Strontium hydroxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

An Extended One Generation Reproductive toxicity study with F2 generation and developmental neurotoxicity was performed on Strontium chloride hexahydrate administered by oral gavage to Sprague-Dawley rats. There was no systemic and reproductive toxicity in the parental generation and in their offspring of both sexes. Their was no systemic toxicity, no developmental neurotoxicity and no neuro-histopathology changes in the adult F1 generation. The NOAEL of Strontium chloride hexahydrate was considered to be 1000 mg/kg bw/day for systemic toxicity, reproduction, and developmental toxicity and neurotoxicity endpoints.

Based on the read-across from Strontium chloride hexahydrate, Strontium hydroxide is considered to be not toxic to reproduction and the corresponding NOAEL is considered to be 456.2 mg Sr(OH)2/kg/day (calculated using the molar mass ratio of SrCl2.6H2O and Sr(OH)2).

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - with F2 generation and developmental neurotoxicity (Cohorts 1A, 1B with extension, 2A and 2B)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 October 2021 to 26 October 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

25 June 2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS :

- Premating exposure duration for parental (P0) animals: 2 weeks

- Basis for dose level selection:
From a “Oral Reproduction Toxicity Study in Wistar rat (male and female fertility/embryo-fetal and postnatal development)” study conducted with the dose levels of 500, 750 and 1000 mg/kg bw/day according to the ICH guideline on Detection of Toxicity to Reproduction of Medical Products (June 24, 1993), the estimated NOAEL for a read-across chemical (i.e., strontium ranelate) for both parental female general/reproductive toxicity and postnatal development of F1 generation was 1000 mg/kg bw/day. The results of this study reveal that, there were no treatment-related maternal toxicity and also no effects on embryo/-fetal development, beside an increase in frequency of delays in skeletal ossification and structural abnormalities at all the 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 20-day old fetuses were completely reversible in 7- to 8-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 basic 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.
A sub-chronic dietary study in Wistar rats with strontium chloride hexahydrate at dose levels of 75, 300, 1200, and 4800 ppm for 90 days was conducted. No differences in clinical chemistry were noted, except of an indication of increased alkaline phosphatase activity in the highest dose group. Urinalysis showed no differences in the groups. The levels of Ca, Mg and P in blood were similar for all dose levels and the Ca/P ratio was constant. In males, thyroid weights were significantly increased in the 1200 and 4800 ppm groups. Although no clear explanation of this finding could be given, it was regarded as treatment-related. In females, pituitary weights were significantly decreased in the 300 and 4800 ppm group, but not in the 1200 ppm group. Glycogen depletion of liver was noted in the highest dose group. However, this might be caused by stress, starvation, or diurnal rhythm and not by treatment with the test substance. Detectable amounts of strontium in blood and muscle were only noticed at the dose of 4800 ppm. The strontium content in bone was increased at all dose levels having a constant level from 4 weeks onwards (steady-state level). No treatment-related changes were observed in the X-ray photographs and on histopathological examination except, slight changes in the liver (glycogen depletion) and thyroid (activation). Thus, no rachitic changes occurred up to the highest dose of 4800 ppm. Considering the increased concentrations of strontium in the bone as a non-toxic effect, a NOAEL of 300 ppm SrCl2 can be derived from this study based on the weight changes of thyroids at the doses of 1200 ppm (LOAEL) and 4800 ppm and thyroid activation at 4800 ppm. According to these estimations, the NOAEL of 300 ppm strontium chloride corresponds to a dose of 22.5 mg/kg bw/day (equal to 12.4 mg Sr/kg bw/day).
Based on the above-mentioned toxicity data of structurally similar or read across chemicals the dose levels of 250, 500 and 1000 mg/kg bw/day were selected as low, mid and high dose levels.

- Inclusion of Cohort 1B and developmental neurotoxicity Cohorts 2A and 2B:
The Cohorts 1A, 1B (with extension to F2 generation), 2A and 2B has been selected for F1 generation assessments based on the available toxicity information of test item.
ECHA considers that concerns in relation with reproductive toxicity are observed in available studies. More specifically, there are indications of one or more modes of action related to endocrine disruption because in the sub-chronic study performed with the analogue substance strontium chloride (EC no 233-971-6), following a protocol similar or equivalent to OECD TG 408 (Kroes et al., 1977), the relative thyroid weights were statistically significantly increased in males at 1200 ppm (corresponding approximately to 50 mg/kg bw/day) and 1400 ppm (by 33% (p>0.01) and 26% (p>0.001), respectively). There were no treatment-related changes in body weights in the study.
In the same study, the relative prostate weights were statistically significantly decreased in males at 75 and 1200 ppm (by 28% (p>0.01) and 21% (p>0.05), respectively), and the relative pituitary weights were statistically significantly decreased in females at 75 and 1200 ppm (by 16% (p>0.05) and 24% (p>0.01), respectively). Although there was no clear dose-response for the decrease in relative prostate and pituitary weights, the findings are statistically significant and thus support triggering of the Extended one generation reproductive toxicity study at Annex IX.
ECHA considers that the criteria in Column 1, Annex IX, section 8.7.3 are met because existing information shows evidence of deviations in hormonally sensitive organs in both sexes without notable general toxicity.

- Termination time for F2: on the day of weaning (PND 21).

- Route of administration:
The vehicle and test item formulations were administered through oral (gavage) route as it is the probable route of exposure to human. The oral gavage route is the preferred route of administration as administration of test item is more effective and precise in gavage studies than in dietary studies. Hence, oral gavage route was selected for dose administration.

- Choice of species:
Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

- Choice of number of animals:
Total Number of Animals - Parental (P) Generation: 200 (100 Males + 100 Females)
Total Number of Animals - First Filial (F1) Generation: A total of 240 males and 240 females were selected and randomly assigned to 4 cohorts on the day of weaning (PND 21).
- Cohort 1A: 160 (80 Males + 80 Females) - [one male and one female per group representing 20 litters from P generation].
- Cohort 1B: 160 (80 Males + 80 Females) - [one male and one female per group representing 20 litters from P generation].
- Cohort 2A: 80 (40 Males + 40 Females) - [one male or one female per group representing 20 litters from P generation].
- Cohort 2B: 80 (40 Males + 40 Females) - [one male or one female per group representing 20 litters from P generation].

- Choice of vehicle:
The test item is soluble in distilled water and forms a clear solution at the concentration of 100 mg/mL, the highest dose concentration selected for the study as per in-house solubility test results. Hence, distilled water was selected as vehicle for the test item formulations.

Specific details on test material used for the study:

- Lot No. : SZBF1770
- Purity: 100.1 %
- Batch Produced by: Honeywell Specialty Chemicals Seelze GmbH, Wunstorferstrasse 40, 30926 Seelze, Germany
- Date of Manufacture : 26 June 2015, retested on June 2021
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility of the test material: The test item is soluble in distilled water and forms a clear solution at the concentration of 100 mg/mL, the highest dose concentration selected for the study.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee, CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA
- Age at receipt (P): 9 to 10 weeks
- Weight at study initiation: (P) Males: 269.20 to 342.75 g; Females: 200.37 to 241.97 g.
- Housing: Animals were housed in a standard polysulphonate cage (size: L 43 x B 28 x H 21 cm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Processed corncob granules were provided as bedding material.
- Diet: Altromin Maintenance diet for rats and mice manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum to the rats throughout the experimental period.
- Water (ad libitum throughout the acclimatization and experimental period): Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: 5 days before initiation of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): targeted temperature 22 ± 3°C, actual range 19.3 to 23.1°C.
- Humidity (%): targeted range between 30 to 70%, actual range 47 to 64%.
- Air changes (per hr): 12 to 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark cycle.

IN-LIFE DATES:
From 03 November 2021 (experimental starting date) to 06 June 2022 (in-life end date).

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Stability and homogeneity: The stability and homogeneity of the test item in dose formulations was established before initiation of the treatment. The stability was determined at room temperature after 24 and 48 hours at dose formulations of 10 and 100 mg/mL in water. It was concluded that the test item in dose formulations was stable up to 48 hours. Homogeneity and dose formulation analysis for dose concentration verification was done during weeks 1, 9, 17, 25 and 30 of the treatment periods. The results were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤ 10%.
- Preparation: The test item was mixed with the vehicle to get desired concentrations of 25, 50 and 100 mg/mL of test item for low, mid and high dose groups, respectively. The test item formulations were prepared within the established stability conditions.

VEHICLE
- Amount of vehicle: 10 mL/kg bw/day.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- The pairs were separated without further continuation of mating in the event of no evidence of mating until 2 weeks of cohabitation period.
- After successful mating, each pregnant female was caged individually during gestation and lactation periods.
- Any deviations from standard protocol: none

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling and analysis of formulations was performed on Week 1, 9, 17, 25 and 30 of the treatment periods.
Approximately 5 mL of samples were collected in duplicates from the vehicle control, low, mid and high dose concentrations.
Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.
The result table is provided in the attached document "Dose formulation analysis".

Duration of treatment / exposure:

Parental (P) Generation Animals:
- Pre-mating: The P animals (both males and females) were treated for a period of 2 weeks during pre-mating period.
- Mating and Post mating:
- Males: The P males were treated for a period of 2 weeks during mating period and with a total period of 12 to 14 weeks until termination (a total treatment period for P males was 87 to 95 days including pre-mating, mating and post-mating periods).
- Females: The P females were treated for a period of 2 weeks during mating period, throughout gestation and lactation periods up to weaning of F1 animals (a total treatment period for non-pregnant P females was 59 to 72 days including pre-mating, mating and until their 26th day from the day of evidence of mating).

F1 Generation Cohort 1A Animals: Direct dosing by oral gavage for the selected C1A males and females was begun from weaning (PND 21) and continued until scheduled necropsy PND 105 [14-weeks age].

F1 Generation Cohort 1B Animals: Direct dosing by oral gavage for the selected C1B males and females was begun from weaning (PND 21) and continued until scheduled necropsy.
- Pre-mating: The C1B animals (both males and females) were treated for a period of 10 weeks during pre-mating period.
- Mating and post-mating:
- Males: The C1B males were treated for a period of 2 weeks during mating period and during post-mating period with a total period of 10 weeks until termination.
- Females: The C1B females were treated for a period of 2 weeks during mating period, throughout gestation and lactation periods up to the weaning sacrifice of F2 pups.

F1 Generation Cohort 2A Animals: Direct dosing by oral gavage for the selected C2A males and females was begun from weaning (PND 21) and continued until scheduled necropsy PND 83.

F1 Generation Cohort 2B Animals: No direct dosing was done for selected C2B males and females and all C2B animals were sacrificed on their PND22.

Frequency of treatment:

Once daily, 7 days each week

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle control

Dose / conc.:

250 mg/kg bw/day

Remarks:

Low dose

Dose / conc.:

500 mg/kg bw/day

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

High dose

No. of animals per sex per dose:

Parental (P) Generation: 25 Males + 25 Females / Group

First Filial (F1) Generation:
- Cohort 1A: 20 Males + 20 Females / Group
- Cohort 1B: 20 Males + 20 Females / Group
- Cohort 2A: 10 Males + 10 Females / Group
- Cohort 2B: 10 Males + 10 Females / Group

Control animals:

yes, concurrent vehicle

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: the animals were fasted overnight.

