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Toxicity to reproduction

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Administrative data

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
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information
Reference
Endpoint:
extended one-generation reproductive toxicity - with F2 generation and developmental neurotoxicity (Cohorts 1A, 1B with extension, 2A and 2B)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included as attachment in Section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Effect level:
328.56 mg/kg bw/day (nominal)
Based on:
element
Remarks:
metal ion based
Sex:
male/female
Basis for effect level:
other: No adverse effects observed for systemic toxicity, reproduction and developmental toxicity endpoints at the highest dose level
Key result
Dose descriptor:
NOAEL
Remarks:
Cohort 1B
Effect level:
328.56 mg/kg bw/day (nominal)
Based on:
element
Remarks:
metal ion based
Sex:
male/female
Basis for effect level:
other: No adverse effects observed for systemic toxicity, reproduction and developmental toxicity endpoints at the highest dose level
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
328.56 mg/kg bw/day (nominal)
Based on:
element
Remarks:
metal ion based
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:
328.56 mg/kg bw/day (nominal)
Based on:
element
Remarks:
metal ion based
Sex:
male/female
Basis for effect level:
other: No adverse effects observed for systemic toxicity and developmental toxicity endpoints at the highest dose level
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 2B)
Effect level:
328.56 mg/kg bw/day (nominal)
Based on:
element
Remarks:
metal ion based
Sex:
male/female
Basis for effect level:
other: No adverse effects observed for developmental toxicity endpoint at the highest dose level
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
328.56 mg/kg bw/day (nominal)
Based on:
element
Remarks:
metal ion based
Sex:
male/female
Basis for effect level:
other: No adverse effects observed for systemic toxicity endpoint at the highest dose level
Key result
Reproductive effects observed:
no
Conclusions:
Based on the read-across from Strontium chloride hexahydrate, Strontium hydroxide is considered to be not toxic to reproduction.
The NOAEL was 328.56 mg Sr/kg/day for systemic toxicity, reproduction and developmental neurotoxicity endpoints, corresponding to 456.2 mg Sr(OH)2/kg/day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
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)
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.

Test material

Constituent 1
Chemical structure
Reference substance name:
Strontium chloride hexahydrate
EC Number:
600-046-7
Cas Number:
10025-70-4
Molecular formula:
Cl2-Sr.6H2-O
IUPAC Name:
Strontium chloride hexahydrate
Test material form:
solid: crystalline
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.

Test animals

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).

Administration / exposure

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%.
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
Doses / concentrationsopen allclose all
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

Examinations

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 and coagulation parameters are listed in Table 1.
- Clinical chemistry parameters are listed in Table 2.

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:
- testis weight, epididymis weight,
- enumeration of cauda epididymis sperm reserves,
- sperm motility,
- sperm morphology,
- testicular spermatid head counts,
- daily sperm production.
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, hair coat development, incisor eruption, eye opening, testes descent.
- Responses for sensory reflexes: surface righting reflex, auditory startle reflex, air righting reflex.
- 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 and coagulation parameters are listed in Table 1.
- Clinical chemistry parameters are listed in Table 2.

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
- Neurological/Functional examination: on PND 64.
a) Home Cage Observations: home cage posture, respiratory pattern, involuntary clonic and tonic movements, vocalization and palpebral closure.
b) Handling Observations: ease to be 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: mobility, gait, arousal, rearing, urination, defecation, tonic involuntary movement, stereotype behavior and grooming.
d) Sensory Observations: approach response, auditory response, touch response, pupil response, tail pinch response and righting reflexes.
e) Neuromuscular Observations: hind limb foot splay, grip strength assessment, motor activity assessment.
f) Physiological Observation: rectal temperature.
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 specified in Table 3 were collected, weighed and preserved.
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 specified in Table 4 were collected, weighed and preserved, but 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.

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 specified in Table 3 were collected, weighed and preserved. 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 specified in Table 5 were collected, weighed and preserved. 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. 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
A quantitative evaluation of ovarian folliclar and corpora lutea was carried out for all females from control and high dose groups of Cohort 1A 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
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) was performed in animals from Cohort 1A.

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

SPLENIC LYMPHOCYTE SUBPOPULATION ANALYSIS
For investigation of pre- and postnatally induced immunotoxic effects, 10 males and 10 females per group from Cohort 1A 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.
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 indicated in Table 6.
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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 in high dose animals of parental animals.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

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):
There were no test item related effects 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 the vehicle control group.
The 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, 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 to be 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.
The 1 (out of 25) male and 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 also no microscopic changes were noted in any of the reproductive organ during conduct of histopathological examination. For the male, 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.
Although not statistically significant:
- the mean post-implantation loss was higher than controls in group G4. This is due to one dam for which there were 6 out of 14 post-implantation losses (i.e. 42.9%).
- the mean post-natal loss was higher than controls in group G4. This is due to 2 dams 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.

