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EC number: 231-133-4 | CAS number: 7440-24-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
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- Nanomaterial pour density
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- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
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- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
Read-across: Strontium metal is highly reactive and instantly oxidizes upon contact with water. It decomposes completely. During the redox-reaction with water, a strong evolution of hydrogen gas and an immediate precipitation of a white, crystalline solid (i.e. Sr(OH)2) is observed (Sr2+ + 2OH- + H2 (g). The amount of dissolved Sr cations is determined by the solubility of the Sr(OH)2 precipitate. According to OECD guideline 105 (1995) and EU method A.6 (2006), the water solubility of strontium was determined to be 6.74 ± 0.14 g/L under the conditions of the test (flask method under protective gas atmosphere; loading of 41 g Sr/L, at 20.0 ± 1.0 °C, pH >13).
Strontium ions are highly mobile, occur only in one valence state (2+), i.e. are not oxidized or reduced, and do not form strong complexes with most inorganic and organic ligands (Krupka et al. 1999. EPA 402-R-99-004B; Salminen et al. 2015; Carbonaro and Di Toro. 2007. Geochim Cosmochim Acta 71 3958–3968; Carbonaro et al. 2011. Geochim Cosmochim Acta 75: 2499-2511 and references therein). Thus, it may be assumed that systemic toxicological effects (not local) are related to the strontium ion. Therefore, the assessment of the systemic toxicity of strontium is based on elemental strontium concentrations. Read-across of systemic toxicity data available for soluble strontium substances is applied since the strontium ions determine the toxicological potential of strontium. Read-across from strontium ranelate or strontium chloride to strontium is possible since as a first surrogate for bioavailability, the solubility of a test substance in water may be applied. All substances (strontium chloride, strontium ranelate and strontium) are soluble (> 5 g/L). Hence, it can be concluded that adverse effects observed with strontium chloride and strontium ranelate are due to the presence of the strontium ion and are relevant for strontium metal.
Male and female fertility was examined in a reproduction toxicity study in male and female Wistar rat with oral administration of dose levels of 500, 750 and 1000 mg/kg bw/d Strontium ranelate. Male and female fertility as well as reproductive performance was not affected at dose level of up to 1000 mg/kg bw/d corresponding to 345 mg/kg bw/d strontium. Females of the female fertility subgroup were treated for 14 days prior to pairing with untreated males and continued throughout pairing and until day 17 of gestation. Males of the subgroup for male fertility assessment were treated for 28 days prior to pairing with untreated females and continued throughout pairing including the day before sacrifice. Treatment of females of this subgroup started after mating on day six of gestation until day 20 of lactation.
Link to relevant study records
- Endpoint:
- two-generation reproductive toxicity
- Remarks:
- based on generations indicated in Effect levels (migrated information)
- Type of information:
- read-across based on grouping of substances (category approach)
- Adequacy of study:
- key study
- Study period:
- 1997-09-23 to 1998-04-08
- Reliability:
- 2 (reliable with restrictions)
- Justification for type of information:
- Strontium metal is highly reactive and instantly oxidizes upon contact with water. It decomposes completely. During the redox-reaction with water, a strong evolution of hydrogen gas and an immediate precipitation of a white, crystalline solid (i.e. Sr(OH)2) is observed (Sr2+ + 2OH- + H2 (g). The amount of dissolved Sr cations is determined by the solubility of the Sr(OH)2 precipitate. According to OECD guideline 105 (1995) and EU method A.6 (2006), the water solubility of strontium was determined to be 6.74 ± 0.14 g/L under the conditions of the test (flask method under protective gas atmosphere; loading of 41 g Sr/L, at 20.0 ± 1.0 °C, pH >13).
Strontium ions are highly mobile, occur only in one valence state (2+), i.e. are not oxidized or reduced, and do not form strong complexes with most inorganic and organic ligands (Krupka et al. 1999. EPA 402-R-99-004B; Salminen et al. 2015; Carbonaro and Di Toro. 2007. Geochim Cosmochim Acta 71 3958–3968; Carbonaro et al. 2011. Geochim Cosmochim Acta 75: 2499-2511 and references therein). Thus, it may be assumed that systemic toxicological effects (not local) are related to the strontium ion. Therefore, the assessment of the systemic toxicity of strontium is based on elemental strontium concentrations. Read-across of systemic toxicity data available for soluble strontium substances is applied since the strontium ions determine the toxicological potential of strontium. - Qualifier:
- according to guideline
- Guideline:
- other: ICH Harmonised Tripartite Guideline - Detection of toxicity to reproduction for medical products, Washington June 24, 1993; ICH Harmonized Tripartite Guideline, Addendum: Toxicity to male fertility, July 1996.
- GLP compliance:
- yes
- Limit test:
- no
- 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 weeksENVIRONMENTAL 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
- 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/vPREPARATION 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 bwThe 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.
- Remarks:
- Doses / Concentrations:0, 500, 750 and 1000 mg/kg/dayBasis:actual ingested
- No. of animals per sex per dose:
- Subgroup B: 25 females/25 males each for the control group and the 3 dose levelsSubgroup 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
- 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: NoBODY WEIGHT: YesTime schedule for examinations:1) Before pairing:- treated males of subgroup B were weighed once weekly2) 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 inclusiveFOOD 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: NoThese 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 GENERATIONSTANDARDISATION OF LITTERS- Performed on day 4 postpartum: noPARAMETERS EXAMINEDThe 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 postpartumPostnatal 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 autopsyF2 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 examinationHISTOPATHOLOGY / 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 femalesThe other organs, including the macroscopic anomalies, were kept in fixative without any further investigation.
- Postmortem examinations (offspring):
- F1 GENERATION AND THEIR F2 LITTERSSACRIFICE- 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
- 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: oestrous cycle:
- not specified
- Reproductive function: sperm measures:
- no effects observed
- Reproductive performance:
- no effects observed
- 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)
- 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
- 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.
- 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
- 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
- Reproductive effects observed:
- not specified
- 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.
Reference
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
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 345 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- The study to examine the effects of strontium ranelate on male and female fertility (K. Momburg, 2001) of rats is regarded as relevant and reliable to evaluate the effects of strontium; i.e., a reliable GLP reproduction toxicity study with strontium ranelate (Oral reproduction toxicity study in the Wistar rat (male and female fertility/embryo-fetal and postnatal development) addressing embryo-fetal and pre-/postnatal development of offspring according to the ICH guideline on Detection of Toxicity to Reproduction of Medicinal Products, June 24, 1993, and with the ICH Guideline Addendum: Toxicity to male fertility, July 1996.
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
According to a final decision on a compliance check (CCH-D-2114527898-30-01/F), an extended one-generation reproductive toxicity test with strontium nitrate according to OECD 443 is ongoing (see study records s_Nextreat_OECD TG422 and s_Nextreat_OECD TG443). Results are expected by the 4th quarter of 2023 and will be included without delay in the REACH dossier for strontium metal.
In a supporting study, no histological changes for uterus, overies, testes and prostate after the administration of up to and including 4800 ppm strontium chloride hexahydrate were observed in an sub-chronic oral repeated dose toxicity study OECD 408 (Kroes, 1977). The authors reported a NOAEL of 4800 ppm, which is equivalent to a strontium concentration of 157.7 mg/kg bw.
Effects on developmental toxicity
Description of key information
Read-across:Strontium metal completely dissolves upon contact and during the reaction with water under a strong evolution of gas and an immediate precipitation of a white crystalline solid, presumably strontium hydroxide (Sr(OH)2). The water solubility test of strontium (OECD TG 105) indicates a high dissolution from strontium metal (6.74 g/L at 20°C, determined as dissolved strontium, separated by filtration from undissolved test item and precipitates), a rapid formation of Sr2+ + 2OH- + H2 (g) and a corresponding increasing solution pH to a pH > 13. Strontium ions are highly mobile, occur only in one valence state (2+), i.e. are not oxidized or reduced, and do not form strong complexes with most inorganic and organic ligands (Krupka et al. 1999. EPA 402-R-99-004B; Salminen et al. 2015; Carbonaro and Di Toro. 2007. Geochim Cosmochim Acta 71 3958–3968; Carbonaro et al. 2011. Geochim Cosmochim Acta 75: 2499-2511 and references therein). Thus, it may be assumed that systemic toxicological effects (not local) are related to the strontium ion. Therefore, the assessment of the systemic toxicity of strontium is based on elemental strontium concentrations. Read-across of systemic toxicity data available for soluble strontium substances is applied since the strontium ions determine the toxicological potential of strontium. Read-across from strontium chloride, strontium ranelate and strontium nitrate to strontium is possible since as a first surrogate for bioavailability, the solubility of a test substance in water may be applied. All substances (strontium chloride, strontium ranelate, strontium nitrate and strontium) are soluble (> 5 g/L). Hence, it can be concluded that adverse effects observed with strontium chloride, strontium ranelate or strontium nitrate are due to the presence of the strontium ion and are relevant for strontium metal.