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS
Twice daily (pre and post dose) throughout the experiment till sacrifice for treatment related clinical signs.
Twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS
On day 1 before treatment and weekly thereafter during experiment.

BODY WEIGHT
- Pre-mating period: on day 0 and once weekly thereafter.
- Cohabitation (mating) period: once weekly.
- Gestation period: on gestation day (GD) 0, 5, 7, 9, 11, 13, 15, 17 and 20.
- Lactation period: on lactation day (LD) 1, 4, 7, 14 and 21.

FOOD CONSUMPTION
- Pre-mating period: once weekly.
- Cohabitation (mating) period: not measured.
- Gestation period: For all the P females during GD 0 to 7, 7 to 14 and 14 to 20.
- Lactation period: For all the P females during LD 1 to 4, 4 to 7, 7 to 14 and 14 to 21.

CLINICAL PATHOLOGY INVESTIGATIONS
- After overnight fasting, blood was collected from 10 randomly selected males and 10 randomly selected females from each group at termination, through retro-orbital plexus puncture method under Isoflurane anaesthesia.
- Haematology, coagulation and clinical chemistry parameters are detailed in the attached document "Details of the observations".

URINALYSIS
- Urine was collected from 10 randomly selected males and females per dose group at termination.
- The animals were kept in urine collection cages overnight and were not given access to feed, but water was provided ad libitum during their stay in urine collection cages.
- Parameters: volume, appearance, color, specific gravity, pH, blood, protein, glucose.
- The remaining urine was centrifuged at 1500 rpm for 3 minutes and subjected to microscopic examination for urine sediments.

THYROID HORMONE LEVELS
Serum T4 and TSH levels were estimated using commercially available ELISA kits from 10 randomly selected males and 10 randomly selected females from each group.

OESTRUS CYCLICITY
See below

REPRODUCTIVE PERFORMANCE EVALUATION
- Mating and fertility index (see below)
- Pre-coital interval (see below)
- Gestation length (see below)
- Gestation index (see below)
- Parturition index (see below)
- Pregnancy index (see below)

DELIVERY AND LITTER OBSERVATIONS
- Live birth index (%) per litter on PND 1 (LD 1)
- Pup survival index (%) per litter between LD 1 to 4, 5 to 7, 8 to 14 and 15 to 21
- Sex ratio (m/f) on LD 1, 4, 7, and 13

Oestrous cyclicity (parental animals):

Observed daily in all P and F1 (cohort 1B) generation females on the following occasions:
- for a period of 2 weeks during pre-mating period before initiation of cohabitation;
- during cohabitation period until evidence of mating;
- at termination (on the day of necropsy).

Observed daily in all F1 (cohort 1A) generation females on the following occasions:
- after the onset of vaginal patency, until the first cornified smear is recorded, in order to determine the time interval between these two events,
- for a period of 2 weeks, starting from PND 75 till sacrifice

Sperm parameters (parental animals):

Parameters examined in all P and F1 (cohort 1A) males:

At termination, testes and epididymis weights (unilateral) were recorded for all P males. One testis and one epididymis (left) were preserved for histopathological examination during necropsy. The other testis and epididymis (right) were used for sperm parameters. The epididymis meant for sperm parameters was used for enumeration of cauda epididymis sperm reserves. In addition, semen from the cauda epididymis was collected for evaluation of sperm motility and morphology.
Sperm motility (%) was evaluated immediately after sacrifice. The percentage of progressively motile sperms was determined subjectively. Samples to evaluate sperm morphology were taken from the suspension used for the sperm motility. Sperm samples were examined as fixed and 200 spermatozoa per sample were classified as either normal (both head and mid-piece/tail appear normal) or abnormal.
The other testis collected from each animal meant for spermatid head counts was preserved at -20ºC±4 ºC and used to evaluation of homogenization resistance spermatid head counts to calculate daily sperm production per animal.

The details of sperm parameters analysis and estimations followed are as mentioned below:

- Sperm motility:
A small segment of distal cauda epididymis was stabbed and transferred to 2 mL of pre-warmed (37±1ºC) DPBS (Dulbecco's Phosphate Buffered Saline). The sperms were allowed to disperse for 5 minutes in DPBS media and sperm suspension was loaded into haemocytometer chamber.
The number of non-motile sperms in the four corner squares of WBC counter were counted under 10X magnification. All the sperms were allowed to become immotile by keeping the haemocytometer on ice pack. The total number of sperms in the same four corner squares of WBC counter were counted. Similar procedure was followed once again as a second trail for each animal. Total no. of non-motile sperms and total sperms for both trials were summed, and the percentage motility was calculated as mentioned below.
Sperm motility (%): (Total no. of sperms - Total no. of non-motile sperms)/Total no. of sperms X 100

- Sperm morphology:
A volume of 1 mL of sperm suspension used to determine sperm motility was transferred to a pre-labelled tube and the volume was made up to 4 mL with DPBS along with 5 to 6 drops of 1 % Eosin Y. The sample was incubated at room temperature for 45 to 60 minutes. One drop of the stained sperm suspension was placed on microscope slide and the smear was prepared. Three slides were prepared for each animal. Slides were air dried overnight and mounted using DPX mountant.
Two hundred sperms per animal were examined for morphology using a normal compound microscope at 10X/40X magnification and morphological abnormalities of head, tail, neck was recorded.

- Testicular spermatid head counts:
The stored testis (right) was thawed to room temperature on the day of evaluation. The Tunica albuginea was removed from testis by longitudinal incision and by peeling away with forceps. One gram of testicular parenchyma was weighed and recorded. The weighed testicular parenchyma was minced and rinsed with 5 mL of Saline Triton-X solution (ST) and subjected for homogenization until formation of uniform suspension. After homogenization the homogenate was made up to 50 mL and allowed to settle and to disappear the foam. The homogenate was loaded into both chambers of the hemacytometer and facilitate to settle all the heads to a common focal plane. Sperm heads under 40X magnification were counted in the four corner squares and middle square in both the chambers of the haemocytometer and the counts were recorded. The total number of sperm heads observed in 5 squares were added for each chamber. A total of 4 chamber counts were performed.
The mean sperm head count obtained from 4 chamber counts was calculated for each animal. Sperm heads count per gram testis was calculated using the following formulae.
Sperm heads per testis (No.): (Mean sperm head count X Dilution volume (50 mL) ) / Volume of counting chamber (0.0001)
Sperm heads per gram of testis (No.): Sperm heads count per testis / Weight of testicular parenchyma (in grams)

The daily sperm production for each animal was calculated by dividing the number of Sperm heads counted by the amount of time (in days) during spermatogenesis while these cells are resistant to homogenization (6.10 days for rats).
Daily sperm production (No.): Spermatid count per gram of testis / Days of resistant to homogenization
The sperm heads count per gram of testis and daily sperm production are presented in millions (X 10^6).

Litter observations:

STANDARDISATION OF LITTERS (F1 and F2)
- Performed on day 4 postpartum: yes
- The litter size was adjusted to 5 of each sex.
- All the sacrificed pups were subjected for gross pathological examination.

OBSERVATION AT BIRTH
F1 and F2 pups: number of pups born (dead and live) in each litter, sex of each pup, sex ratio (male/female), externally visible abnormalities (including cleft palate; subcutaneous haemorrhages; abnormal skin colour or texture; presence of umbilical cord; lack of milk in stomach; presence of dried secretions), live birth index (%) per litter.

OBSERVATION DURING LACTATION
F1 and F2 pups:
- Behavioral changes, number of alive, dead and cannibalized pups.
- Occurence of post-natal developmental land marks: pinna unfolding (point of pinna detachment from the head) from PND1, hair coat development (appearance of fur on the body) from PND 2, incisor eruption from PND 6, eye opening (separation of upper eye lid from the lower eye lid in both eyes ) from PND 10, testes descent (full descend of both testes into the scrotal sac) from PND 19.
- Responses for sensory reflexes: surface righting reflex from PND 4, auditory startle reflex from PND 10, air righting reflex from PND 17.
- Anogenital distance (AGD) measurement: on PND 4, in each live pup from each litter.
- Appearance of nipples/areolae: on PND 13, in each male pup from each litter.

POST-WEANING OBSERVATIONS
- Cage side observation (all animals): Twice daily throughout the experiment till sacrifice for treatment related clinical signs (only once daily on PND 21 and 22 for Cohort 2B animals) and twice daily for mortality and morbidity.
- Detailed clinical examination (animals from Cohorts 1A, 1B and 2A): Once weekly after weaning until sacrifice.

BODY WEIGHT
- Pup weight: on PND 1, 4, 7, 14 and 21
- Cohorts 1A and 2A: on the day of weaning (initiation of treatment), every 2 days for the first 2 weeks following weaning, then once weekly throughout the experimental period until sacrifice.
- Cohort 1B: on the day of weaning (initiation of treatment), once weekly during the pre-mating period, on the day of sexual maturation (i.e. balano-preputial separation for males and vaginal patency for females), once weekly during the cohabitation (mating) period, then on GD 0, 5, 7, 9, 11, 13, 15, 17 and 20 and on LD 1, 4, 7, 14 and 21.
- Cohort 2B: on PND 21 and 22.

FOOD CONSUMPTION
- Cohorts 1A and 2A: once weekly.
- Cohort 1B: once weekly during the pre-mating period, then during GD 0 to 7, 7 to 14 and 14 to 20, and during LD 1 to 4, 4 to 7, 7 to 14 and 14 to 21. The food consumption was not measured during the cohabitation period.

SEXUAL MATURATION
- Cohorts 1A, 1B and 2A
- For females: the vaginal opening was evaluated daily starting from PND 22
- For males: the balano-preputial separation was evaluated daily starting from PND 35.

CLINICAL PATHOLOGY INVESTIGATIONS (COHORT 1A)
- After overnight fasting, blood was collected from 10 randomly selected males and 10 randomly selected females from each group at termination, through retro-orbital plexus puncture method under Isoflurane anaesthesia.
- Haematology, coagulation and clinical chemistry parameters are detailed in the attached document "Details of the observations".

URINALYSIS (COHORT 1A)
- Urine was collected from 10 randomly selected males and females per dose group at termination.
- The animals were kept in urine collection cages overnight and were not given access to feed, but water was provided ad libitum during their stay in urine collection cages.
- Parameters: volume, appearance, color, specific gravity, pH, blood, protein, glucose.
- The remaining urine was centrifuged at 1500 rpm for 3 minutes and subjected to microscopic examination for urine sediments.

THYROID HORMONE LEVELS
- Surplus F1 pups: T4 on PND 4 / T4 and TSH on PND 21
- Cohort 1A: Serum T4 and TSH levels were estimated using commercially available ELISA kits from 10 randomly selected males and 10 randomly selected females from each group.