Details on results (P0)

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.
Reproductive indices are detailed in Table 7, Uteri-observations are detailed in Table 9, Thyroid Hormone Levels are detailed in Table 10 and Sperm parameters are detailed in Table 11.

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.
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.
Delivery and litter observations are detailed in Table 8.

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.

Effect levels (P0)

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 toxicity, reproduction and developmental toxicity endpoints

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity was recorded 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):
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-C1B and G3-C1B (males),
- lower mean body weight on PND 49 and mean percent change in body weight gain during PND 21 to 49 in group G4-C1B (males),
- higher mean body weight on PND 77, 84, 91 and 98 in group G3-C1B (females),
- lower mean percent change in body weight gain during PND 21 to 56 in group G2-C1B (females),
- higher mean gestational body weight on GD 0, 7, 9 and 11 in group G3-C1B,
- lower mean percent change in body weight gain during GD 7 to 9 in group G4-C1B.
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):
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-C1B; 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
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P1)

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

Details on results (P1)

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 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.
When compared with controls, the mean number of female live pups born per dam was statistically significantly higher in group G3-C1B and the mean number and percentage of post-implantation losses per dam was statistically significantly lower in groups G2-C1B and G3-C1B. However, these difference were considered as incidental and toxicologically not relevant.

Reproductive indices are detailed in Table 7, Delivery and litter observations are detailed in Table 8, Uteri-observations are detailed in Table 9 and Occurrence of sexual maturity is detailed in Table 15.

Effect levels (P1)

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 toxicity, reproduction and developmental toxicity endpoints

Results: F1 generation

General toxicity (F1)

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, and no mortality/morbidity was recorded 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-C1A on PND 99 and 100 and one female from group G4-C1A 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.

F1 adults - Cohort 2A:
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.
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:
At birth:
- 1 male and 1 female pup from the vehicle control group,
- 3 males from the low dose group,
- 2 males and 1 female pup from the mid dose group and one cannibalized pup was found with undetermined sex,
- 7 male and 6 female pups from the high dose group.
On PND 4, 3 male and 2 female pups from the G4 group were found dead.
These incidences lacked dose correlation and gross findings and hence considered as incidental findings without any relation to administration of test item.

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:
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; it was 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-C1A (males).
- higher mean body weight on PND 21 in all the tested C1A groups (females),
- higher mean body weight on PND 28 in group G3-C1A (females),
- lower mean percent change in body weight gain during PND 21 to 28 in group G4-C1A (females),
- lower mean percent change in body weight gain during PND 21 to 49 in group G3-C1A (females),
- lower mean percent change in body weight gain during PND 21 to 104 in all the tested C1A 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-C2A (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-C1A (males),
- lower mean feed consumption during week 4 in group G2-C1A (males),
- higher mean feed consumption during weeks 3 and 5 in group G3-C1A (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-C2A (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-C1A 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):
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-C1A 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 occurrences of sexual maturation (day of occurrence of vaginal patency/opening) and mean body weight on the day of sexual maturation in any of the tested dose group females 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.

Cohorts 1B and 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:
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-C1A - higher absolute and relative weight of mandibular lymph nodes (males).
- Group G4-C1A - 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-C2B and G4-C2B (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/C1B/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):
Thyroid hormones - F1 pups:
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.

Thyroid hormones - Cohort 1A:
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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Auditory startle test - Cohort 2A:
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 - Cohort 2A:
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-C2A (females) and the mean no. of movement counts in groups G3-C2A and G4-C2A (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 (F1)

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 C1A groups of both sexes when compared with vehicle control group.

Details on results (F1)

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.
Anogenital distance (AGD) on PND 4 of F1 pups is detailed in Table 12 and The Occurrence of Postnatal Developmental Landmarks and Response to Sensory Refexes are detailed in Table 13.

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.
Thyroid Hormone Levels are detailed in Table 10, Sperm parameters are detailed in Table 11, Splenic lymphocyte sub-populations are detailed in Table 14, and Occurrence of sexual maturity is detailed in Table 15.

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.
Occurrence of sexual maturity is detailed in Table 15.

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.

Effect levels (F1)

open allclose all
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

Results: F2 generation

General toxicity (F2)

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):
At birth: 1 female pup from the low dose group was found dead.
On PND 2:
- 1 male and 2 female pups from the vehicle control group,
- 2 male pups from the low dose group,
- 3 female pups from mid dose group.
On PND 4:
- 3 males and 2 female pups from the low dose group,
- 3 males from the high dose group.
On PND 5, a female pup from the high dose group was found dead.
These incidences lacked dose correlation and gross findings and hence considered as incidental findings without any relation to administration of test item.
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

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Details on results (F2)

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.
Anogenital distance (AGD) on PND 4 of F1 pups is detailed in Table 12 and The Occurrence of Postnatal Developmental Landmarks and Response to Sensory Refexes are detailed in Table 13.