Based on the outcome of a prenatal developmental toxicity assay, strontium chloride hexahydrate did induce adverse developmental effects in a GLP OECD TG414 study performed in pregnant Wistar rats from gestation day 6 (GD6) to gestation day 19 (GD19) administered by oral gavage at dose levels of 105, 420, and 1681 mg SrCl2.6H2O/kg bw/d (Nextreat, 2021). These developmental effects were seen in the absence of maternal toxicity effects.
No mortality or clinical signs in the dams were observed under the conditions of this study.
Based on the results of thyroid hormones analysis, thyroid weights, histopathological evaluation of thyroids and the measurement of anogenital distance of fetuses, no endocrine disruptor effect was observed in the study.
Treatment at 1681 mg SrCl2.6H2O/kg bw/day was associated with maternal toxicity effects, such as reduced body weight and reduced body weight gain and reduced food consumption of the dams, as well as with developmental toxicity effects, such as increased number of dead fetuses, increased post implantation loss, increased intrauterine mortality and growth retardation of the fetuses. Consequently, this led also to a reduced litter weight and reduced gravid uterine weight.
In addition, the test item at this dose level caused ossification disturbances on the whole skeletal system in the fetuses, which was expressed as: incomplete ossification of the whole skull, unossified sternebrae, wavy and marked wavy ribs, unossified thoracic, lumbar and sacral vertebrae, unossified metacarpal and metatarsal bones, unossified pubis and/or ischium, misshapen, bent and/or short scapula, bent and/or short clavicula, humerus, radius, ulna, femur, tibia and fibula.
Treatment at 420 mg SrCl2.6H2O/kg bw/day no evidence of adverse maternal effect was observed, but was associated with developmental toxicity effects, such as increased number of dead fetuses and post implantation loss. In addition, the test item at this dose level caused skeletal variations in the fetuses such as incomplete ossification of the skull, wavy and marked wavy ribs, and skeletal malformations, identified as bent and/or short scapula, humerus, femur, tibia and fibula.
No adverse maternal or developmental toxicity effect was observed at 105 mg SrCl2.6H2O/kg bw/day.
LOAELmaternal toxicity: 1681 mg SrCl2.6H2O/kg bw/day
NOAELmaternal toxicity: 420 mg SrCl2.6H2O/kg bw/day
LOAELdevelopmental toxicity: 420 mg SrCl2.6H2O/kg bw/day
NOAELdevelopmental toxicity: 105 mg SrCl2.6H2O/kg bw/day
LOAELmaternal toxicity: 552.4 mg Sr/kg bw/day
NOAELmaternal toxicity: 138.1 mg Sr/kg bw/day
LOAELdevelopmental toxicity: 138.1 mg Sr/kg bw/day
NOAELdevelopmental toxicity: 34.5 mg Sr/kg bw/day
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- read-across based on grouping of substances (category approach)
- Adequacy of study:
- key study
- Study period:
- ARRIVAL OF ANIMALS: 26 May 2021 START OF MATING: 01 July 2021 START OF EXPERIMENT: 01 July 2021 START OF DOSING: 07 July 2021 END OF DOSING: 28 July 2021 END OF EXPERIMENT: 29 July 2021
DATE OF FINAL REPORT: 18 November 2021 - Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Strontium metal is highly reactive and instantly oxidizes upon contact with water. It decomposes completely. During the redox-reaction with water, a strong evolution of hydrogen gas and an immediate precipitation of a white, crystalline solid (i.e. Sr(OH)2) is observed (Sr2+ + 2OH- + H2 (g). The amount of dissolved Sr cations is determined by the solubility of the Sr(OH)2 precipitate. According to OECD guideline 105 (1995) and EU method A.6 (2006), the water solubility of strontium was determined to be 6.74 ± 0.14 g/L under the conditions of the test (flask method under protective gas atmosphere; loading of 41 g Sr/L, at 20.0 ± 1.0 °C, pH >13).
Strontium ions are highly mobile, occur only in one valence state (2+), i.e. are not oxidized or reduced, and do not form strong complexes with most inorganic and organic ligands (Krupka et al. 1999. EPA 402-R-99-004B; Salminen et al. 2015; Carbonaro and Di Toro. 2007. Geochim Cosmochim Acta 71 3958–3968; Carbonaro et al. 2011. Geochim Cosmochim Acta 75: 2499-2511 and references therein). Thus, it may be assumed that systemic toxicological effects (not local) are related to the strontium ion. Therefore, the assessment of the systemic toxicity of strontium is based on elemental strontium concentrations. Read-across of systemic toxicity data available for soluble strontium substances is applied since the strontium ions determine the toxicological potential of strontium. - Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- OECD Guidelines for Testing of Chemicals No. 414 (2018)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: VWR International Kft., 20E284111
- Purity: 100%
- Sr content: 32.86%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15-25°C) - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Han:WIST rat
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Toxi-Coop Zrt., H-1122 Budapest, Magyar Jakobinusok tere 4B
- Age at study initiation: Young adult rats, nulliparous and non-pregnant, 13 weeks old
- Weight at study initiation: Did not exceed ± 20% of the mean weight at onset of treatment and were in the range of 201-249 g
- Fasting period before study: no
- Housing: The animals were housed individually in T3H polycarbonate cages. “SAFE 3/4-S-FASERN” certified wooden chips (batch number: 03027210315, expiry date: 15 March 2024) produced by J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) and “Sizzle pet” nest material (batch number: 201016/02, expiry date: 01 December 2023) produced by LBS (Serving Biotechnology) Ltd. (Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH, United Kingdom) were available to animals during the study. Fresh bedding was provided for the animals as frequently as appropriate/practical, but at least twice weekly. Copies of the Certificates of Analyses are archived with the raw data.
Cages were arranged in such a way that possible effects due to cage placement are minimised.
- Diet (e.g. ad libitum): ad libidum
- Water (e.g. ad libitum): ad libidum
- Acclimation period: At least 36 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24 °C (target: 22 ± 3 °C)
- Humidity (%): 39 – 64 % (target: 30-70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): lighting period 12 hours daily, from 6.00 a.m. to 6.00 p.m.
The minimum and maximum temperature and relative humidity values were recorded daily during the study. - Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- distilled water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
formulation PREPARATION: by gavage administration
- Rate of preparation (frequency): The test item was formulated daily in the vehicle (distilled water), as an aqueous solution at the appropriate concentrations.
VEHICLE
- Justification for use and choice of vehicle: distilled water, based on results of pilot developmental study performed at the Test Facility with the test item
- Lot/batch no. (if required): Parma Produkt, batch number: 2104-5527 / 2105-5513
expire data: 27 October 2021 / 13 November 2021
A constant volume of 10 mL/kg body weight was administered to all dose groups, including the control. The individual volume of the treatment was based on the most recent individual body weight of the animals.
The dose levels were selected by the Sponsor in consultation with the Study Director, based on the results from pilot developmental toxicity study [3] with the aim of inducing toxic effects (developmental and/or maternal toxicity) but no death or suffering at the highest dose, and to obtain a No Adverse Effect Dose Level (NOAEL) at the lowest dose.
The oral route was selected since it is one of the routes of administration requested by the regulatory authorities, and it is considered suitable to provide the exposure required for this developmental toxicology study.
The control or test item formulations were administered to mated, sperm positive assumed pregnant female rats daily by oral gavage on a 7 days/week basis, approximately at similar times with minor variations as practical, from GD6 to GD19. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Stability of the test item in the vehicle was assessed during the analytical method validation: Validation of an Analytical Method for the Determination of Strontium in Water, FumoPrep Ltd. (Study code: FPBSTUDY-241-VAL1). 2021 (GLP). In that study, the formulation samples in the 10-170 mg/mL concentration range (using distilled water as vehicle) were proven as being stable for at least 8 days when stored at room temperature 20±5°C.
Concentration and homogeneity of the dosing formulations were determined twice during the study (13 and 27 July 2021).
Based on the results, all test item formulations were shown to be homogeneous and they were found to be in the range of 91.1 to 105.5% of nominal concentrations, as detailed in the table below. No test item was detected in the negative (vehicle) control sample. Based on these results, formulations were considered suitable for the study purposes.
See Table 1: Analytical results, in 'Any other information on material and methods' - Details on mating procedure:
The oestrus cycle of female animals was examined a day before start of pairing. After acclimation, the females were paired according to their oestrus cycle with males in the morning for approximately 2 hours (1 male : 1 female) until at least 22 sperm positive females/group were attained. The mating of siblings were avoided and the mating partners of the females were randomly chosen. After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of sperm in the vaginal smear was considered as evidence of copulation (GD0). Sperm positive females were separated and caged individually.