REPRODUCTIVE TOXICITY
- C1A and C1B females: Oestrus cyclicity (see above).
- C1B females: Reproductive performance (see Parental observations and examinations and Reproductive indices)
- C1B females: Delivery and litter observations (see Parental observations and examinations and Offspring viability indices)

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY (Cohort 2A):
- Auditory startle test: on PND 25
The day of testing was counterbalanced across treated and control groups. Each session consisted of
10 trials (5 blocks of 10 trials) and the mean response amplitude was calculated. The auditory startle reflex was noted as a sudden flinch or cessation of ongoing movement followed by auditory stimulus. The whole-body response such as flinching, jumping, and freezing of activity was considered as positive signal for meeting the criterion for startle response.
An auditory startle test was performed using a separate isolation chamber/area with known dimensions using a fixed sound level meter. Animals were placed in an isolated chamber and were avoided from vicinity to any of the external objects or sounds which might have interfere the stimulus. The sound level meter was adjusted to level range of Hi (60 to 130 dB), time weighting of FAST (125mS) set on slow response (“A” weighting) for measuring the intensity of the acoustic stimuli. The background noise in the procedure room was recorded on each day before conducting the test. An acoustic sound was made using a clicker to deliver noise and the produced sound level was recorded each time. The frequency of sound and auditory startle response for each trail were recorded for each animal.
- Neurological/Functional examination: on PND 64.
a) Home Cage Observations: Animals in the cage were observed for home cage posture, respiratory pattern, involuntary clonic and tonic movements, vocalization and palpebral closure and the scores were recorded.
b) Handling Observations: Animals were observed for their ease of removal from cage, ease of handling, red and crusty deposits around eyes/nose/mouth, lacrimation, salivation, fur appearance, piloerection, eye prominence and muscle tone.
c) Open Field Observations: Animals were placed in an open field arena and observed for their mobility, gait, arousal, rearing, urination, defecation, tonic involuntary movement, stereotype behavior and grooming.
d) Sensory Observations: Animals were observed for approach response, auditory response, touch response, pupil response, tail pinch response and righting reflexes.
e) Neuromuscular Observations:
Hind limb foot splay: Animals were dropped onto a recording paper sheet from a height of approximately 30 cm with painted paws to record the hind limb foot splay.
Grip strength assessment: Animals were assessed for hind limb and fore limb grip strength using a grip strength meter.
Motor activity assessment: Animals were assessed for motor activity using a locomotor activity monitoring system.
f) Physiological Observation (rectal temperature): The physiological temperature of the animals was recorded using a calibrated digital thermometer.

Postmortem examinations (parental animals):

SACRIFICE
All the P and C1B males and females were fasted overnight prior to scheduled necropsy. The animals were euthanized using CO2 exposure followed by exsanguination.
- Male animals: All the surviving P and C1B males were sacrificed after completion of mating procedure.
- Maternal animals: All the surviving littered P and C1B females were sacrificed on LD 22. The females not confirmed with mating were sacrificed on 26th day from the day of termination of cohabitation process. The females evidenced with mating but not littered were sacrificed on 26th day from the day of confirmation of mating.

GROSS NECROPSY
Gross pathological examination was performed on all the P and C1B males and females sacrificed terminally.
A special attention was paid to the organs of the reproductive system of both sexes.

HISTOPATHOLOGY / ORGAN WEIGHTS
- For the P generation:
The organs collected, weighed and preserved for histopathological examination are listed in the attached document "Details of the observations".
Histopathological examination were conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) sacrificed at termination. The histopathological investigations were not extended to the lower dose groups as there were no treatment related effects noted at the high dose level during microscopic examinations.
Additionally, reproductive organs of all animals suspected of reduced fertility (those that failed to mate, conceive and sire) from all the dose groups were subjected to histopathological examination.

- For the C1B generation:
The reproductive and endocrine organs collected, weighed and preserved are listed in the attached document "Details of the observations". They were processed at block stage and were not examined for histopathology as there were no suspected reproductive or endocrine toxicants or no equivocal results obtained from C1A.

UTERI OBSERVATIONS
The number of implantation sites was recorded during conduct of necropsy for each P and C1B dam/litter .
♦ Post-Implantation Loss (%) = (No. of Implantations - No. of viable pups) / (No. of implantations) x 100
♦ Post-implantation Loss (No.) = (No. of implantations) - (No. of live births)
♦ Total postnatal Loss (%) = (Total no. of pups dead/cannibalized during postnatal period) / (Total no. of live pups delivered) x 100
♦ Total postnatal loss (No.) = (Total no. of live pups delivered) / (Total no. of pups survived during postnatal period)

OVARIAN FOLLICULAR ASSESSMENT
A quantitative ovarian follicle assessment was carried out for all P females from control and high dose groups and the assessments were not extended to lower dose groups as there were no treatment-related changes noted in any of the high dose group animals.
The fixed ovaries from each female were cut into 3 equal parts and middle third portion of the ovary was embedded and sectioned. Five sections from each ovary were selected at 100 µm apart and placed on same slide by numbering 1, 2, 3, 4 and 5. Sections of ovaries were stained by routine H&E or PAS staining technique.
For each female, both the ovaries were employed for counting of follicles by light microscopy under higher magnification such as 20X /40X. The primordial follicles and primary follicles in each section of both ovaries were counted and recorded.

SPERM PARAMETERS
Sperm parameters were evaluated for all P males (see above).

Postmortem examinations (offspring):

SACRIFICE
All the animals were fasted overnight prior to scheduled necropsy. The animals were euthanized using CO2 exposure followed by exsanguination.
- The F1 offsprings not selected as parental animals were sacrificed as follows:
- Surplus pups not allocated to any of the cohorts: on PND 21
- Cohort 1A: on PND 105
- Cohort 2A: on PND 83
- Cohort 2B: on PND 22
- The F2 offsprings were sacrificed on PND 21.

GROSS NECROPSY
Special attention was paid to the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGTHS
Surplus F1 pups not allocated to any of the cohorts: brain, spleen, and thymus were collected, weighed and preserved, and mammary tissues were collected and preserved.

Cohort 1A : the organs collected, weighed and preserved are listed in the attached document "Details of the observations". The organs and tissues of all high dose and control animals were examined for histopathology. The histopathological investigations were not extended to the lower dose groups as there were no treatment related effects noted at the high dose level during microscopic examinations.

Cohort 2A: the organs collected, weighed and preserved are listed in the attached document "Details of the observations". The organs and tissues of all high dose and control animals were examined for histopathology, but the examination was not extended to lower dose groups as there were no microscopic changes noted in high dose group animals.

Cohort 2B: the brain was collected, weighed and preserved are listed in the attached document "Details of the observations". The brain of all high dose and control animals was examined for neuro-histopathology, but the examination was not extended to lower dose groups as there were no microscopic changes noted in high dose group animals.

OVARIAN FOLLICULAR ASSESSMENT (COHORT 1A)
A quantitative evaluation of ovarian folliclar and corpora lutea was carried out for all females from control and high dose groups and the assessments were not extended to lower dose groups as there were no treatment related changes noted in any of the high dose group animals.

ORGAN-TYPIC DEVELOPMENT (COHORT 1A)
Organ-typic development (i.e. caput, corpus and cauda of the epididymis and the vas deferens for males and ovary with oviduct, uterus, and vagina for females) was assessed in animals from Cohort 1A.

SPERM PARAMETERS (COHORT 1A)
Measured for all males (see above).

SPLENIC LYMPHOCYTE SUBPOPULATION ANALYSIS (COHORT 1A)
For investigation of pre- and postnatally induced immunotoxic effects, 10 males and 10 females per group were subjected to the following assessment at termination:
The spleen was collected, weighed and cut in to cross section to yield the distal and proximal halves. One half was transferred to PBS medium and stored at -80±10°C until analysis for splenic lymphocyte subpopulation (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells) using flow cytometry.

NEUROPATHOLOGY OF BRAIN (COHORT 2A & 2B)
Gross morphometric assessment of brains was performed by linear measurements of cerebrum and cerebellum using calibrated digital Vernier callipers. Five sections were examined from the brain to allow examination of olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain, brainstem, and cerebellum.

Statistics:

The raw data were subjected to computer statistical processing. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05), indicated by the aformentioned tests designated by the superscripts throughout the report as follows: * Statistically significant (P<0.05) change than the vehicle control group. The statistical analysis performed for the different parameters are listed in the attached document "Details of the observations".
Data of dead/moribund animals, non-pregnant animals, females mated but not littered and lactation data of females with total litter loss were excluded from statistical analysis.

Reproductive indices:

MATING AND FERTILITY INDEX:
♦ Male mating index (%) = (No. of males with confirmed mating/Total No. of males cohabited) ×100
♦ Male fertility index (%) = (No. of males impregnating a female/Total No. of males with evidence of mating) ×100
♦ Female mating index (%) = (No. of sperm-positive females/Total No. of females cohabited) ×100
♦ Female fertility index (%) = (No. of females with evidence of implantation sites/No. of sperm-positive females) ×100

PRE-COITAL INTERVAL:
♦ Pre-coital interval (days) = (Date of confirmation of mating) - (Date of initiation of cohabitation)

GESTATION LENGTH:
♦ Gestation Length (days) = (Date of parturition) - (Date of evidence of mating (GD 0))

GESTATION INDEX:
♦ Gestation Index (%) = (No. of females with live born) / (No. of females with evidence of pregnancy) x 100

PARTURITION INDEX:
♦ Parturition Index (%) = (No. of females littered) / (No. of females with evidence of pregnancy) x 100

FEMALE FECUNDITY OR PREGNANCY INDEX:
♦ Pregnancy Index (%) = (No. of females with evidence of prensence of live/dead fetuses/pups) / (No. of females with evidence of mating) x 100

Offspring viability indices:

DELIVERY AND LITTER OBSERVATIONS:
♦ Sex ratio (m/f) on LD 1/4/7/14/21 = (No. of male offspring) / (No. of female offspring)
♦ Live Birth Index (%) per litter = (No. of pups born alive) / (Total no. of pups born) x 100
♦ Pup survival index (%) on LD 4/7/14/21 = (Total No. of live pups on LD 4/7/14/21) / (No. of pups born/4/7/14) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity in any of the animals of both sexes from all the tested dose groups throughout the experimental period.
The detailed clinical examination did not reveal any behavioural changes in any of the animals of both sexes from all the tested dose and vehicle control groups.

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity noted in any of the animals of both sexes from all the tested dose groups throughout the experimental period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in any of the tested dose groups of both sexes throughout the experimental period, including gestation and lactation periods, when compared with vehicle control group.
The mean gestational body weight gain was statistically significantly higher than controls between GD 15 and 17 in group G4. However, the difference was slight and occasional, and was considered to be not related to treatment and not toxicologically relevant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related changes noted in mean feed consumption in any of the tested dose groups of both sexes throughout the experimental period, including gestation and lactation periods, when compared with vehicle control group.
The mean feed consumption was statistically significantly lower than controls between GD 14 and 20 in group G3; however, the difference is slight and occasional, and was considered to be not related to treatment and not toxicologically relevant.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in the mean haematological values in any of the tested dose groups of both sexes when compared with the vehicle control group.
When compared with controls, the mean total leucocyte count, total erythrocyte count, haemoglobin, haematocrit levels were statistically significantly lower in group G3 females and the mean absolute basophils were statistically significantly lower in group G2 and G3 females. However, since the differences were slight and the values remain within in-house historical control range of same species and strain, they were considered to be not related to treatment and not toxicologically relevant.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test item changes noted in the mean clinical chemistry values in any of the tested dose groups of both sexes when compared with the vehicle control group.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no test item related changes noted in mean serum Thyroxine (T4) and Thyroxine Stimulating Hormone (TSH) levels in any of the tested dose groups of both sexes when compared with vehicle control group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in the urine parameters in any of the tested dose groups of both sexes when compared with the vehicle control group.
The mean volume of urine collected was statistically significantly higher than controls in groups G2 and G3 females; however, the difference was slight and considered to be not related to treatment and not toxicologically relevant.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related histopathological findings noted in high dose animals of both sexes and no changes noted in ovarian follicle count in high dose females.

