Strontium bis(2-ethylhexanoate)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Strontium chloride hexahydrate

EC Number:

600-046-7

Cas Number:

10025-70-4

Molecular formula:

Cl2-Sr.6H2-O

IUPAC Name:

Strontium chloride hexahydrate

Test material form:

solid

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)

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

105 mg/kg bw/day (nominal)

Remarks:

Sr: 34.5 mg/kg bw/d

Doses / concentrations 3

Dose / conc.:

420 mg/kg bw/day (nominal)

Remarks:

Sr: 138.1 mg/kg bw/d

Doses / concentrations 4

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'

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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.

Effect levels (maternal animals)

1

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

2

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

3

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

4

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

5

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

Results (fetuses)

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

Effect levels (fetuses)

1

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

2

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

3

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

4

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

Overall developmental toxicity

1

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

2

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

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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)