In case of unsuccessful pairing, the oestrus cycle determination was extended until the next pro-oestrus and the female was re-mated.
The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that group averages of the body weight were as similar as possible. Females inseminated by the same male were evenly distributed across groups.- Duration of treatment / exposure:
- The control or test item formulations were administered to mated, sperm positive assumed pregnant female rats daily by oral gavage on a 7 days/week basis, approximately at similar times with minor variations as practical, from GD6 to GD19
- Duration of test:
- Acclimatisation period: at least 36 days
Gestation days (GD):
-1: Oestrus cycle evaluation during mating period (up to the day of sperm-positive vaginal smear, defined as GD0)
0-5: No dose administration
6-19: Daily treatment
20: Caesarean section and necropsy - Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 105 mg/kg bw/day (nominal)
- Remarks:
- Sr: 34.5 mg/kg bw/d
- Dose / conc.:
- 420 mg/kg bw/day (nominal)
- Remarks:
- Sr: 138.1 mg/kg bw/d
- Dose / conc.:
- 1 681 mg/kg bw/day (nominal)
- Remarks:
- Sr: 552.4 mg/kg bw/d
- No. of animals per sex per dose:
- 89 sperm positive female animals; 22, 22, 22 and 23 mated female animals in the control, low, mid and high dose groups, respectively. The surplus sperm positive animals are assigned to the high dose group, because this seems to be the most important from the toxicological point of view.
30 male animals for mating - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director, based on the results from pilot developmental toxicity study with the aim of inducing toxic effects (developmental and/or maternal toxicity) but no death or suffering at the highest dose, and to obtain a No Adverse Effect Dose Level (NOAEL) at the lowest dose. Study: Strontium chloride hexahydrate - Oral (Gavage) Dose Range Finding Toxicity Study in Pregnant Hannover Wistar Rats – Nextreat Laboratories, Study code: N20016-414P, 2021
- Rationale for animal assignment (if not random): The mating of siblings were avoided and the mating partners of the females were randomly chosen. The sperm-positive, assumed pregnant females were allocated to each experimental group (on each mating day) in such a way that group averages of the body weight were as similar as possible. Females inseminated by the same male were evenly distributed across groups. 22, 22, 22 and 23 sperm positive mated female animals in the control, low, mid and high dose groups, respectively. The surplus sperm positive animals are assigned to the high dose group, because this seems to be the most important from the toxicological point of view.
- Fasting period before blood sampling for (rat) dam thyroid hormones: no
- Time of day for (rat) dam blood sampling: before 11:30 AM, to avoid the diurnal variation
Mated assumed pregnant Hannover Wistar rats were treated as follows: see table 2 in 'Any other information on material and methods' - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: morbidity and mortality, twice daily (at the beginning and end of each working day)
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations daily. Detailed clinical observations were made on all animals at the onset of treatment (GD6) then on GD13 and at necropsy (GD20). Observation was performed on the skin, fur, eyes and mucous membranes, occurrence of secretions and excretions, autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as presence of clonic or tonic movements, stereotypes, bizarre behaviour was also observed. Special attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
On GD13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).
BODY WEIGHT: Yes
- Time schedule for examinations: on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20
Body weight gain of pregnant females was calculated for each interval, including
GD 0-6, GD 6-20 and GD 0-20.
FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Food consumption was measured on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption of pregnant females was calculated for each interval, including GD0-6, GD6-20 and GD0-20.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:
The dams’ viscera were examined macroscopically for any structural abnormalities or pathological changes. All gross findings are retained in 10% buffered formalin solution.
The weight of the thyroid gland with parathyroid glands for all dams was measured with a precision of 0.001 g. As a paired organ, it was weighed individually, but reported together. Absolute organ weights were measured, and relative paired organ weights to the body weights were calculated and reported. The organs are retained in 10% buffered formalin solution.
The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. If no implantation sites were evident but corpora lutea were present, the uterus was stretched and hold in front of a light source to clearly identify the implantation sites. Uteri that appeared non-gravid were further examined to confirm the non-pregnant status by Salewski staining method.
The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percent of pre- and post-implantation losses was calculated. The degree of resorption (early, late) was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.
Histopathology:
All thyroid and parathyroid glands and the retained kidneys - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
The ovaries and uterus were removed and the pregnancy status ascertained. The uterus including the cervix was weighed and examined for early and late embryonic or foetal deaths and for the number of live foetuses. If no implantation sites were evident but corpora lutea were present, the uterus was stretched and hold in front of a light source to clearly identify the implantation sites. Uteri that appeared non-gravid were further examined to confirm the non-pregnant status by Salewski staining method.
The number of corpora lutea in each ovary and implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death were counted, the number and percent of pre- and post-implantation losses was calculated. The degree of resorption (early, late) was described in order to estimate the relative time of death of the conceptus. The placentas were examined macroscopically.
Animals were checked for early delivery or abortion also, but no signs of these phenomenons were noted. - Blood sampling:
- - Plasma: Yes
- Serum: Yes
- Volume collected: at least 1 mL each (serum, plasma)
For thyroid hormone analysis, blood samples were taken by cardiac puncture into two tubes, one containing lithium heparin as anticoagulant (to be processed for plasma) and one containing no anticoagulant (to be processed for serum) at study termination (at least 1 mL each). The samples were taken before 11:30 AM each day, to avoid the diurnal variation of the hormone concentration among animals. Blood samples from non-pregnant females were not pooled with pregnant dams.
The processed plasma was divided into two aliquots and the serum into three aliquots. The first plasma aliquots were assessed for T4, the first and second serum aliquots for T3 and TSH, respectively.
Thyroxine (T4)
The T4 levels were determined from the plasma samples by IDEXX Catalyst One Chemistry Analyzer (Catalyst Total T4 Test slide, batch: 708094, exp.date: 11 May 2022
measurable range: 6.4-257.4 nmol/L).
Triiodothyronine (T3)
Total Triiodothyronine (T3) ELISA kit (Cat. No.: RCD025R, produced by BioVendor – Laboratorní medicína a.s., batch number: X21-090, exp.date: 28 February 2022, measurable range: 0.2-10.0 ng/mL) was used for T3 level determination from the serum samples. BMG FLUOstar Omega Microplate Reader was used for the optical density measurements.
Thyroid-stimulating hormone (TSH)
Rat Thyroid Stimulating Hormone (TSH) ELISA kit (Cat. No.: abx156194, produced by Abbexa Ltd., batch number: E2108560W, exp.date: 31 March 2022, measurable range: 24-15000 pg/mL) was used for TSH level determination from the serum samples.
BMG FLUOstar Omega Microplate Reader was used for the optical density measurements.
The samples were analysed on the day of sampling or stored in a freezer until analysis. - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Anogenital distance of all live rodent pups: Yes
After ensuring humane death, each fetus was weighed individually (accuracy ±0.01 g) and subjected to external examination. The sex of each fetus was determined and the anogenital distance was determined of all live fetuses. Thereafter, the fetuses were individually identified; approximately half of each litter was subjected to detailed visceral examination, and the other half was processed for skeletal examination.
Particular attention was paid to the reproductive tract which was examined for signs of altered development. External fetal sex (as determined by gross examination) was compared with internal (gonadal) sex in all fetuses (examined for both skeletal and soft tissue malformations). In addition, indication of incomplete testicular descent/cryptorchidism was noted in male fetuses.
For the fetuses subjected to visceral examination, the abdominal and thoracic region were opened and the thymus and great arteries were freshly examined by means of a dissecting microscope. The rest of the body was fixed in Sanomiya mixture, then after fixation the body was micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method.
For the fetuses subjected to skeletal examination, the abdominal region was opened and the viscera and skin of fetuses were removed and the cadaver was fixed in alcian-blue - acetic acid - ethanol mixture. After fixation in isopropanol, the skeletons were stained by KOH-Alizarin red-S method and examined by means of a dissecting microscope.
All abnormalities (variations, malformations and retardations) found during the fetal examinations were recorded. - Statistics:
Descriptive statistics (mean, standard deviation, %versus control) were calculated for the continuous variables. Frequency and percentage were calculated for categorical variables in Microsoft Excel.
Statistical analysis was performed for the continuous variables using an automated decision tree within the R software.
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate.
If either of the Shapiro-Wilk or Levene tests shows significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis is required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
The Chi-squared test was used for non-continuous data. For datasets with values ≤ 5 the Fisher-exact test was applied.
Nine sperm positive, but non-pregnant females (#1522 in the control, #2521, #2522 in the Low dose, #3520, #3521, #3522 in the Mid dose, #4521, #4522, #4523 in the High dose group) and one female in the control group (#1521) with ≤ 5 implantation sites were excluded from statistical analysis; however, the individual report tables contains all data of these animals.