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no test item-related irregularities observed in oestrus cyclicity of any of the tested dose group females during treatment period. The mean length of oestrus cycle per female during treatment period was unaffected by the test item administration in any of the tested dose groups.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

- Sperm motility:
There were no test item-related effects noted in mean sperm motility (%) in any of the tested dose groups when compared with vehicle control group.

- Sperm morphology:
There were no test item-related sperm morphological changes noted in any of the tested dose groups when compared with vehicle control group.
The noted sporadic statistically significant changes such as, higher number and percentage of normal sperms, lower number and percentage of total abnormal sperms and sperms with neck (mid piece) and tail abnormalities in groups G2 and G3 are considered to be incidental and toxicologically not relevant.

- Spermatid head count and Daily sperm production:
There were no test item-related effects noted in mean spermatid head count/concertation or daily testicular sperm production per gram and per animal in any of the tested dose groups when compared with the vehicle control group.
The noted sporadic statistically significant higher mean Spermatids per Gram of Testis (all dose groups), Daily Sperm Production per gram of Testis (all dose groups) and Daily Sperm Production per gram of animal (groups G2 and G3) are considered as incidental and toxicologically not relevant.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There were no effects on male and female reproductive performance/indices noted from P generation in any of the tested dose groups when compared with vehicle control group.

- Male mating index:
A total of 24 (out of 25), 24 (out of 25), 25 (out of 25) and 24 (out of 25) males were evidenced with mating during the cohabitation period (14 days) with a mating index of 96.0%, 96.0%, 100.0% and 96.0%, for the dose groups G1, G2, G3 and G4 respectively.
The 1 (out of 25) male with no evidence of mating from groups G1, G2 and G4 did not reveal any gross pathological changes during conduct of necropsy and also no microscopic changes were noted in any of the reproductive organ during conduct of histopathological examination. There were no effects noted in sperm parameters from any of these animals. Hence, these incidences are considered as incidental and un-related to treatment.
There were no statistically significant differences noted for male mating index in any of the tested dose groups when compared with the vehicle control group.

- Male fertility index:
A total of 22 (out of 24), 21 (out of 24), 21 (out of 25) and 21 (out of 24) males noted with positive signal during cohabitation were confirmed as fertile by impregnating a female or siring a litter with a fertility index of 91.67%, 87.50%, 84.00% and 87.50% from the dose groups G1, G2, G3 and G4, respectively.
There were no statistically significant differences noted for male fertility index in any of the tested dose groups when compared with the vehicle control group.

- Pre-coital interval/Copulatory interval/Mean time to mating:
A total of 25 pairs were left for cohabitation from each group. The mean pre-coital interval was 3.75, 4.29, 5.64 and 2.88 days for groups G1, G2, G3 and G4, respectively.
There were no statistically significant differences noted for mean pre-coital interval in any of the tested dose groups when compared with the vehicle control group.

- Gestation length/Duration of pregnancy:
The mean gestation length [day of confirmed as successfully mated to day of parturition] was 22.64, 22.24, 22.62 and 22.33 days for groups G1, G2, G3 and G4, respectively.
There were no statistically significant differences noted for mean gestation length in any of the tested dose groups when compared with the vehicle control group.

- Female mating index:
A total of 24 (out of 25), 24 (out of 25), 25 (out of 25) and 24 (out of 25) cohabitated females were confirmed as mated with evidence of sperms in vaginal smear during cohabitation period (14 days) with a mating index of 96.00%, 96.00%, 100.00% and 96.00%, from the dose groups G1, G2, G3 and G4, respectively.
The 1 (out of 25) female with no evidence of mating from groups G1, G2 and G4 did not reveal any gross pathological changes during conduct of necropsy and no microscopic changes noted in any of the reproductive organs during conduct of histopathological examination. Hence, these incidences are considered as incidental and un-related to treatment.
There were no statistically significant differences noted for female mating index in any of the tested dose groups when compared with the vehicle control group.

- Female fertility index:
A total of 22 (out of 24), 21 (out of 24), 21 (out of 25) and 21 (out of 24) females with evidence of sperms in vaginal smear were confirmed with presence of implantations / presence of live or dead pups / evidence of parturition with a fertility index of 91.67%, 87.50%, 84.00% and 87.50% from groups G1, G2, G3 and G4, respectively.
There were no statistically significant differences noted for female fertility index in any of the tested dose groups when compared with the vehicle control group.

- Fecundity or Pregnancy index:
A total of 22 (out of 24), 21 (out of 24), 21 (out of 25) and 21 (out of 24) females with evidence of sperms in vaginal smear were confirmed as pregnant / with evidence of implantation sites with a fecundity index of 91.67%, 87.50%, 84.00% and 87.50% from groups G1, G2, G3 and G4, respectively.
There were no statistically significant differences noted for female fecundity index in any of the tested dose groups when compared with the vehicle control group.

- Gestation index:
A total of 22 (out of 22), 21 (out of 21), 21 (out of 21) and 21 (out of 21) pregnant females were confirmed with live born pups with a gestation index of 100.0% for all the tested dose groups and vehicle control group.

- Parturition index:
A total of 22 (out of 22), 21 (out of 21), 21 (out of 21) and 21 (out of 21) pregnant females were confirmed with parturition with a parturition index of 100.0% for all the tested dose groups and vehicle control group.

- Implantation sites and viable pups:
A mean number of 12.50, 11.14, 10.57 and 12.33 implantation sites and mean number of 12.41, 10.95, 10.24 and 11.67 viable pups were noted from groups G1, G2, G3 and G4, respectively.
There were no statistically significant changes noted for both mean implantation sites and viable pups in any of the tested dose groups when compared with the vehicle control group.

- Post-implantation loss:
A mean number of 0.09, 0.19, 0.33 and 0.67 post-implantation losses with a percentage of 0.53%, 1.36%, 3.12% and 5.21% were noted from groups G1, G2, G3 and G4, respectively.
There were no statistically significant changes noted for mean number and percentage of occurred post-implantation losses in any of the tested dose groups when compared with the vehicle control group. However, although not statistically significant, the mean post-implantation loss was higher than controls in group G4. This is due to one dam (no. Rg2857) for which there were 6 out of 14 post-implantation losses (i.e., 42.9%). This was considered as an isolated case and not treatment-related.

- Postnatal loss:
A mean number of 0.00, 0.00, 0.00 and 0.24 postnatal losses with a percentage of
0.00, 0.00, 0.00 and 1.89 were noted from groups G1, G2, G3 and G4, respectively.
There were no statistically significant changes noted for mean number and percentage of occurred postnatal losses in any of the tested dose groups when compared with the vehicle control group. However, although not statistically significant, the mean post-natal loss was higher than controls in group G4. This is due to 2 dams (i.e., Rg2856 and Rg2864) for which there were 4 out of 14 (i.e., 28.6%) and 1 out of 9 (i.e., 11.1%) post-natal losses, respectively. This was considered as isolated cases and not treatment-related.

There were no test item related changes noted in any of the systemic and reproductive endpoints of both sexes, at any of the dose levels tested.

- Delivery and Litter observations:
There were no test item related changes or effects observed in birth parameters such as, total litter size, number of live pups born, sex ratio (m/f) and live birth index in any of the tested dose groups when compared with the vehicle control group.
However, 1 male and 1 female pup from group G1, 3 male pups from group G2, 2 males and 1 female pup from group G3, and 7 males and 6 female pups from group G4 were found dead at birth. This incidence of slightly higher mortality from group G4 was due to noted 43% of mortalities i.e., 4 males and 2 females from a single litter (Rg2857) which is an incidental and un-related to treatment. One cannibalized pup was found with undetermined sex at birth from G3 group. The mean live birth index (%) per dam was statistically significantly lower than controls in group G4. This is due to one dam (i.e. Rg2857) for which the live birth index was 57.14% (which is correlated with the post-implantation loss). This was considered as an isolated case and not treatment-related.
In addition, the pup survival index per litter during lactation period was unaffected by the test item in all the tested dose groups when compared with the vehicle control group.
Although not statistically significant, the pup survival index was lower than controls in group G4 between LD1 and LD 4. This is due to 2 dams for which the pup survival index was 71.43% and 88.89%, respectively. This was considered as isolated cases and not treatment-related.

Detailed results are provided in the attached document "Results tables - Parental generation".

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the highest dose level for systemic and reproduction toxicity endpoints in P0 generation and for developmental toxicity endpoints in F1 generation

Clinical signs:

no effects observed

Description (incidence and severity):

Cohort 1B:
There were no clinical signs of toxicity in any of the animals of both sexes from all the tested dose groups throughout the experimental period.
The detailed clinical examination did not reveal any changes in any of the animals of both sexes from all the tested dose and vehicle control groups.

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity was recorded in any of the animals of both sexes from all the tested dose groups throughout the experimental period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1B:
There were no test item related changes noted in mean body weight and percent change in mean body weight gain with respect to PND 21 in any of the tested dose groups of both sexes throughout the experimental period, including gestation and lactation periods, when compared with vehicle control group.

The following statistically significant changes were noted across the dose groups when compared with vehicle control group:
- higher mean body weight on PND 32 in groups G2 and G3 (males),
- lower mean body weight on PND 49 and mean percent change in body weight gain during PND 21 to 49 in group G4 (males),
- higher mean body weight on PND 77, 84, 91 and 98 in group G3 (females),
- lower mean percent change in body weight gain during PND 21 to 56 in group G2 (females),
- higher mean gestational body weight on GD 0, 7, 9 and 11 in group G3,
- lower mean percent change in body weight gain during GD 7 to 9 in group G4.

However, since these differences were slight and occasional and since the mean values remain within in-house historical control range of same species and strain, these changes were considered to be incidental and un-related to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1B:
There were no test item related changes noted in mean feed consumption in any of the tested dose groups of both sexes throughout the experimental period, including gestation and lactation periods, when compared with vehicle control group.
The mean feed consumption during GD 14 to 20 was statistically significantly lower than controls in group G4; however, the difference is slight and occasional and is considered as incidental and un-related test item exposure.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item related changes noted in mean absolute/relative organ weights and terminal body weight in any of the tested dose groups of both sexes when compared with vehicle control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross pathological changes observed during necropsy in any of the adult animals of the F1 generation - Cohort 1B.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Cohort 1B:

- Sexual maturation:
There were no effects noted in mean occurrences of sexual maturation (day of occurrence of balanopreputial separation in males and day of occurrence of vaginal opening in females) and no changes noted in mean body weight on the day of sexual maturation in any of the tested dose groups when compared with vehicle control group.