Effect levels (F2)

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 examined endpoints at the highest dose level

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 7. Reproductive indices of the P generation and F1 adult genenration (Cohort 1B)

















































































































































Dose level (mg/kg/day)



0



250



500



1000



P generation



Female mating index (%)


96.0096.0010096.00

Female fertility index (%)


91.6787.5084.0087.50

Male mating index (%)


96.0096.0010096.00

Male fertility index (%)


91.6787.5084.0087.50
Pre-coital interval / Copulatory interval3.754.295.642.88

Gestation length (days)


22.6422.2422.6222.33

Pregnancy index (%)


91.6787.5084.0087.50

Gestation index (%)


100100100100

Parturition index (%)


100100100100

F1 adult generation (Cohort 1B)



Female mating index (%)


100100100100

Female fertility index (%)


85.0080.0080.0080.00

Male mating index (%)


100100100100

Male fertility index (%)


85.0080.0080.0085.00

Pre-coital interval / Copulatory interval


6.907.357.056.20

Gestation length (days)


23.2423.5023.1323.35

Pregnancy index (%)


85.0080.0080.0085.00

Gestation index (%)


100100100100

Parturition index (%)


100100100100

 


Table 8. Delivery and litter observations of the P generation and F1 adult genenration (Cohort 1B)































































































































































Dose level (mg/kg/day)



0



250



500



1000


P generation
Live birth index (%)99.4798.9898.2895.09
Sex ratio (male/female) at birth1.321.051.001.20
Pup survival index between LD 1 to 4 (%)10010010098.11
Sex ratio (male/female) on LD 41.321.051.001.19
Pup survival index between LD 5 to 7 (%)100100100100
Sex ratio (male/female) on LD 71.061.041.011.01
Pup survival index between LD 8 to 14 (%)100100100100
Sex ratio (male/female) on LD 141.061.041.011.01
Pup survival index between LD 15 to 21 (%)100100100100
Sex ratio (male/female) on LD 211.061.041.011.01
F1 adult generation (Cohort 1B)
Live birth index (%)99.3599.4399.3897.65
Sex ratio (male/female) at birth1.361.210.831.45
Pup survival index between LD 1 to 4 (%)97.9495.9898.2795.42
Sex ratio (male/female) on LD 41.361.160.881.38
Pup survival index between LD 5 to 7 (%)10010010094.12
Sex ratio (male/female) on LD 71.291.130.951.35
Pup survival index between LD 8 to 14 (%)100100100100
Sex ratio (male/female) on LD 141.291.130.951.35
Pup survival index between LD 15 to 21 (%)100100100100
Sex ratio (male/female) on LD 211.291.130.951.35

 


Table 9. Uteri-observations of the P generation and F1 adult genenration (Cohort 1B)







































































































Dose level (mg/kg/day)



0



250



500



1000


P generation
Number of implantations12.5011.1410.5712.33
Number of viable (live) pups12.4110.9510.2411.67
Post-implantation loss (no.)0.090.190.330.67
Post-implantation loss (%)0.531.363.125.21
Postnatal loss (no.)0.000.000.000.24
Postnatal loss (%)0.000.000.001.89
F1 adult generation (Cohort 1B)
Number of implantations7.769.639.508.65
Number of viable (live) pups7.069.509.388.18
Post-implantation loss (no.)0.710.13*0.13*0.47
Post-implantation loss (%)13.121.26*1.25*7.44
Postnatal loss (no.)0.180.440.190.24
Postnatal loss (%)2.064.021.736.54

* = mean value of group is significantly different from control


 


Table 10. Thyroid hormone concentrations in P generation, F1 pups and F1 Cohort 1A animals









































































































Dose level (mg/kg/day)



0



250



500



1000



Males P generation



T4 (ng/mL)


85.04780.84385.93380.070

TSH (µIU/mL)


1.0070.8911.4351.072

Females P generation



T4 (ng/mL)


67.21366.67067.36767.104

TSH (µIU/mL)


0.9790.8971.2830.715

F1 pups



T4 (ng/mL) - PND 4


62.02158.91657.74058.892

T4 (ng/mL) - PND 21


56.19155.32777.191*73.033*

TSH (µIU/mL) - PND 21


1.0620.8610.9631.036

Males Cohort 1A



T4 (ng/L)


51.36057.743*61.697*58.091*

TSH (µIUg/mL)


1.0510.8570.7971.034

Females Cohort 1A



T4 (ng/L)


73.26774.70372.68653.442*

TSH (µIUg/mL)