The limit for growth retarded fetuses was calculated from the average body weight of the vehicle control fetuses. A fetus was considered as growth retarded if the deviation from the mean control values was greater than minus two fold standard deviation of all control fetuses. Cut-off: 2.6945 g.- Indices:
- Caesarean Section and Necropsy Data:
- Number of corpora lutea: mean ± S.D.
- Number of implantations: mean ± S.D.
- Number and percentage of live fetuses: mean ± S.D.
- Number and percentage of intrauterine mortality: mean ± S.D.
Classified according to time of death: preimplantation loss, postimplantation loss, early and late embryonic loss as well as fetal death.
- Preimplantation loss: %, group mean
(Number of corpora lutea-Number of implantations x100)/Number of corpora lutea
- Postimplantation loss: %, group mean
(Number of implantations-Number of live fetuses x100)/Number of implantations
Fetal Data:
- Total numbers of litters
- Number of fetuses (alive and dead) per sex and dam
- Sex distribution: %, group mean
(Number of male (female) fetuses x100)/Number of fetuses
- Fetal body weight (male, female and combined sexes) (accuracy 0.01 g): mean * S.D.
- Anogenital distance, (accuracy 0.01 mm) group mean by sex ± S.D.
- External, visceral and skeletal abnormalities/litter: %, group mean
(Number of fetuses with abnormality x100)/Number of fetuses - Historical control data:
- Historical control data for OECD 414 studies from the test facility Nextreat laboratories Kft. were provided.
Data included:
Body weight, body weight gain, gravid uterine weight, corrected body weight and corrected body weight gain
Food consumption
Thyroid and parathyroid weight, Thyroid hormones (T3, T4, TSH)
Intrauterine mortality and viable fetuses data
Fetal examination finding
Mean, SD, Min, Max, n, CV(%)
n litters: 41
Historical control data for OECD TG 414: see Table 7 in attached background material: Summary tables_N20016-414.docx - Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Compared to the control, lower mean body weight could be observed in the high dose dams (1681 mg/kg bw/day) during the treatment period, which attained statistically significance on GD20 (-4.3 %; p˂0.05). However, if the body weight was corrected for gravid uterine weight the lower body weight did not attain statistical significance (-2.6 %).
Stagnation of body weight development was observed in 3 of 20 dams and minimal body weight loss was noted in seven out of twenty dams between GD6 and GD8 in the high dose group (1681 mg/kg bw/day), furthermore one animal lost body weight of 10.6 % between GD6 and GD10. Body weight development was statistically significantly lower for this period when compared to control animals (-113.2%, p˂0.05). In addition, statistically significantly lower mean body weight gain could be observed between GD18 and GD20 (-27.5%, p˂0.05). The cumulative body weight gain values from GD0 to GD20 and the corrected mean body weight gain value were also significantly lower (-13.8 % and -15.7 %; p<0.01 and p<0.05, respectively) than control. These differences could be attributed to the treatment with the test item.
Summary of body weight, body weight gain, gravid uterine weight, corrected body weight and corrected body weight gain: see Table 1: Summary of body weight, body weight gain, gravid uterine weight, corrected body weight and corrected body weight gain in attached background material: Summary tables_N20016-414.docx - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- In the high dose group (1681 mg/kg bw/day) the food consumption was statistically significant lower than in the control group between GD6-8 (-14.5 %; p˂0.01), between GD18-20
(-8.1 %; p˂0.05) and the cumulative mean food consumption value between GD6-20 (-6.6 %) and between GD0-20 (-6.4 %) was also statistically significant lower than controls (p˂0.05, respectively).
The food consumption of the low and mid dose groups (105 and 420 mg/kg bw/day, respectively) was comparable with the control group.
Summary of food consumption: see Table 2 in attached background material: Summary tables_N20016-414.docx - Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Endocrine findings:
- no effects observed
- Description (incidence and severity):
- thyroid weights and hormones (TSH, T4, T3)
No test item-related difference was observed for thyroid and parathyroid gland weight between the control group and treatment groups (105, 420 and 1681 mg/kg bw/day). Neither absolute nor relative thyroid and parathyroid gland weights of any of the treatment groups were statistically significantly different from that of the control group.
No test item-related differences were observed for thyroid hormones TSH, T4 and T3 between the control group and the treatment groups. The mean TSH, T4 and T3 levels were comparable in the control and test item treated groups.
Summary of thyroid weight and thyroid hormone values: see Table 4 in attached background material: Summary tables_N20016-414.docx - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Gravid uterine weight
In the high dose group (1681 mg/kg bw/day), the gravid uterine weight was statistically significantly lower (-12.4%; p<0.05) compared to the control group.
No test item related changes were observed in gravid uterine weight of the low and mid dose group animals (105 and 420 mg/kg bw/day) when compared to control data.
Summary of gravid uterine weight: see Table 1 in attached background material: Summary tables_N20016-414.docx
thyroid weights
No test item-related difference was observed for thyroid and parathyroid gland weight between the control group and treatment groups (105, 420 and 1681 mg/kg bw/day). Neither absolute nor relative thyroid and parathyroid gland weights of any of the treatment groups were statistically significantly different from that of the control group.
Summary of thyroid weight: see Table 4 in attached background material: Summary tables_N20016-414.docx - Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- No test item-treatment related macroscopic or microscopic findings were noted for any dose level (105, 420 and 1681 mg/kg bw/day) at necropsy.
No structure/morphology effect was noted in the thyroid and parathyroid glands during histopathological evaluation.
Unilateral or bilateral pelvic dilatation was observed in one control (1510), in one low dose (2518), in one mid dose (3510) and in two high dose (4511, 4516) female dams. As the observed pyelectasia were without degenerative, inflammatory or other histopathological lesions and being a common background change in rats, these findings are not considered as test item-related effect.
Summary of pathological findings: see Table 3 in attached background material: Summary tables_N20016-414.docx - Histopathological findings: neoplastic:
- no effects observed
- Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- effects observed, treatment-related
- Description (incidence and severity):
- In maternal reproductive parameters, there were no test item-related differences in the number of corpora lutea, in pre-implantation loss, in number of implantations, in early embryonic loss and in late embryonic loss between the control group and the treatment groups (105, 420 and 1681 mg/kg bw/day).
The absolute and litter mean number and the percentage of dead fetuses were statistically significant higher in the mid and high dose groups (p<0.05 or p<0.01) compared to the control and were out of the historical control data. The differences were dose-dependent and considered test item related.
Dose-dependent increase in the post implantation loss was also observed in the mid and high dose group, which was not statistically significant upon analysis of the litter mean number and percentage, but attained statistical significance by the analysis of the absolute numbers (p<0.05) compared to the control.
The pattern was similar in case of total intrauterine mortality, however, it was not statistically significant upon analysis of the litter mean number and absolute numbers, but attained statistical significance by the analysis of the percentage of intrauterine mortality of the high-dose group (p<0.05) compared to the control. The difference was considered to be test item-related.
The total and mean numbers of viable fetuses were slightly lower in the mid and high dose groups compared to the control, but the differences did not attain statistically significance.
There was no test item related effect in the maternal reproductive parameters in the low dose group.
Summary of intrauterine evaluation:see Table 1 in Any other information on results incl. tables, and see Table 5 in attached background material: Summary tables_N20016-414.docx - Total litter losses by resorption:
- not specified
- Early or late resorptions:
- effects observed, treatment-related
- Description (incidence and severity):
- In maternal reproductive parameters, there were no test item-related differences in the number of corpora lutea, in pre-implantation loss, in number of implantations, in early embryonic loss and in late embryonic loss between the control group and the treatment groups (105, 420 and 1681 mg/kg bw/day).
The absolute and litter mean number and the percentage of dead fetuses were statistically significant higher in the mid and high dose groups (p<0.05 or p<0.01) compared to the control and were out of the historical control data. The differences were dose-dependent and considered test item related.
Dose-dependent increase in the post implantation loss was also observed in the mid and high dose group, which was not statistically significant upon analysis of the litter mean number and percentage, but attained statistical significance by the analysis of the absolute numbers (p<0.05) compared to the control.
The pattern was similar in case of total intrauterine mortality, however, it was not statistically significant upon analysis of the litter mean number and absolute numbers, but attained statistical significance by the analysis of the percentage of intrauterine mortality of the high-dose group (p<0.05) compared to the control. The difference was considered to be test item-related.
The total and mean numbers of viable fetuses were slightly lower in the mid and high dose groups compared to the control, but the differences did not attain statistically significance.
There was no test item related effect in the maternal reproductive parameters in the low dose group.