- Oestrus cycle evaluation:
There were no test item-related irregularities observed in oestrus cyclicity of any of the tested dose group females during treatment period. The mean length of oestrus cycle per female during treatment period was unaffected by the test item administration in any of the tested dose groups.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no test item-related irregularities observed in oestrus cyclicity of any of the tested dose group females during treatment period.
The mean length of oestrus cycle per female during treatment period was unaffected by the test item administration in any of the tested dose groups.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Cohort 1B:
There were no effects on male and female reproductive performance/indices noted from F1 generation in any of the tested dose groups when compared with vehicle control group.

- Male mating index:
All the males i.e., 20 (out of 20) were evidenced with mating during the cohabitation period with a mating index of 100.0% from all the dose groups and vehicle control group.

- Male fertility index:
A total of 17 (out of 20), 16 (out of 20), 16 (out of 20) and 17 (out of 20) males noted with positive signal during cohabitation were confirmed as fertile by impregnating a female or siring a litter with a fertility index of 85.00%, 80.00%, 80.00% and 85.00% from the dose groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for male fertility index in any of the tested dose groups when compared with the vehicle control group.

- Pre-coital interval/Copulatory interval/Mean time to mating:
A total of 20 pairs were left for cohabitation from each group. The mean pre-coital interval was 6.90, 7.35, 7.05 and 6.20 days for groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for mean
pre-coital interval in any of the tested dose groups when compared with the vehicle control group.

- Gestation length/Duration of pregnancy:
The mean gestation length [day of confirmed as successfully mated to, day of parturition] was 23.24, 23.50, 23.13 and 23.35 days for groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for mean gestation length in any of the tested dose groups when compared with the vehicle control group.

- Female mating index:
All the females i.e., 20 (out of 20) were confirmed as mated with evidence of sperms in vaginal smear during cohabitation period (within 14 days) with a mating index of 100.0% from all the dose groups and vehicle control group.

- Female fertility index:
A total of 17 (out of 20), 16 (out of 20), 16 (out of 20) and 17 (out of 20) females with evidence of sperms in vaginal smear were confirmed with presence of implantations / presence of live or dead pups / evidence of parturition with a fertility index of 85.00%, 80.00%, 80.00% and 85.00% from the dose groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for female fertility index in any of the tested dose groups when compared with the vehicle control group.

- Fecundity or pregnancy index:
A total of 17 (out of 20), 16 (out of 20), 16 (out of 20) and 17 (out of 20) females with evidence of sperms in vaginal smear were confirmed as pregnant / with evidence of implantation sites with a fecundity index of 85.00%, 80.00%, 80.00% and 85.00% from groups G1, G2, G3 and G4, respectively.
There were no statistically significant differences noted for female fecundity index in any of the tested dose groups when compared with the vehicle control group.

- Gestation index:
A total of 17 (out of 17), 16 (out of 16), 16 (out of 16) and 17 (out of 17) pregnant females were confirmed with live born pups with a gestation index of 100.0% for all the tested dose groups and vehicle control group.

- Parturition index:
A total of 17 (out of 17), 16 (out of 16), 16 (out of 16) and 17 (out of 17) pregnant females were confirmed with parturition with a parturition index of 100.0% for all the tested dose groups and vehicle control group.

- Implantation sites and viable pups:
A mean number of 7.76, 9.63, 9.50 and 8.65 implantation sites and mean number of 7.06, 9.50, 9.38 and 8.18 viable pups were noted from groups G1, G2, G3 and G4, respectively.
There were no statistically significant changes noted for both mean implantation sites and viable pups in any of the tested dose groups when compared with the vehicle control group.

- Post-implantation loss:
A mean number of 0.71, 0.13, 0.13 and 0.47 post-implantation losses with a percentage of 13.12, 1.26, 1.25 and 7.44 were noted from groups G1, G2, G3 and G4, respectively.
The noted statistically significant decrease in mean post-implantation loss (no. and %) in groups G2 and G3 when compared with the vehicle control group is considered as incidental and toxicologically irrelevant.

- Postnatal loss:
A mean number of 0.18, 0.44, 0.19 and 0.24 postnatal losses with a percentage of
2.06, 4.02, 1.73 and 6.54 were noted from groups G1, G2, G3 and G4, respectively.
There were no statistically significant changes noted for mean number and percentage of occurred postnatal losses in any of the tested dose groups when compared with the vehicle control group.

There were no test item related changes noted in any of the systemic and reproductive endpoints of both sexes.

- Delivery and litter observations:
There were no test item related changes or effects observed in birth parameters such as, total litter size, number of live pups born, sex ratio (m/f) and live birth index in any of the tested dose groups when compared with the vehicle control group. Also, the pup survival index per litter during lactation period was unaffected by the test item in all the tested dose groups when compared with the vehicle control group. However, one female pup from G2 group was found dead at birth, which is an incidental and un-related to treatment.
When compared with controls, the mean number of female live pups born per dam was statistically significantly higher in group G3 and the mean number and percentage of post-implantation losses per dam was statistically significantly lower in groups G2 and G3. However, these difference were considered as incidental and toxicologically not relevant.

Detailed results are provided in the attached document "Results tables - Cohort 1B".

Key result

Dose descriptor:

NOAEL

Remarks:

Cohort 1B

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the highest dose level for systemic and reproduction toxicity endpoints in F1 parental generation and for developmental toxicity endpoints in F2 generation

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation pups:
There were no external abnormalities or behavioural changes noted in any of the pups during daily observation from all the tested dose and vehicle control group during postnatal period. All the pups had normal behaviour during daily observations.

F1 adults - Cohort 1A:
There were no clinical signs of toxicity in any of the animals of both sexes from all the tested dose groups throughout the experimental period.
Lethargy was observed in one female from group G1 on PND 99 and 100 and one female from group G4 on PND 100 and 102. However, these animals became normal later in the treatment period. These observations are considered as incidental and un-related to test item exposure. The detailed clinical examination did not reveal any changes in any of the animals of both sexes from all the tested dose groups and vehicle control groups.

F1 adults - Cohort 2A:
There were no clinical signs of toxicity in any of the animals of both sexes from all the tested dose groups throughout the experimental period.
The detailed clinical examination did not reveal any changes in any of the animals of both sexes from all the tested dose and vehicle control groups.

F1 adults - Cohort 2B:
There were no clinical signs of toxicity recorded in any of the animals of both sexes from all the tested dose groups throughout the experimental period.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F1 generation pups:
In group G4, a total of 5 pups were found dead during PND 1 to 4. These mortalities were noted in 2 (out of 21) litters of which 2 males and 2 females from one dam (Rg2856) and 1 male pup from second dam (Rg2864). The other 19 (out of 21) litters from the same dose level, didn’t reveal any such incidences. Moreover, the other surviving pups from the same litters did not show any behavioural changes during the entire postnatal period. Also, the pup survival is 98.11% at this dose level which is well within the acceptable limits.

Cohorts 1A, 2A and 2B:
There were no mortality/morbidity recorded in any of the animals of both sexes from all the tested dose groups throughout the experimental period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation pups:
There were no test item-related effects noted in mean pup [both male and female] weight per litter in all any of the tested dose groups when compared with the vehicle control group, recorded on PND 1, 4, 7, 14 and 21.
On PND 21, the mean pup weight was statistically significantly higher than controls in female groups G2 and G4 and in male group G4. However, the difference was slight and the mean value are within the in-house historical control range of same species and strain and therefore considered to be incidental.

Cohort 1A:
There were no test item related changes noted in mean body weight and percent change in mean body weight gain with respect to postnatal day (PND) 21 in any of the tested dose groups of both sexes throughout the experimental period when compared with vehicle control group.
The following statistically significant changes were noted across the dose groups when compared with vehicle control group:
- lower mean percent change in body weight gain during PND 21 to 28, PND 21 to 32 in group G4 (males).
- higher mean body weight on PND 21 in all the tested groups (females),
- higher mean body weight on PND 28 in group G3 (females),
- lower mean percent change in body weight gain during PND 21 to 28 in group G4 (females),
- lower mean percent change in body weight gain during PND 21 to 49 in group G3 (females),
- lower mean percent change in body weight gain during PND 21 to 104 in all the tested groups (females).
However, these differences were slight and occasional and the mean values remain within the in-house historical control range of same species and strain. Therefore, these differences are considered to be incidental and un-related test item exposure.

Cohort 2A:
There were no test item related changes noted in mean body weight and percent change in mean body weight gain with respect to postnatal day (PND) 21 in any of the tested dose groups of both sexes throughout the experimental period when compared with vehicle control group.
The mean body weight was statistically significant higher than controls on PND 63 in group G4 (males); however, since the mean values are well within the in-house historical control range of same species and strain, this difference is considered as incidental and un-related test item exposure.

Cohort 2B:
There were no test item related changes noted in mean body weight and percent change in mean body weight gain with respect to PND 21 in any of the tested dose groups of both sexes throughout the experimental period when compared with vehicle control group.
Statistically significant differences were noted from controls; however, they were slight and considered to be toxicologically not relevant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A:
There were no test item related changes noted in mean feed consumption in any of the tested dose groups of both sexes throughout the experimental period when compared with vehicle control group.
The following statistically significant changes were noted across the dose groups when compared with vehicle control group:
- lower mean feed consumption during week 3 in group G4 (males),
- lower mean feed consumption during week 4 in group G2 (males),
- higher mean feed consumption during weeks 3 and 5 in group G3 (females).
However, these differences were slight and occasional and the mean values remain within the in-house historical control range of same species and strain. Therefore, these differences are considered to be incidental and un-related test item exposure.

Cohort 2A:
There were no test item related changes noted in mean feed consumption in any of the tested dose groups of both sexes throughout the experimental period when compared with vehicle control group.
The mean feed consumption was statistically significant lower during week 6 and 7 in group G4 (females); however, since the mean values are well within the in-house historical control range of same species and strain, these slight differences are considered as incidental and un-related test item exposure.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A:
There were no test item-related changes noted in the obtained mean haematological values in any of the tested dose groups of both sexes when compared with the vehicle control group.
The mean absolute and percentage of monocytes was statistically significantly higher than controls in group G3 males. However, since the differences were slight and occasional and since the mean values remain within in-house historical control range of same species, these changes are considered to be incidental and un-related to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A:
There were no test item-related changes noted in the obtained mean clinical chemistry values in any of the tested dose groups of both sexes when compared with the vehicle control group.
The mean Alkaline phosphatase levels was statistically significantly higher than controls in group G4 females. However, since this difference was slight and occasional and since the mean value remains within in-house historical control range of same species, this change is considered to be incidental and un-related to treatment.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes noted in the urine parameters in any of the tested dose groups of both sexes when compared with the vehicle control group.

