high dose groups did not detect any abnormal finding; adjacent osseous and cartilaginous structures were also normal
- the systemic histopathological examination of ovaries and oviducts from non-pregnant females (F1 breeders), as well as that of the complete genital tract from the corresponding males, did not reveal any abnormal finding

OTHER FINDINGS (OFFSPRING)
- mean body weight of the F2 pups at birth was similar for treated and control groups
- the sex-ratio of F2 pups from treated females was within the normal range, and therefore, was not modified by the test substance
- the external macroscopic examination of F2 pups revealed only one live newborn control pup with a shortened tail due to maternal cannibalism

3) F2 PUPS
VIABILITY
- treatment of F0 females with S 12911-2, from implantation to weaning of F1 pups, had no impact on F2 pup viability during the early postnatal phase until Lactation Day three
- no litter loss was observed in any of the groups
- the viability indes was not adversely affected

CLINICAL SIGNS
- F2 pups from all treated groups did not show any particular clinical sign

BODY WEIGHT
- mean body weight, as well as changes in mean body weight of F2 pups, from birth to Lactation Day three, were similar for treated and control groups

GROSS PATHOLOGY (OFFSPRING)
Examination of F2 pups found dead before terminal sacrifice:
- skeletal examination of dead F2 pups resulted in a compression of the right transverse processes of the third to the sixth lumber vertebrae in a F2 control pup
- one wavy rib in a F2 pup from the 1000 mg/kg/day dose group is not attributed to any effect of the test substance, because of its spontaneous occurrence in this rat strain
Examination of F2 pups at terminal sacrifice
- macroscopic external examination of F2 pups at terminal sacrifice (between Lactation Day three and five) revealed no abnormality
- systematic histological examination of the femoro-tibial joints of F2 pups from the control and high dose groups did not detect any abnormal finding; adjacent osseous and cartilaginous structures were also normal

Effect levels (F1)

1

Dose descriptor:

NOAEL

Remarks:

(postnatal development)

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on postnatal development up to 1000 mg/kg bw/d corresponding on LD13 to F1 pup strontium plasma concentration of 7.5 µg/mL just after the 24 h time point for dams.

2

Dose descriptor:

NOAEL

Remarks:

(fertility)

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on male and female fertility

Results: F2 generation

Effect levels (F2)

Dose descriptor:

NOAEL

Remarks:

(pre- and early postnatal development)

Generation:

F2

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on pre- and early postnatal development

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

TOXICOKINETIC EVALUATION

- after administration of S 12911 -2 to F0 females sugroup D, strontium and ranelate were detected in the plasma of all animals; this proves that the compound was absorbed at each dose level

- it appears that males were more exposed to strontium than non-pregnant females; as dosing of males before pairing covered a period twice as long as that of females (28 versus 14 days), steady state may not have been reached by Day 14

- no gender differences were observed for the systemic exposure to ranelate

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

- systemic exposure (AUC24) of males to strontium and maximal plasma concentrations (Cmax) increased significantly less than dose

- no correlation was found between dose and AUC24- or Cmax-values for ranelate

- AUC24 -values of strontium in females increased proportionally to dose on MD-1 and Lactation Day 12, 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 and Lactation Day 12

- it appears that at all dose levels, mean strontium plasma levels measured at about 24 hours post-dosing of dams on Lactation Day 12 were about 2 -fold higher in the untreated F1 pups than in the corresponding F0 dams

- the determination of strontium concentrations in the bone of F1 pups revealed a dose-related increase which was consistent with that observed for strontium plasma levels in F1 pups on Lactation Day 13; consequently, the percentage of strontium in F1 rat bones (calculated as the molar ratio between strontium and the sum of strontium + calcium) also increased with dose, and in a very similar proportion to the strontium plasma levels

Applicant's summary and conclusion

Conclusions:

No observed adverse effect levels (NOAELs) of S 12911-2 in the rat:

The NOAEL for F0 female general toxicity and reproductive performance is 1000 mg/kg/day, corresponding on LD12 to maternal AUC24 and C24h-values of 441 µg.h/mL and 3.5 µg/ml of strontium, respectively, and 41.4 µg.h/mL and 0.3 µg/ml of ranelate, respectively, and on the last day of the pre-pairing period to AUC24-values for males of 376 µg.h/mL of strontium and 16.5 µg.h/mL of ranelate.

The NOAEL for postnatal development of the F1 generation is 1000 mg/kg/day, corresponding on LD13 to F1 pup strontium plasma concentration of 7.5 µg/mL just after the 24 h time point for dams. The lower percentage of pups with incisior eruption was regarded as not adverse because it had no consequential negative effect on growth.

The NOAEL for F1 male and female fertility is 1000 mg/kg/day.

The NOAEL for pre-and early postnatal development of the F2 generation is 1000 mg/kg/day.