Summary of intrauterine evaluation:see Table 1 in Any other information on results incl. tables, and see Table 5 in attached background material: Summary tables_N20016-414.docx - Dead fetuses:
- effects observed, treatment-related
- Description (incidence and severity):
- Dose-dependent increase of total intrauterine mortality, however, it was not statistically significant upon analysis of the litter mean number and absolute numbers, but attained statistical significance by the analysis of the percentage of intrauterine mortality of the high-dose group (p<0.05) compared to the control. The difference was considered to be test item-related.
Summary of intrauterine evaluation:see Table 1 in Any other information on results incl. tables, and see Table 5 in attached background material: Summary tables_N20016-414.docx - Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- Eighty-nine sperm positive females were included in the study (22, 22, 22 and 23 in the control, low, mid and high dose group, respectively). The number of confirmed pregnant, evaluated dams in the dose groups treated at 0, 105, 420 and 1681 mg/kg/day was 20, 20, 19 and 20, respectively. In the control group one animal had 5 implantation sites and was excluded from the evaluation.
- Other effects:
- no effects observed
- Description (incidence and severity):
- No macroscopic abnormalities were observed in the placentas in any of the experimental groups in the study.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 138.1 mg/kg bw/day
- Based on:
- element
- Remarks:
- Sr
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 552.4 mg/kg bw/day
- Based on:
- element
- Remarks:
- Sr
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 420 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 1 681 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 420 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- dead fetuses
- pre and post implantation loss
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Compared to the control, statistically significant decreased mean fetal weights (-12.5 %) and mean litter weights (-21.8 %) were observed (p<0.01) in the high dose group (1681 mg/kg bw/day). These findings correlated with the lower gravid uterine weight in this group and could be attributed as test item-related effect. No test item related differences in mean fetal weight and mean litter weight were observed for the low and mid dose groups (105 and 420 mg/kg bw/day, respectively).
The total number of body weight retarded fetuses (evaluated as external variation) was statistically significant increased in the high dose group (n = 34; p<0.01), compared to the control (n = 7). This resulted in a higher incidence of fetuses with retarded body weight (23.05 %; p<0.01). Similar reduction was also present if the results were analyzed sex-wise. This correlated with the reduction of the mean fetal weights and considered test item related. No test item related differences were observed for the low and mid dose groups (105 and 420 mg/kg bw/day, respectively).
Summary of body weight of fetuses: see Table 2 in Any other information on results incl. tables, and see Table 6 in attached background material: Summary tables_N20016-414.docx - Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- The total and mean numbers of viable fetuses were slightly lower in the mid and high dose groups compared to the control, but the differences did not attain statistically significance.
Summary of intrauterine evaluation and viable fetuses: see Table 1 in Any other information on results incl. tables, and see Table 5 in attached background material: Summary tables_N20016-414.docx - Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- There was no toxicologically significant difference in the sex distribution of fetuses between of the control and treatment groups.
Summary of sex distribution of fetuses: see Table 2 in Any other information on results incl. tables, and see Table 6 in attached background material: Summary tables_N20016-414.docx - Changes in litter size and weights:
- effects observed, treatment-related
- Description (incidence and severity):
- Compared to the control, statistically significant decreased mean fetal weights (-12.5 %) and mean litter weights (-21.8 %) were observed (p<0.01) in the high dose group (1681 mg/kg bw/day). These findings correlated with the lower gravid uterine weight in this group and could be attributed as test item-related effect. No test item related differences in mean fetal weight and mean litter weight were observed for the low and mid dose groups (105 and 420 mg/kg bw/day, respectively).
Summary of litter weight of fetuses: see Table 2 in Any other information on results incl. tables, and see Table 6 in attached background material: Summary tables_N20016-414.docx - Anogenital distance of all rodent fetuses:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In the high dose group (1681 mg/kg bw/day), the body weight-normalized mean anogenital distance was higher than control mean in both sexes (by 12.7% in males and 11.2% in females) and attained statistical significance (p<0.01). As the absolute anogenital distance values were similar to the controls, these differences could be attributed to the retardation of the body weight of the fetuses and not described as test item related endocrine effect.
No statistically significant difference in body weight-normalized mean anogenital distance or absolute anogenital distance was observed for the low and mid dose groups (105 and 420 mg/kg bw/day).
Summary table: see Table 6 in attached background material: Summary tables_N20016-414.docx - Changes in postnatal survival:
- not examined
- External malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- Malformations:
1681 mg/kg bw/day: External malformations were observed in the forelimbs of the foetuses. Short forelimbs were observed in 30 out of 179 fetuses in the 1681 mg/kg bw/day group (15.8 %; p<0.01) and were graded as test item related malformation. This findings caused statistically significant higher externally malformed fetuses in this group (p<0.01).
420 mg/kg bw/day: No external malformations were observed in foetuses of the 420 mg/kg bw/day groups (0 %).
105 mg/kg bw/day: No external malformations were observed in foetuses of the 105 mg/kg bw/day groups (0 %).
Variations:
No external variations (except for retarded fetal body weight, as described above) could be observed in any of the control or test item treated groups (105, 420 and 1681 mg/kg bw/day).
Summary table of external fetal data: see Table 3 in Any other information on results incl. tables, and Table 6 in attached background material: Summary tables_N20016-414.docx - Skeletal malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- Malformations:
1681 mg/kg bw/day: The test item treatment with the high dose of 1681 mg/kg bw/day caused ossification disturbances in the skeletal system, as follows: whole skull (incomplete ossification), scapula (misshapen, bent and or short), clavicula (bent and/or short), humerus (bent and/or short), radius and/or ulna (bent and/or short), femur (bent and/or short) and tibia and/or fibula (bent and/or short). All these findings were statistically significantly higher than control evaluated in litter mean, percentage mean and absolute numbers (p < 0.01, respectively).
420 mg/kg bw/day: At the mid dose of 420 mg/kg bw/day, skeletal malformations, such as bent and/or short scapula, humerus, femur, tibia and fibula could be observed. The higher incidences of these changes reached statistical significance compared to the control only in total number of bent/and or short humerus and femur (p<0.05). Even though the mean number and percentage of the abnormalities did not attain statistical significance in all cases compared to the control, based on the clear dose dependency and profound test item related effect on the bones of scapula, humerus, femur, tibia and fibula in the high dose, these findings were considered as test item specific malformations.
105 mg/kg bw/day: No skeletal malformations were noted in the low dose group (105 mg/kg bw/day).
Variations:
1681 mg/kg bw/day: The test item treatment with the high dose of 1681 mg/kg bw/day caused ossification disturbances in the skeletal system, as follows: skull bones (incomplete ossification (≤ 3 bones)), sternum (unossified (≥ 4)), ribs (wavy or marked wavy), thoracic vertebrae (unossified (≥ 2)), lumbar vertebrae (unossified (≥ 1)), sacral vertebrae (unossified (≥ 1)), pubis and/or ischium (unossified), metacarpal bones (ossified (< 3) and metatarsal bones (ossified (< 4)). All of these findings were statistically significantly higher than control evaluated in litter mean, percentage mean and absolute numbers and were out of the historical control data (except for skull bones (incomplete ossification (≤ 3 bones)).
420 mg/kg bw/day: Skeletal variations were observed. Compared to the control, statistically significant higher mean (p<0.01), percentage mean and absolute number of incomplete ossification of skull bones (1 to 3 bones) was noted. In addition, the total number of fetuses with wavy and/or marked wavy ribs was also statistically significant higher (p<0.05) than control. All of these findings were out of the historical control range.
105 mg/kg bw/day: A higher incidence of total number of fetuses with wavy ribs was noted, which attained statistical significance (p<0.05) compared to the control. As there were no statistically significant differences in the mean number and in the percentage compared to the control, and as wavy ribs are known as transient and reversible variation [Kast, 1994; 9], and no other statistically significant skeletal abnormalities were noted in this group, this finding was not considered as an adverse effect of the test item.
Summary table of fetal skeletal examination data: see Table 4 in Any other information on results incl. tables, and Table 6 in attached background material: Summary tables_N20016-414.docx - Visceral malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- Malformations:
No visceral malformations were observed in the control and treatment groups (105, 420 and 1681 mg/kg bw/day).
Variations:
1681 mg/kg bw/d: Statistically significant higher mean, percentage and total number of thymic cord was observed in the high dose group (p<0.05), resulting in statistically lower mean number of viscerally intact fetuses (p<0.05). The incidence was above those of the historical control data, therefore the relationship with test item cannot be excluded.
420 mg/kg bw/d: A short brachiocephalic trunk was observed in one female fetus of the mid dose group (420 mg/kg bw/day). This finding was not considered to be test item-related, since it was only observed once in the control group and mid dose group.
0 mg/kg bw/d: A short brachiocephalic trunk was observed in one male fetus of the control group. This finding was not considered to be test item-related, since it was only observed once in the control group and mid dose group.
No other visceral variations were observed in the control group and treatment groups (105, 420 and 1681 mg/kg bw/day).