Sexual maturation:

no effects observed

Description (incidence and severity):

Cohort 1A:
There were no test item-related effects noted in mean occurrence of sexual maturation (day of occurrence of balanopreputial separation in males and day of occurrence of vaginal opening in females) and no changes noted in mean body weight on the day of sexual maturation in any of the tested dose groups when compared with vehicle control group.
There were no test item-related changes or delays noted for mean occurrence of first cornified cells (days) and mean time interval between vaginal patency to occurrence of first cornified cells in any of the tested dose groups when compared with vehicle control group.

Cohort 2A:
There were no effects noted in mean occurrences of sexual maturation (day of occurrence of balanopreputial separation in males and day of occurrence of vaginal opening in females) and no changes noted in mean body weight on the day of sexual maturation in any of the tested dose groups when compared with vehicle control group.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no test item-related changes in mean pup [both male and female] anogenital distance measurement (mm) recorded on PND 4 and its ratio per litter in any of the tested dose groups when compared with the vehicle control group.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no occurrences or evidence of retention of nipples in any of the male pups examined on PND 13 from all the tested dose groups and vehicle control group litters.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

PND 21 surplus pups:
There were no test item-related changes noted in mean absolute/relative organ weights of PND 21 surplus pups (litter wise) in any of the tested dose groups of both sexes when compared with vehicle control group.
The difference from controls was occasionally statistically significant for the mean absolute and/or relative spleen weight in males and/or females from groups G2 and G4 and for mean relative brain weight in G4 males. However, the values are within in-house historical control range of same species and strain and no gross pathological changes were noted in any of these organs. Therefore, these differences were considered to be unrelated to treatment.

Cohort 1A:
There were no test item-related changes noted in mean absolute/relative organ weights and terminal body weight in all the tested dose groups of both sexes when compared with vehicle control group.
However, the following statistically significant changes were noted in all the tested dose groups when compared with vehicle control group:
- Group G2: higher absolute and relative weight of mandibular lymph nodes (males).
- Group G4:
higher absolute and relative weight of mandibular lymph nodes (males).
higher absolute weight of thyroid along with parathyroid (males).
higher relative weight of spleen (females).
lower absolute weight of thymus (females).
However, since there were no correlation with gross pathological changes (all dose groups) or microscopic changes (high dose group) in any of these organs and the mean values are within in-house historical control range of same species and strain, these changes are considered to be incidental and un-related to treatment.

Cohort 2A:
There were no test item related changes noted in mean absolute/relative brain weights and terminal body weight in all the tested dose groups of both sexes when compared with vehicle control group.

Cohort 2B:
There were no test item related changes noted in mean absolute/relative brain weights in all the tested dose groups of both sexes when compared with vehicle control group.
The mean relative brain weight was statistically significantly lower than controls in groups G3 and G4 (males); however, this difference being related to the higher mean terminal body weight observed in the same groups, it is considered as incidental and un-related to test item exposure.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no gross pathological changes observed during necropsy in any of the pups of the F1 generation.
There were no test item-related gross pathological changes observed during necropsy in any of the adult animals of the F1 generation (C1A/C2A/C2B). A single isolated incidence of unilateral small-sized testis was observed in group G4-C1A, which was microscopically correlated as mild, diffuse, unilateral tubular atrophy of testis.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related histopathological findings in high dose animals of cohorts of F1 animals (C1A, C2A and C2B).
In C1A males, caput, corpus and cauda of epididymides and vas deferens were examined for appropriate organ-typic development and were found to be within normal histological limits.
In C1A females, ovary with oviduct, uterus and vagina were examined for organ-typic development and were found to be within normal histological limits.
Few of the microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.
Quantitative ovarian follicular assessment (primordial and primary follicles) in randomly selected parental and C1A females of control and high dose groups did not reveal any test item related variations. In addition, quantitative evaluation of corpora luteal count in C1A females of control and high dose group did not reveal any test item related variations.
In all groups of C2A and C2B animals, the linear measurements of the cerebrum and cerebellum were found to be within normal ranges. There were no test item-related variations in measured parameters, except an incidental and alone occurrence of statistically significant increase in mean length of cerebrum in group G4-C2B (males) when compared with vehicle control group.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

F1 PUPS:

- Postnatal developmental landmarks:
There were no test item-related changes/delays noted in mean occurrences of postnatal developmental landmarks of F1 pups in any of the tested dose groups when compared with vehicle control group.

- Sensory reflexes:
There were no test item-related changes/delays noted in mean responses of sensory reflexes of F1 pups in any of the tested dose groups when compared with vehicle control group.

- Thyroid hormones:
There were no test item-related changes noted in mean serum Thyroxine (T4) levels of PND 4 pups (pooled per litter) in any of the tested dose groups when compared with the vehicle control group. There were no test item-related changes noted in mean serum Thyroxine (T4) and Thyroxine Stimulating Hormone (TSH) levels of PND 21 pups (pooled per litter) in any of the tested dose groups when compared with the vehicle control group. Mean serum Thyroxine (T4) levels were statistically significantly higher than controls in groups G3 and G4. However, the values remain within the in-house historical control range and these changes are considered to be incidental and un-related to treatment.

COHORT 1A:

- Thyroid hormones:
There were no test item related changes noted in mean serum Thyroxine (T4) and Thyroxine Stimulating Hormone (TSH) levels in any of the tested dose groups of both sexes when compared with vehicle control group. The mean serum T4 levels were statistically significantly higher than controls in all the dose groups (males) and lower than controls in group G4 (females). However, since these differences were slight and occasional and since the values remain within the in-house historical control range of same species and strain, these changes are considered to be incidental and un-related to treatment.

- Oesturs cycle evaluation:
There were no test item-related changes observed in oestrus cyclicity in any of the tested dose group females during treatment period. The mean length of oestrus cycle per female during treatment period was unaffected by the test item administration in any of the tested dose groups and comparable with the vehicle control group.

- Sperm parameters:
There were no changes noted in mean sperm motility (%) in any of the tested dose groups when compared with vehicle control group.
There were no test item-related sperm morphological changes noted in any of the tested dose groups when compared with vehicle control group. The noted statistically significant decrease in number and percentage of sperms with head abnormalities in group G2 is considered as incidental and toxicologically in-significant.
There were no test item-related changes noted in mean spermatid head count/concertation or daily testicular sperm production per gram and per animal in any of the tested dose groups when compared with the vehicle control group.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 2A:

- Auditory startle test:
There were no test item related changes noted in mean response amplitude in any of the blocks conducted on PND 25 from all the tested dose groups of both sexes when compared with vehicle control group.

- Neurological/functional observations:
The neurological/functional observations such as, home cage, handling, open-field, sensory, physiological observations did not reveal any changes in any of the animals of both sexes from all the tested dose groups. There were no changes noted in mean fore/hind limb grip strengths, mean motor activity assessments, and mean hind limb foot splay in all tested dose groups of both sexes when compared with vehicle control group.
The mean no. of urine pools observed during open filed test in group G3 (females) and the mean no. of movement counts in groups G3 and G4 (females) were statistically significantly lower than controls; however, these differences were of slight magnitude and considered as incidental and un-related test item exposure.

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

Cohort 1A:
There were no changes noted in mean splenic sub-populations of T "helper" (CD4+) cells, T cytotoxic (CD8+) cells, natural killer (NK) cells and B lymphocytes in any of the tested dose groups of both sexes when compared with vehicle control group.

F1 PUPS:
There were no test item related changes in growth parameters, postnatal developmental landmarks, sensory reflexes, thyroid hormone levels in F1 generation pups, at any of the dose levels tested. No gross pathological changes were noted in any of the F1 pups of both sexes.

F1 ADULTS - COHORT 1A:
There were no test item related changes noted in any of the systemic and reproductive endpoints of both sexes, at any of the dose levels tested.
There were no test item related changes observed in oestrus cyclicity in any of the tested dose group females during treatment period. The mean length of oestrus cycle per female during treatment period was unaffected by the test item administration in any of the tested dose groups and comparable with the vehicle control group.
There were no changes noted in mean sperm motility (%), sperm morphology, mean spermatid head count/concertation or daily testicular sperm production per gram and per animal in any of the tested dose groups when compared with vehicle control group.
There were no changes noted in sub-populations of splenic lymphocytes in any of the tested dose groups. There were no test item-related histopathological findings noted in the high dose animals of both sexes and no changes noted in ovarian follicle and corpora luteal counts in the high dose females. The organ-typic development examination did not reveal any changes in the high dose animals.

F1 ADULTS - COHORT 2A:
There were no test item related changes noted in any of the systemic and neurotoxicity endpoints of both sexes, at any of the dose levels tested. There were no test item-related neuropathological changes noted in any of the high dose animals of both sexes.

F1 ADULTS - COHORT 2B:
There were no test item related changes noted in systemic and neuropathological changes in both sexes, at any of the dose levels tested.

Detailed results are provided in the attached documents "Results tables - F1 generation pups", "Results tables - Cohort 1A", Results tables - Cohort 2A", Results tables - Cohort 2B".

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1A)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed for systemic toxicity endpoint at the highest dose level

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 2A)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed for systemic toxicity and developmental neurotoxicity endpoints at the highest dose level

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 2B)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed for developmental neurotoxicity endpoint at the highest dose level

Clinical signs:

no effects observed

Description (incidence and severity):

There were no external abnormalities or behavioural changes noted in any of the F2 pups during daily observation from all the tested dose and vehicle control group during postnatal period. All the pups had normal behaviour during daily observations.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

On PND 2, 1 male and 2 female pups from G1-C1B group, 2 male pups from G2-C1B group, and 3 female pups from G3-C1B group were also found dead.
On PND 4, 3 males and 2 female pups from the G2-C1B group, as well as 3 males from the G4-C1B group, were found dead. On PND 5, a female pup from G4-C1B group was found dead.
These mortalities are considered incidental and not treatment-related as the other litters from the same dose levels didn’t reveal any such incidences. Moreover, the other surviving pups from the same litters did not show any behavioural changes during the entire postnatal period from all the dose groups. Also, the pup survival from groups G2, G3 and G4 is 95.98%, 98.27% and 95.42%, respectively which are well within the acceptable limits and comparable control group (97.94%).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean F2 generation pup [both male and female] weight per litter in any of the tested dose groups when compared with the vehicle control group, recorded on postnatal day (PND) 1, 4, 7, 14 and 21.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There were no changes in mean F2 pup [both male and female] anogenital distance measurement (mm) and its ratio per litter were noted in any of the tested dose groups when compared with the vehicle control group, recorded on PND 4.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no occurrences or evidence of retention of nipples in any of the F2 male pups examined on the PND 13 from all the tested dose and vehicle control group litters.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes observed during necropsy in any of the pups of the F2 generations.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- Postnatal developmental landmarks:
There were changes/delays noted in mean occurrences of postnatal developmental landmarks of F2 pups such as pinna unfold, fur development, incisor eruption, eye opening and testes descent evaluated during postnatal period in any of the tested dose groups when compared with vehicle control group.

- Sensory reflexes:
There were no changes/delays noted in mean responses of sensory reflexes of F1 pups such as surface righting, auditory startle and air righting performed during postnatal period in any of the tested dose groups when compared with vehicle control group.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

There were no test item related changes in growth parameters, postnatal developmental landmarks, sensory reflexes, in F2 generation pups. No gross pathological changes were noted in any of the F2 pups of both sexes.