Summary table of fetal visceral examination data: see Table 5 in Any other information on results incl. tables, and Table 6 in attached background material: Summary tables_N20016-414.docx - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 34.5 mg/kg bw/day
- Based on:
- element
- Remarks:
- Sr
- Sex:
- male/female
- Basis for effect level:
- skeletal malformations
- other: increased number of dead fetuses and post implantation loss and skeletal variations: incomplete ossification of the skull, wavy an marked wavy ribs
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 138.1 mg/kg bw/day
- Based on:
- element
- Remarks:
- Sr
- Sex:
- male/female
- Basis for effect level:
- skeletal malformations
- other: increased number of dead fetuses and post implantation loss and skeletal variations: incomplete ossification of the skull, wavy an marked wavy ribs
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 105 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- skeletal malformations
- other: increased number of dead fetuses and post implantation loss skeletal variations: incomplete ossification of the skull, wavy an marked wavy ribs
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 420 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- skeletal malformations
- other: increased number of dead fetuses and post implantation loss and skeletal variations: incomplete ossification of the skull, wavy an marked wavy ribs
- Key result
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 138.1 other: mg Sr/kg bw/day
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects in the absence of maternal toxicity effects
- Dose response relationship:
- yes
- Relevant for humans:
- yes
- Key result
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 420 other: mg SrCl2.6H2O
- Treatment related:
- yes
- Relation to maternal toxicity:
- developmental effects in the absence of maternal toxicity effects
- Dose response relationship:
- yes
- Relevant for humans:
- yes
- Conclusions:
- NOAELmaternal toxicity: 420 mg SrCl2.6H2O/kg bw/day, 138.1 mg Sr/kg bw/day
NOAELdevelopmental toxicity: 105 mg SrCl2.6H2O/kg bw/day, 34.5 mg Sr/kg bw/day - Executive summary:
In summary, daily administration of the Strontium chloride hexahydrate by oral gavage to pregnant Hannover Wistar rats from gestation day 6 (GD6) to gestation day 19 (GD19) at dose levels of 105, 420 and 1681 mg SrCl2.6H2O/kg bw/day did not result any mortality or clinical signs of the dams under the conditions of this study.
Based on the results of thyroid hormones analysis, thyroid weights, histopathological evaluation of thyroids and the measurement of anogenital distance of fetuses, no endocrine disruptor effect was observed in the study.
Treatment at 1681 mg SrCl2.6H2O/kg bw/day was associated with maternal toxicity effects, such as reduced body weight and reduced body weight gain and reduced food consumption of the dams, as well as with developmental toxicity effects, such as increased number of dead fetuses, increased intrauterine mortality and growth retardation of the fetuses. Consequently, this led also to a reduced litter weight and reduced gravid uterine weight.
In addition, the test item at this dose level caused ossification disturbances on the whole skeletal system in the fetuses, which was expressed as: incomplete ossification of the whole skull, unossified sternebrae, wavy and marked wavy ribs, unossified thoracic, lumbar and sacral vertebrae, unossified metacarpal and metatarsal bones, unossified pubis and/or ischium, misshapen, bent and/or short scapula, bent and/or short clavicula, humerus, radius, ulna, femur, tibia and fibula.
Treatment at 420 mg SrCl2.6H2O/kg bw/day no evidence of adverse maternal effect was observed, but was associated with developmental toxicity effects, such as increased number of dead fetuses and post implantation loss. In addition, the test item at this dose level caused skeletal variations in the fetuses such as incomplete ossification of the skull, wavy and marked wavy ribs, and skeletal malformations, identified as bent and/or short scapula, humerus, femur, tibia and fibula.
No adverse maternal or developmental toxicity effect was observed at 105 mg SrCl2.6H2O/kg bw/day.
Based on the reduces maternal body weight and maternal body weight gain and food consumption at the 1681 mg SrCl2.6H2O/kg bw/day (552.4 mg Sr/kg bw/day) dose level, the NOAEL for maternal toxicity is, as follows:
NOAELmaternal toxicity: 420 mg SrCl2.6H2O/kg bw/day (138.1 mg Sr/kg bw/day)Based on increased post implantation loss and skeletal variations at the 420 mg SrCl2.6H2O/kg bw/day (138.1 mg Sr/kg bw/day) dose level, the NOAEL for foetotoxicity toxicity is, as follows: NOAEL foetotoxicity: 105 mg SrCl2.6H2O/kg bw/day (34.5 mg Sr/kg bw/day)
Based on skeletal malformations at the 420 and 1681 mg SrCl2.6H2O/kg bw/day (552.4 mg Sr/kg bw/day and 138.1 mg Sr/kg bw/day) dose levels, the NOAEL for developmental toxicity is, as follows:
NOAEL developmental toxicity: 105 mg SrCl2.6H2O/kg bw/day (34.5 mg Sr/kg bw/day)
Reference
Table 1: Summary of intrauterine evaluation
Parameters | Dose (mg/kg bw/day) |
| |||
0 | 105 | 420 | 1681 |
| |
Number of evaluated dams | 20 | 20 | 19 | 20 |
|
Mean number of corpora lutea | 11.15 | 11.10 | 11.11 | 10.80 | NS |
Total number of corpora lutea | 223 | 222 | 211 | 216 | NS |
Mean number of implantations | 10.70 | 10.65 | 10.58 | 10.35 | NS |
Total number of implantations | 214 | 213 | 201 | 207 | NS |
Pre-implantation loss, mean | 0.45 | 0.50 | 0.53 | 0.45 | NS |
Pre-implantation loss (%), mean | 3.94 | 4.25 | 4.74 | 4.29 | NS |
Pre-implantation loss, total | 9 | 10 | 10 | 9 | NS |
Early embryonic loss, mean | 0.35 | 0.10 | 0.26 | 0.35 | NS |
Early embryonic loss (%), mean | 3.34 | 0.87 | 3.03 | 3.15 | NS |
Early embryonic loss, total | 7 | 2 | 5 | 7 | NS |
Late embryonic loss, mean | 0.05 | 0.10 | 0.32 | 0.25 | NS |
Late embryonic loss (%), mean | 0.50 | 0.91 | 2.69 | 2.17 | NS |
Late embryonic loss, total | 1 | 2 | 6 | 5 | NS |
Dead fetuses, mean | 0.20 | 0.10 | 0.68* | 0.80* | DU |
Dead fetuses (%), mean | 1.78 | 1.01 | 5.96* | 7.49* | DU |
Dead fetuses, total | 4 | 2 | 13* | 16** | CS |
Post-implantation loss, mean | 0.60 | 0.30 | 1.26 | 1.40 | NS |
Post-implantation loss (%), mean | 5.62 | 2.79 | 11.67 | 12.81 | NS |
Post-implantation loss, total | 12 | 6 | 24* | 28* | CS |
Total intrauterine mortality, mean | 1.05 | 0.80 | 1.79 | 1.85 | NS |
Total intrauterine mortality (%), mean | 9.56 | 7.04 | 16.41 | 17.10* | DU |
Total intrauterine mortality, total | 21 | 16 | 34 | 37 | NS |
Viable fetuses, mean | 10.10 | 10.35 | 9.32 | 8.95 | NS |
Viable fetuses, total | 202 | 207 | 177 | 179 | NS |
Statistical significance compared to control: * = p<0.05, ** = p<0.01
DN: Dunnett’s Multiple Range Test, DU: Dunn test, CS: Chi square test, NS: Statistically not significant compared to control
Table 2: Summary of body weight, litter weight and sex distribution of fetuses
Parameters | Dose (mg/kg bw/day) |
| |||
0 | 105 | 420 | 1681 |
| |
Number of evaluated litters | 20 | 20 | 19 | 20 |
|
Examined fetuses, total | 202 | 207 | 177 | 179 |
|
Mean fetal weight (g) | 3.359 | 3.394 | 3.387 | 2.940** | DN |
Mean body weight of male fetuses (g) | 3.443 | 3.501 | 3.509 | 3.017** | DN |
Mean body weight of female fetuses (g) | 3.281 | 3.288 | 3.293 | 2.879** | DN |
Mean litter weight (g) | 33.9 | 35.11 | 31.35 | 26.51** | DN |
Fetuses with retarded body weight (%), (runts) | 3.15 | 3.60 | 3.11 | 23.05** | DU |
Number of fetuses with retarded body weight | 7 | 8 | 5 | 34** | CS |
% of male fetuses with retarded body weight (D<2 +- SD of control) | 1.7 | 2.5 | 0.0 | 17.2** | DU |
% of female fetuses with retarded body weight (D<2 +- SD of control) | 3.9 | 3.8 | 4.8 | 27.0** | DU |
Ratio of male fetuses (%) | 49.3 | 46.1 | 43.5 | 45.7 | NS |
Ratio of female fetuses (%) | 50.7 | 53.9 | 56.5 | 54.3 | NS |
Table 3: Summary of External Fetal Data
Parameters | Dose (mg/kg bw/day) |
| |||
0 | 105 | 420 | 1681 |
| |
Number of evaluated litters | 20 | 20 | 19 | 20 |
|
Examined fetuses, total | 202 | 207 | 177 | 179 |
|
Externally intact fetuses, mean | 9.8 | 10.0 | 9.1 | 5.9** | DU |
Externally intact fetuses (%), mean | 96.9 | 96.4 | 96.9 | 63.8** | DU |
Externally intact fetuses, total | 195 | 199 | 172 | 118* | CS |
Fetuses with external variation, mean& | 0.4 | 0.4 | 0.3 | 1.6** | DU |
Fetuses with external variation (%), mean& | 3.2 | 3.6 | 3.1 | 20.5** | DU |
Fetuses with external variation, total& | 7 | 8 | 5 | 31** | CS |
Fetuses with external malformation, mean§ | 0.0 | 0.0 | 0.0 | 1.5** | DU |
Fetuses with external malformation (%), mean§ | 0.0 | 0.0 | 0.0 | 15.8** | DU |
Fetuses with external malformation, total§ | 0 | 0 | 0 | 30** | CS |
Malformations |
| ||||
Short forelimbs, mean | 0.0 | 0.0 | 0.0 | 1.5** | DU |
Short forelimbs (%), mean | 0.0 | 0.0 | 0.0 | 15.8** | DU |
Short forelimbs, total | 0 | 0 | 0 | 30** | CS |
Statistical significance compared to control: * = p<0.05, ** = p<0.01
DN: Dunnett’s Multiple Range Test, DU: Dunn test, CS: Chi square test, FE: Fisher-exact test
NS: Statistically not significant compared to control
&: All external variations are resulting from the fetuses with retarded body weight.
§: All external malformations are resulting from the fetuses with short forelimbs.
Table 4: Summary of skeletal fetal data
Parameters | Dose (mg/kg bw/day) |
| |||
0 | 105 | 420 | 1681 |
| |
Number of evaluated litters | 20 | 20 | 19 | 20 |
|
Examined fetuses, total | 105 | 106 | 91 | 95 |
|
Skeletally intact fetuses, mean | 4.7 | 4.5 | 2.7** | 0.0** | DU |
Skeletally intact fetuses (%), mean | 88.7 | 85.6 | 59.3** | 0.0** | DU |
Skeletally intact fetuses, total | 93 | 90 | 52 | 0** | CS |
Fetuses with skeletal variation, mean | 0.6 | 0.8 | 1.7** | 0.1 | DU |
Fetuses with skeletal variation, (%), mean | 10.5 | 14.4 | 34.9** | 1.0 | DU |
Fetuses with skeletal variation, total | 11 | 16 | 33** | 1** | CS |
Fetuses with skeletal malformation, mean | 0.1 | 0.0 | 0.3 | 4.7** | DU |
Fetuses with skeletal malformation (%), mean | 0.9 | 0.0 | 5.8 | 99.0** | DU |
Fetuses with skeletal malformation, total | 1 | 0 | 6* | 94** | CS |
Malformations |
| ||||
Skull |
|
|
|
|
|
Skull bones, incomplete ossification (whole skull), mean | 0.0 | 0.0 | 0.0 | 4.7** | DU |
Skull bones, incomplete ossification (whole skull) (%), mean | 0.0 | 0.0 | 0.0 | 99.0** | DU |
Skull bones, incomplete ossification (whole skull), total | 0 | 0 | 0 | 94** | CS |
Pectoral girdle |
|
| |||
Scapula misshapen, bent and/or short, mean | 0.0 | 0.0 | 0.2 | 4.7** | DU |
Scapula misshapen, bent and/or short (%), mean | 0.0 | 0.0 | 3.2 | 99.0** | DU |
Scapula misshapen, bent and/or short, total | 0 | 0 | 3 | 94** | CS |
Clavicula bent and/or short, mean | 0.0 | 0.0 | 0.0 | 3.9** | DU |
Clavicula bent and/or short (%), mean | 0.0 | 0.0 | 0.0 | 81.7** | DU |
Clavicula bent and/or short, total | 0 | 0 | 0 | 77** | CS |
Forelimb |
|
| |||
Humerus bent/and or short, mean | 0.0 | 0.0 | 0.2 | 4.7** | DU |
Humerus bent/and or short (%), mean | 0.0 | 0.0 | 4.1 | 99.0** | DU |
Humerus bent and/or short, total | 0 | 0 | 4* | 94** | CS |
Radius and/or ulna bent/and or short, mean | 0.0 | 0.0 | 0.0 | 4.5** | DU |
Radius and/or ulna bent or short (%), mean | 0.0 | 0.0 | 0.0 | 94.8** | DU |
Radius and/or ulna bent and/or short, total | 0 | 0 | 0 | 90** | CS |
Hindlimb |
|
| |||
Femur bent/and or short, mean | 0.0 | 0.0 | 0.3 | 4.6** | DU |
Femur bent or short (%), mean | 0.0 | 0.0 | 4.9 | 97.4** | DU |
Femur bent and/or short, total | 0 | 0 | 5* | 92** | CS |
Tibia and/or fibula bent/and or short, mean | 0.0 | 0.0 | 0.1 | 4.6** | DU |
Tibia and/or fibula bent or short (%), mean | 0.0 | 0.0 | 1.7 | 97.2** | DU |
Tibia and/or fibula bent and/or short, total | 0 | 0 | 2 | 92** | CS |
Parameters | Dose (mg/kg bw/day) |
| |||
0 | 105 | 420 | 1681 |
| |
Variations |
| ||||
Skull |
|
| |||
Skull bones, incomplete ossification (≤ 3 bones), mean | 0.2 | 0.1 | 1.7** | 0.1 | DU |
Skull bones, incomplete ossification (≤ 3 bones) (%), mean | 4.2 | 1.3 | 31.4** | 1.0 | DU |
Skull bones, incomplete ossification (≤ 3 bones), total | 4 | 1 | 32** | 1 | CS |
Sternum |
|
| |||
Sternebrae, unossified (≥ 4), mean | 0.3 | 0.2 | 0.4 | 4.2** | DU |
Sternebrae, unossified (≥ 4), (%), mean | 4.6 | 3.0 | 7.2 | 88.9** | DU |
Sternebrae, unossified (≥ 4), total | 5 | 3 | 7 | 83** | CS |
Ribs |
|
| |||
Wavy, mean | 0.0 | 0.3 | 0.2 | 0.4** | DU |
Wavy (%), mean | 0.0 | 5.8 | 3.5 | 8.8** | DU |
Wavy, total | 0 | 6* | 4* | 7** | CS |
Marked wavy, mean | 0.0 | 0.1 | 0.3 | 4.1** | DU |
Marked wavy (%), mean | 0.0 | 0.9 | 5.8 | 84.9** | DU |
Marked wavy, total | 0 | 1 | 5* | 82** | CS |
Vertebrae |
|
| |||
Thoracal, unossified (≥2), mean | 0.3 | 0.2 | 0.1 | 2.7** | DU |
Thoracal, unossified (≥2) (%), mean | 5.0 | 3.0 | 1.7 | 56.1** | DU |
Thoracal, unossified (≥2), total | 5 | 3 | 2 | 53** | CS |
Lumbar, unossified (≥1), mean | 0.1 | 0.0 | 0.3 | 3.1** | DU |
Lumbar, unossified (≥1) (%), mean | 1.3 | 0.0 | 4.9 | 65.1** | DU |
Lumbar, unossified (≥1), total | 1 | 0 | 5 | 61** | CS |
Sacral, unossified (≥1), mean | 0.3 | 0.2 | 0.4 | 4.3** | DU |
Sacral, unossified (≥1) (%), mean | 5.4 | 3.0 | 8.4 | 90.6** | DU |
Sacral, unossified (≥1), total | 6 | 3 | 8 | 86** | CS |
Pelvic girdle |
|
| |||
Pubis and/or ischium, unossified, mean | 0.2 | 0.2 | 0.3 | 4.1** | DU |
Pubis and/or ischium, unossified (%), mean | 2.9 | 3.0 | 5.5 | 85.7** | DU |
Pubis and/or ischium, unossified, total | 3 | 3 | 6 | 81** | CS |
Forelimb |
|
| |||
Metacarpal bones, ˂ 3 ossified, mean | 0.1 | 0.1 | 0.3 | 3.4** | DU |
Metacarpal bones, ˂ 3 ossified (%), mean | 1.3 | 1.0 | 4.5 | 72.3** | DU |
Metacarpal bones, ˂ 3 ossified, total | 1 | 1 | 5 | 67** | CS |
Hindlimb |
|
| |||
Metatarsal bones, ˂ 4 ossified, mean | 0.1 | 0.1 | 0.1 | 3.6** | DU |
Metatarsal bones, ˂ 4 ossified (%), mean | 1.3 | 1.0 | 0.9 | 75.6** | DU |
Metatarsal bones, ˂ 4 ossified, total | 1 | 1 | 1 | 71** | CS |
Statistical significance compared to control: * = p<0.05, ** = p<0.01
DN: Dunnett’s Multiple Range Test, DU: Dunn test, CS: Chi square test, FE: Fisher-exact test
NS: Statistically not significant compared to control
Table 5: Summary of Visceral Fetal Data
Parameters | Dose (mg/kg bw/day) |
| |||
0 | 105 | 420 | 1681 |
| |
Number of evaluated litters | 20 | 20 | 19 | 20 |
|
Examined fetuses, total | 97 | 101 | 86 | 84 |
|
Viscerally intact fetuses, mean | 4.8 | 5.0 | 4.3 | 4.0* | DU |
Viscerally intact fetuses (%), mean | 99.0 | 98.3 | 96.2 | 95.7 | NS |
Viscerally intact fetuses, total | 96 | 99 | 82 | 80 | NS |
Fetuses with visceral variation, mean | 0.1 | 0.1 | 0.2 | 0.2 | NS |
Fetuses with visceral variation (%), mean | 1.0 | 1.7 | 3.8 | 4.4 | NS |
Fetuses with visceral variation, total | 1 | 2 | 4 | 4 | NS |
Fetuses with visceral malformation, mean | 0.0 | 0.0 | 0.0 | 0.0 | NS |
Fetuses with visceral malformation (%), mean | 0.0 | 0.0 | 0.0 | 0.0 | NS |
Fetuses with visceral malformation, total | 0 | 0 | 0 | 0 | NS |
Variations |
| ||||
Thymic cord, mean | 0.0 | 0.10 | 0.16 | 0.20* | DN |
Thymic cord (%), mean | 0.0 | 1.7 | 2.5 | 4.4* | DU |
Thymic cord, total | 0 | 2 | 3 | 4* | FE |
Short brachiocephalic trunk, mean | 0.1 | 0.0 | 0.1 | 0.0 | NS |
Short brachiocephalic trunk (%), mean | 1.0 | 0.0 | 1.3 | 0.0 | NS |
Short brachiocephalic trunk, total | 1 | 0 | 1 | 0 | NS |
Statistical significance compared to control: * = p<0.05, ** = p<0.01
DN: Dunnett’s Multiple Range Test, DU: Dunn test, CS: Chi square test, FE: Fisher-exact test
NS: Statistically not significant compared to control
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 34.5 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- High quality (GLP compliant guideline study)
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