Detailed results are provided in the attached document "Results tables - F2 generation pups".

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed for the developmental toxicity endpoints at the highest dose level

Key result

Reproductive effects observed:

no

Conclusions:

- The test item exposure to maternal P females did not impose any systemic and developmental toxicity in their offspring (F1 generation) of both sexes during postnatal period in any of the tested dose levels. Therefore, the no-observed-adverse-effect-level (NOAEL) of Strontium chloride hexahydrate is considered to be 1000 mg/kg bw/day, for systemic and reproduction toxicity endpoints in P generation and for developmental toxicity endpoints in F1 generation.

- The repeated exposure of test item, Strontium chloride hexahydrate, from the day of weaning (i.e., PND 21) by oral gavage route with the dose levels of 250, 500 and 1000 mg/kg bw/day to F1 generation, C1A animals, did not produce any systemic toxicity in any of the tested dose groups. Therefore, the NOAEL of Strontium chloride hexahydrate is considered to be 1000 mg/kg bw/day for systemic toxicity endpoints.

- The repeated exposure of test item, Strontium chloride hexahydrate, from the day of weaning (i.e., PND 21) by oral gavage route with the dose levels of 250, 500 and 1000 mg/kg bw/day to F1 generation, C1B animals, did not produce any systemic and reproductive toxicity in any of the tested dose groups. The test item exposure to maternal C1B females did not impose any systemic and developmental toxicity in their offspring (F2 generation) of both sexes during postnatal period in any of the tested dose levels. Therefore, the NOAEL of Strontium chloride hexahydrate is considered to be 1000 mg/kg bw/day, for systemic and reproduction toxicity endpoints in F1 parental generation (C1B) and for developmental toxicity endpoints in F2 generation.

- The repeated exposure of test item, Strontium chloride hexahydrate, from the day of weaning (i.e., PND 21) by oral gavage route with the dose levels of 250, 500 and 1000 mg/kg bw/day to F1 generation, C2A animals, did not produce any systemic toxicity and developmental neurotoxicity in any of the tested dose groups. Therefore, the NOAEL of Strontium chloride hexahydrate is considered to be 1000 mg/kg bw/day for developmental neurotoxicity endpoints.

- The repeated exposure of test item, Strontium chloride hexahydrate, to P animals throughout pre-mating, mating and post-mating (gestation and lactation) by oral gavage route with the dose levels of 250, 500 and 1000 mg/kg bw/day did not produce any developmental neurotoxicity and no neuro-histopathology changes in any of the C2B tested dose groups. Therefore, the NOAEL of Strontium chloride hexahydrate is considered to be 1000 mg/kg bw/day for developmental neurotoxicity endpoints.

Executive summary:

In this Extended One-Generation Reproductive Toxicity (EOGRT) study in Sprague Dawley rats, a total of 200 (100 males + 100 females) Sprague Dawley rats were selected for parental (P) generation and distributed to four groups. Each parental (P) generation group (G1, G2, G3 and G4) consisted of 25 males and 25 females. A total of 240 males and 240 females were selected on the day of weaning (i.e., postnatal day (PND) 21) and randomly assigned to four cohorts [Cohort 1A (80 males + 80 females), Cohort 1B (80 males + 80 females), Cohort 2A (40 males + 40 females) and Cohort 2B (40 males + 40 females)]. Each C1A cohort group (G1-C1A, G2-C1A, G3-C1A and G4-C1A) consisted of 20 males and 20 females (1 male and 1 female/litter, representing 20 P litters), each C1B cohort group (G1-C1B, G2-C1B, G3-C1B and G4-C1B) consisted of 20 males and 20 females (1 male and 1 female/litter, representing 20 P litters), each C2A cohort group (G1-C2A, G2-C2A, G3-C2A and G4-C2A) consisted of 10 males and 10 females (1 male or 1 female/litter, representing 20 P litters) and each C2B cohort group (G1-C2B, G2-C2B, G3-C2B and G4-C2B) consisted of 10 males and 10 females (1 male or 1 female/litter, representing 20 P litters).
The animals in groups, G1/G1-C1A/G1-C1B/G1-C2A were administered with vehicle (distilled water), the animals in groups, G2/G2-C1A/G2-C1B/G2-C2A, G3/G3-C1A/G3-C1B/G3-C2A and G4/G4-C1A/G4-C1B/G4-C2A groups were administered with the dose levels of 250, 500 and 1000 mg/kg bw/day with concentrations of 25, 50 and 100 mg/mL, respectively, in an equi-volume of 10 mL/kg bw/day. The animals from Cohort C2B (G1-C2B, G2-C2B, G3-C2B and G4-C2B) were not treated with test item and sacrificed on PND 22 for neurotoxicity assessments.

 

The stability of the test item in dose formulations was established before initiation of the treatment. The formulations were stable up to 48 hours at room temperature with dose concentrations of 10 mg/mL and 100 mg/mL in water. The analytical verification of dose formulations was done during weeks 1, 9, 17, 25 and 30 of the treatment periods. The results were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤ 10%.

 

All the P generation animals were observed twice daily (pre- and post-dose) for clinical signs, twice daily for mortality/morbidity and once weekly for detailed clinical examination. The body weights (throughout the experimental period) and feed consumption (throughout the experimental period, except during cohabitation period) were recorded once weekly, except for body weights which were recorded twice weekly during the gestation period. The assessment for haematology, clinical chemistry, urinalysis, and thyroid hormonal levels [thyroxine (T4) hormone and thyroid stimulating hormone (TSH)] was conducted for 10 (out of 25) randomly selected males and 10 (out of 25) randomly selected females from each group at termination. Sperm parameters (motility, morphology, and sperm concentration/daily sperm production) were evaluated for all P males. Oestrus cyclicity was evaluated during pre-mating, cohabitation period and at termination for all P females. The gross pathology and organ weighing was performed on the day of termination. Histopathological examination was conducted on all the tissues collected from vehicle control and high dose group animals. A quantitative ovarian follicle assessment was carried out for all P females from control and high dose groups. All the P males were evaluated for reproductive performance or indices such as, mating and fertility index and all the P females were evaluated for mating and fertility index, pre-coital interval, gestation length, fecundity index, gestation index, parturition index, post-implantation loss and postnatal loss. All P females were observed for birth parameters (number of live/dead pups born, litter size, sex ratio, and live birth index per litter) and for litter observations (number of live/dead pups during lactation period, sex ratio and pup survival index per litter).
In all the tested dose groups (G2, G3 and G4), there were no test item-related changes noted in any of the systemic and reproductive endpoints of both sexes. There were no test item-related histopathological findings noted in high dose animals of both sexes and no changes noted in ovarian follicle count in high dose females.

 

All the surviving F1 generation pups from all the tested groups were observed once daily for external examinations and twice daily for mortalities till termination, weighed individually on postnatal day (PND) 1, 4, 7, 14 and 21. All the surviving F1 generation pups from all the tested groups, were observed for occurrences of postnatal developmental landmarks, for responses towards to sensory reflexes during postnatal period, measured for anogenital distance on PND 4, and observed for retention of any nipples/areolae in male pups on PND 13. In the surplus pups, the assessment for serum T4 levels and serum T4 and TSH levels were conducted on PND 4 (i.e., day of litter standardization) and PND 21 (i.e., day of termination of the surplus pups not allocated to any of the cohorts), respectively. Gross pathological observations were performed in surplus pups at termination (i.e., PND 4 or PND 21).
In all the tested dose group (G2, G3 and G4) litters, there were no test item-related changes in growth parameters, postnatal developmental landmarks, sensory reflexes, thyroid hormone levels in F1 generation pups. No gross pathological changes were noted in any of the F1 pups of both sexes.

 

All the Cohort 1A (C1A) animals were observed twice daily (pre- and post-dose) for clinical signs, twice daily for mortality/morbidity and once weekly for detailed clinical examination. The body weights were recorded every two days once for the first 2 weeks followed by weaning and once weekly thereafter. The average feed consumption was recorded once weekly. All the animals were evaluated for occurrence of sexual maturation (i.e., balano-preputial separation for males and vaginal patency for females) and the body weight of each animal was recorded on the day of evidence of sexual maturation. All the C1A females were evaluated for mean occurrence of first cornified cells and time interval between vaginal patency and occurrence of first cornified cells. All C1A females were evaluated for oestrus cyclicity from PND 75 to until sacrifice. The assessment for haematology, clinical chemistry, urinalysis, thyroid hormonal levels (T4 and TSH) and flow cytometric analysis of splenic samples for assessment of splenic lymphocyte sub-populations of T "helper" (CD4+) cells, T cytotoxic (CD8+) cells, natural killer (NK) cells and B lymphocytes was conducted for 10 (out of 20) randomly selected males and 10 (out of 20) randomly selected females from each group at termination. Sperm parameters (motility, morphology, and sperm concentration/daily sperm production) were evaluated for all C1A males. The gross pathology and organ weighing was performed on the day of termination. Histopathological examination was conducted on all the tissues collected from vehicle control and high dose group animals, including a quantitative evaluation of ovarian follicular and corpora lutea and an examination of the organ-typic development (i.e., caput, corpus and cauda of the epididymis and the vas deferens for C1A males and ovary with oviduct, uterus, and vagina for C1A females).
In all the tested dose groups (G2-C1A, G3-C1A and G4-C1A), there were no test item- related changes noted in any of the systemic and reproductive endpoints of both sexes. There were no changes noted in sub-populations of splenic lymphocytes in any of the tested dose groups. There were no test item-related histopathological findings noted in the high dose animals of both sexes and no changes noted in ovarian follicle and corpora luteal counts in the high dose females. The organ-typic development examination did not reveal any changes in the high dose animals.

 

All the Cohort 1B (C1B) animals were observed twice daily (pre- and post-dose) for clinical signs, twice daily for mortality/morbidity and once weekly for detailed clinical examination. The body weights were recorded every two days once for the first 2 weeks followed by weaning and once weekly therafter, except for body weights which were recorded twice weekly during the gestation period. The average feed consumption was recorded once weekly, except during cohabitation period. Oestrus cyclicity was evaluated during pre-mating, cohabitation period and at termination for all C1B females. The gross pathology and organ weighing was performed on the day of termination. All the C1B males were evaluated for reproductive performance or indices such as, mating and fertility index and all the C1 females were evaluated for mating and fertility index, pre-coital interval, gestation length, fecundity index, gestation index, parturition index, post-implantation loss and postnatal loss. All C1B females were observed for birth parameters (number of live/dead pups born, litter size, sex ratio, and live birth index per litter) and for litter observations (number of live/dead pups during lactation period, sex ratio and pup survival index per litter).
In all the tested dose groups (G2-C1B, G3-C1B and G4-C1B), there were no test item-related changes noted in any of the systemic and reproductive endpoints of both sexes.

 

All the surviving F2 generation pups from all the tested groups were observed once daily for external examinations and twice daily for mortalities till termination, weighed individually on PND 1, 4, 7, 14 and 21. All the surviving F2 generation pups from all the tested groups, were observed for occurrences of postnatal developmental landmarks, for responses towards to sensory reflexes during postnatal period, measured for anogenital distance on PND 4, and observed for retention of any nipples/areolae in male pups on PND 13. Gross pathological observations were performed in all F2 pups on PND 21.
In all the tested dose groups (G2-C1B, G3-C1B and G4-C1B) litters, there were no test item-related changes in growth parameters, postnatal developmental landmarks, sensory reflexes in F2 generation pups. No gross pathological changes were noted in any of the F2 pups of both sexes.

 

All the Cohort 2A (C2A) animals were observed twice daily (pre- and post-dose) for clinical signs, twice daily for mortality/morbidity and once weekly for detailed clinical examination. The body weights were recorded every two days once for the first 2 weeks followed by weaning and once weekly thereafter. The average feed consumption was recorded once weekly. All the animals were evaluated for occurrence of sexual maturation (i.e., ano-preputial separation for males and vaginal patency for females) and the body weight of each animal was recorded on the day of evidence of sexual maturation. An auditory startle test was performed on PND 25 for all C2A animals. Neurological/functional observations were performed for all C2A animals on PND 64. The gross pathology and brain weighing was performed on the day of termination. The histopathological examination of brain, eyes with optic nerve, peripheral nerve, skeletal muscle and spinal cord was performed from vehicle control and high dose group animals. The gross morphometry was performed on brains collected from all C2A animals.
In all the tested dose groups (G2-C2A, G3-C2A and G4-C2A), there were no test item-related changes noted in any of the systemic and neurotoxicity endpoints of both sexes. There were no test item-related neuropathological changes in any of the high dose animals of both sexes.

 

All the Cohort 2B (C2B) animals were observed once daily for clinical signs and twice daily for mortality/morbidity on PND 21 and 22. The body weights were recorded on PND 21 and 22 (at termination). The gross pathology and brain weighing was performed on the day of termination (i.e., PND 22). The histopathological examination of brain was performed for vehicle control and high dose group animals. The gross morphometry was performed on brains collected from all C2B animals.
In all the tested dose groups (G2-C2B, G3-C2B and G4-C2B), there were no test item-related changes noted in any of the systemic and neurotoxicity endpoints of both sexes. There were no test item-related neuropathological changes in any of the high dose animals of both sexes.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

456.2 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

No reproductive toxicity data are available for strontium hydroxide itself. However, an analogue approach is used for the read-across of reproductive toxicity properties of strontium hydroxide from strontium chloride, based on the hypothesis that properties are likely to be similar as a result of the presence of a common metal ion Sr2+. Further information on the read-across justification is included as attachment in Section 13.

 

In absence of overt general toxicity, the available 90-day repeated dose toxicity study (Kroes, 1977) in rats with an analogue sustance, Strontium chloride, showed evidence of deviations in organ weights of hormonally sensitive organs (i.e. effects were observed on relative organ weights of prostate and thyroid in males and in pituitary of females). Since these effects might be indications for a mode of action related to endocrine disruption and developmental neurotoxicity, an EOGRT study was required for further investigations.

In the EOGRT study (Manjunath, 2022), there were no adverse test item-related changes noted in mean absolute/relative organ weights possibly related to endocrine effects, in the thyroid hormones of parental and cohorts of F1 adult animals in any of the tested dose groups of both sexes. There were no test item-related histopathological findings in high dose animals of parental and cohorts of F1 adult animals. There were no test-item related changes noted in neurotoxicity endpoints of F1 adult animals in any of the tested dose groups of both sexes, and no neuropathological changes in any of the high dose animals of both sexes. No adverse effects were observed on reproduction and developmental toxicity parameters which could be indicative for an endocrine mode of action or endocrine disturbance.

Therefore, based on these results, any concern related to thyroid toxicity, developmental neurotoxicity, or mode(s) of action related to endocrine disruption can be excluded.

Effects on developmental toxicity

Description of key information

Strontium ranelate was not teratogenic in rats and rabbits. Only an increase in the frequency of delays in skeletal ossification and structural abnormalities (way ribs, bent bones, shortened and thickened humerus and misshapen clavicle) was observed in the rat at all dose levels. 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. 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. 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.
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.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-09-23 to 1998-04-08

Reliability:

2 (reliable with restrictions)

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S5 - Detection of toxicity to reporduction for medical products to male fertility, Washington June 24, 1993; ICH Harmonized Tripartite Guideline, Addendum: Toxicity to male fertility; July 1996.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

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 Caesarian section were eleven weeks old and their body weight ranged from 217.9 to 261.2 g. The F0 females intended for delivery of their offspring were thirteen weeks old at the beginning of the pairing period and their body weight ranged from 215.1 to 305.1 g on Gestation Day zero.
- Housing: except within the pairing period, the treated F0 males of subgroups B and D, as well as all females, were housed in individual cages. The untreated males of subgroups A and C were regrouped up to three per cages. After delivery, F0 dams were kept with their F1 offspring in the same cage until weaning. After weaning, all F1 animals from the same litter, not selected as breeders, were housed together by sex until sexual maturation. The F1 breeders were regrouped up to three per cage for each sex from weaning to pairing. After successful mating in F1 males remained regrouped up to three per cage, while the F1 females were housed in individual cages.
- Cage measurements: length =45 cm, width = 30 cm, height = 20 cm
- Diet (ad libitum): U.A.R (Usine Alimentation Rationnelle, Epinay-sur-Orge, France) sterilized feed pellets
- Water (ad libitum): sterilized drinking water
- Acclimation period: about two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 1°C
- Humidity: 55 ± 15%
- Air changes: 14 to 19 times per hour
- Photoperiod (hrs dark / hrs light): 12/12
- Dust level at filter outlet: less than 40 000 particles ≥ 0.5 µm per m^2 an no particle > 5 µm

Route of administration:

oral: gavage

Vehicle:

other: hydroxyethylcellulose

Details on exposure:

VEHICLE
- Batch no.: EI 922
- Physical state: powder
- Stability: until April 2000
- Dissolved in demineralized water at the concentration of 1% w/v

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

The dose volume was calculated on the basis of 10 mL/kg bw (body weight not recorded).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Tests of stability and homogeneity, carried out before the start of the study (Ginot, Y.M., 1990)*, showed that preparations of S12911-2 in 1% hydroxyethylcellulose were stable for twenty-six days when stored in stoppered flasks at room temperature, at concentrations spanning those administered.
Chemical analysis of the preparations administered during the study showed that measured concentrations were close to the intended values.
The pH of the test substance preparations was checked and values were found equal to 7.6 for the first set of preparations.

*Reference:
- Ginot, Y.M. Technologie Servier. Stability Study no.: PA.R. TOX.G04.R02.12911.01, 1990.
Homogeneity Study No.: PA.R. TOX.G02.R02.12911.01, 1990

Details on mating procedure:

- M/F ratio per cage: 1:1 ratio (subgroup B and D: males were treated)
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of gestation (GD0)

Duration of treatment / exposure:

F0 animals:
Female fertility and embryofetal development:
- subgroup A (females): fourteen days prior to pairing with untreated males from the same subgroup and continued throughout pairing and until day seventeen of gestation inclusive

Toxicokinetics during pre-mating and gestation periopds:
- subgroup C (females): fourteen days prior to pairing with untreated males from the same subgroup and continued throughout pairing and until day seventeen of gestation inclusive

Frequency of treatment:

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

Duration of test:

Until gestation day 20

No. of animals per sex per dose:

Subgroup A: 20 females each for the control group and the 3 dose levels (males were also included)
Subgroup C: in each treated group five supplementary females made up subgroup C for toxicokinetic investigations
(Groups B and D: see section 7.8.1 Toxicity to reproduction)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels were chosen on the basis of the results from previous reproductive and general toxicity studies in the rat with S 12911-2.
A study on male fertility, carried out by a laboratory (Müller, W. and Semich, R., 1994)* showed that the reproductive performance were not altered when S 12911-2 was administered by gavage at 825 mg/kg/day for eighty days before pairing with untreated female rats. The test substance was well tolerated. No treatment-related mortality was observed.

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

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

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

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

Maternal examinations:

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

DETAILED CLINICAL OBSERVATIONS: No

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

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

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

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

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

HISTOPATHOLOGY / ORGAN WEIGHTS
- no histopathological examination was carried out on organs with macroscopic anomalies, which were sampled from F0 animals at terminal sacrifice.
Microscopic examination was performed on:
- testes and epididymides of high dose and control F0 males
- complete genital tract of any F0 males which did not fertilize their corresponding females
- ovaries and oviducts of F0 females which were not pregnant (no implantation site detected after uterus staining)
The other organs, including the macroscopic anomalies, were kept in fixative without any further investigation.

Ovaries and uterine content:

EXAMINATION OF UTERINE CONTENTS (F0 FEMALES OF SUBGROUP A)
- after hysterectomy, the corpora lutea, the implantation sites, the live foetuses and the resorptions were counted
- an external macroscopic examination was performed on the foetuses and the placentas, which were then weighed individually
- the sex of each foetus was determined either during the organ inspection or during the evisceration of those intended for the skeletal examination

Fetal examinations:

Examination of foetuses of F0 females of subgroup A)
Foetuses were killed and half of the foetuses from each litter were allocated to skeletal examination, and half of them were allocated to soft tissue inspection
- skeletal examination was performed on foetuses of all groups
- organ examination included only foetuses of the high dose group and control group because no biologically significant difference was observed in the findings between these two groups.
- the sex of each foetus was determined either during the organ inspection or during the evisceration of those intended for the skeletal examination.

Statistics:

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

Indices:

Fertility Index

Details on maternal toxic effects:

Maternal toxic effects:no effects

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

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

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

GROSS PATHOLOGY
- autopsy revealed no macroscopic organ anomaly

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

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

Dose descriptor:

NOAEL

Remarks:

(F0 female)

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: other:

Dose descriptor:

NOAEL

Effect level:

< 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: other:

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

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

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

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

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

TOXICOKINETIC EVALUATION

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

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

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

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

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

Conclusions:

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

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

118 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study to examine the effects of strontium ranelate on embryo-fetal and pre-/postnatal development (K. Momburg, 2001) of rats is regarded as relevant and reliable to evaluate the effects of strontium. In addition, a study on the embryo-fetal development of rabbits (K. Momberg 1999) with strontium ranelate is available to complete the database for this endpoint.

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

No developmental toxicity / teratogenicity data are available for strontium hydroxide itself. However, an analogue approach is used for the read-across of developmental toxicity / teratogenicity properties of strontium hydroxide from strontium ranelate, based on the hypothesis that properties are likely to be similar as a result of the presence of a common metal ion Sr2+. Further information on the read-across justification is included as attachment in Section 13.

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 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.

In addition, in the study published by Lansdown et al. (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 indicated 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 which corresponds to an external dose of about 83 mg Sr/kg bw/d (equal to 115 mg Sr(OH)₂/kg bw/d) to female rats. However, the study was conducted only in a small number of females (p=3) per group and only a limited number of parameters were 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 overall evaluation of the available data for strontium on reproduction and developmental toxicity, no classification and labelling for reproduction is deemed to be justified according to regulation (EC) 1272/2008.