In a supporting study, the effects of strontium ranelate on the embryo-fetal and pre-/postnatal development was investigated in a GLP compliant toxicity study according to the ICH guideline on Detection of Toxicity to Reproduction of Medical Products, June 24, 1993 (K. Momberg 2001). Groups of pregnant female rats were either exposed from day 14 prior to mating with untreated males until day 17 of gestation (group A) or after mating with treated males from day 6 of gestation until day 20 of lactation (group B). Embryo-fetal development was evaluated in group A and effects on parturition and pre-/post natal development in group B. Animals received the test compound by oral gavage at dose levels of 500, 750 and 1000 mg/kg bw/d. Control animals were treated with the vehicle. Additional subgroups (group C and D) were included for toxicokinetic investigations. No treatment-related maternal toxicity was observed in group A and B females. No effects on embryo/-fetal development were observed beside an increase in the frequency of delays in skeletal ossification and structural abnormalities (way ribs, bent bones, shortened and thickened humerus and misshapen clavicle) at all dose levels. Although the percentage of affected fetuses by a delay of ossification was higher in the treated groups than in the control, there was no dose-response relationship and values were within the normal historical control range of this strain. In addition, the structural abnormalities observed in twenty day old fetuses were completely reversible in seven to eight week old F1 animals, because all these anomalies were no longer visible during postnatal development as shown by X-ray radiography. These transitory findings were regarded as not effecting basical development of offspring but were related to retarded ossification. It was also discussed that these findings appear not relevant in the case of human exposure during organogenesis, because the skeletal development at parturition in humans is much more advanced than in rodent species. In conclusion, these findings were not considered as true congenital skeletal malformations, because they were reversible and were therefore considered as variations without any functional consequences. Although in the context of the study, the lowest dose level of 500 mg/kg bw/d strontium ranelate corresponding to 172 mg/kg bw/d strontium does not represent an NOAEL, but a LOAEL, the findings at this dose level were of transient nature and regarded as not relevant for human embryo/fetal development, and thus did not provide evidence of an adverse effect on the development of offspring. The structural abnormalities were also not present in theoral embryo-fetal development study with strontium ranelate (K. Momberg 1999) in rabbits at up to 1500 mg/kg bw/d. In the post-natal development part of the study, a delay of incisor eruption was seen on lactation day 11 mainly in offspring of the 1000 mg/kg bw/d group, but had no negative effect on animal growth which is directly related to feed intake and use of teeth for gnawing of feed pellets. In addition, the relevance of these effects for humans was deemed as low, because tooth development in man differ from rat with incisor eruption occurring after weaning at 6-24 months after birth. Therefore, the NOAEL for postnatal development was established at the highest dose group of 1000 mg/kg bw/d strontium ranelate corresponding to 345 mg/kg bw/d strontium.
In addition, in the study published by Lansdown et al. (1972) (see 7.8.2 "s_Lansdown_1972) groups of 3 female Wistar rats were treated subcutaneously with 25, 50, 100 or 200 mg strontium nitrate/kg bw/d in 1 ml distilled water from day 9 to 19 of pregnancy when they were killed. Control animals received distilled water only. The progeny from strontium-treated mothers did not differ from that of controls in size or body weight. The litter sizes were normal and the number of resorption sites was not increased. No histological changes were detected in the soft tissues and the skeletal tissues exhibited the characteristic degree of ossification for 19-day old rat fetuses. The results indicate that high doses of strontium nitrate (up to and including 200 mg/kg bw/d s. c.) are not teratogenic. Thus, the dose of 200 mg/kg bw/d can be considered as a NOAEL for developmental toxicity corresponding to an external dose of about 83 mg/kg bw/d strontium to female rats. However, the study was conducted in a small number of females (p=3) per group and a limited number of parameters was evaluated. In addition, the subcutaneous route of administration is not relevant route of exposure for risk assessment purposes. Nevertheless, the study by Lansdown et al. (1972) on pregnant rats is regarded as appropriate to support the evaluation of the effects of strontium on embryo-fetal development.
Justification for classification or non-classification
Based on the outcome of a prenatal developmental toxicity assay and a sub-chronic repeated dose toxicity test, strontium does not impair sexual function or fertility . Therefore, no classification is required for fertility.
Based on the outcome of a prenatal developmental toxicity study according to the OECD 414 guideline (Nextreat, 2021), clear evidence was shown of adverse effects on development induced by strontium chloride hexahydrate in the absence of maternal toxic effects. These adverse effects were: skeletal malformations (bent and/or short scapula, humerus, femur, tibia, and fibula), next to increased number of dead fetuses and post implantation loss, and skeletal variations (incomplete ossification of the skull, wavy and marked wavy ribs). A clear dose-response was observed.
LOAEL developmental = 420 mg SrCl2.6H2O/kg bw/d (138.1 mg Sr/kg bw/d)
NOAEL developmental = 105 mg SrCl2.6H2O/kg bw/d (34.5 mg Sr/kg bw/d)
LOAEL maternal = 1681 mg SrCl2.6H2O/kg bw/d (552.4 mg Sr/kg bw/d) based on decreased body weight and body weight gain, decreased food consumption of the dams
NOAEL maternal = 420 mg SrCl2.6H2O/kg bw/d (138.1 mg Sr/kg bw/d)
Based on the information from the key study OECD TG 414 study (Nextreat, 2021), the hazard classification for SrCl2.6H2O and Sr is re-evaluated and updated.
According to the Guidance on the application of CLP criteria, and the clear adverse effects on the development of the offspring, with a clear dose-response relationship, and in the absence of maternal toxicity, the substance SrCl2.6H2O and Sr meet the criteria laid down in Annex I Section 3.7.2.1.1 for classification as toxic to reproduction category 1B 'Presumed human reproductive toxicant' (evidence for classifcation is from animal data).
Label elements in accordance with table 3.7.3 of Annex I: 3.7.4.1:
Classification: 1B
GHS Pictograms: GHS08: Health Hazard
Signal word: Danger
Hazard statement: H360D May damage the unborn child
Additional information
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