1.0880.8390.9321.132

* = mean value of group is significantly different from control


 


Table 11. Sperm parameters in P generation and F1 Cohort 1A animals





















































































































Dose level (mg/kg/day)



0



250



500



1000



Males P generation


Sperm motility (%)83.2482.7282.7683.22
Sperms with abnormality (no.)7.805.84*5.92*7.72
Sperms with abnormality (%)3.902.92*2.96*3.86
Normal sperms (no.)192.20194.16*194.08*192.28
Normal sperms (%)96.1097.08*97.04*96.14
Average sperm head count (no.)154.44163.34162.08161.79
Daily sperm production per animal43.0 million47.9 million*46.9 million*45.3 million

Males Cohort 1A


Sperm motility (%)89.389.290.089.3
Sperms with abnormality (no.)7.206.606.707.40

Sperms with abnormality (%)


3.603.303.353.70

Normal sperms (no.)


192.80193.40193.30192.60

Normal sperms (%)


96.4096.7096.6596.30

Average sperm head count (no.)


180.58177.76181.11177.06

Daily sperm production per animal


48.5 million46.3 million50.0 million48.3 million

* = mean value of group is significantly different from control


 


Table 12. Anogenital distance (AGD) on PND 4 and Retention of nipple/areolae on PND13 of F1 and F2 pups





























































Dose level (mg/kg/day)



0



250



500



1000



F1 pups



AGD ratio - males


2.152.172.152.16

AGD ratio - females


1.171.181.191.18

Retention of nipple/areolae - males


0.000.000.000.00

F2 pups



AGD ratio - males


2.102.152.152.11

AGD ratio - females


1.201.191.201.22

Retention of nipple/areolae - males


0.000.000.000.00

 


Table 13. Occurence (PND) for Postnatal Developmental Landmarks and Response to Sensory Reflexes of F1 and F2 pups



































































































































Dose level (mg/kg/day)



0



250



500



1000



F1 pups


Pinna unfolding2.392.292.362.29
Hair coat/fur development4.514.514.544.42
Incisor Eruption10.1810.3110.2310.22
Eye opening13.3113.3413.2913.28
Testes descent19.4119.3619.4319.37
Surface righting reflex4.274.234.284.24
Auditory startle reflex12.6312.7012.6512.62
Air righting reflex17.5217.5817.5317.59

F2 pups


Pinna unfolding2.392.462.512.43
Hair coat/fur development4.985.055.064.81
Incisor Eruption9.8410.109.969.91
Eye opening13.7313.9113.9413.62
Testes descent19.4019.6219.4919.66
Surface righting reflex4.414.424.494.40
Auditory startle reflex12.7412.8212.8412.81
Air righting reflex17.6017.6717.7317.70

 


Table 14. Splenic lymphocyte subpopulations of Cohort 1A











































































Dose level (mg/kg/day)



0



250



500



1000



Cohort 1A males


B lymphocytes 31.1132.0631.3330.91
T helper cells (Th/CD4+)58.2759.0261.8961.31
T cytotoxic cells (Tc/CD8+)33.2435.5232.2333.07
Natural killer cells (NK)2.912.472.632.55

Cohort 1A females


B lymphocytes 32.6233.6332.3732.72
T helper cells (Th/CD4+)62.8162.2759.4255.74
T cytotoxic cells (Tc/CD8+)31.6231.6835.1439.32
Natural killer cells (NK)2.392.492.432.20

 


Table 15. Occurrence (PND) of sexual maturity of Cohorts 1A, 1B and 2A







































































Dose level (mg/kg/day)



0



250



500



1000



Cohort 1A


Occurrence of balano-preputial separation (males)43.0542.2042.4042.40
Occurrence of vaginal opening (females)33.6033.2033.4033.64
Occurrence of first cornified cells (females)34.7034.0034.1534.55

Cohort 1B


Occurrence of balano-preputial separation (males)42.3542.7042.9042.55
Occurrence of vaginal opening (females)33.3533.6533.7034.10
Cohort 2A
Occurrence of balano-preputial separation (males)43.1043.0041.8042.50
Occurrence of vaginal opening (females)35.1034.1035.1033.70

 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of the EOGRT study:
- The repeated exposure of test item, Strontium chloride hexahydrate, by oral gavage route with the dose levels of 250, 500 and 1000 mg/kg bw/day to parental (P) generation did not produce any systemic and reproductive toxicity in any of the tested dose groups. 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, reproduction and developmental 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, 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, reproduction and developmental 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, 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:

The objective of this Extended One-Generation Reproductive Toxicity (EOGRT) study in Sprague Dawley rats was to evaluate the effects of test item, Strontium chloride hexahydrate, by oral gavage route in Sprague-Dawley rats as a result of pre- and postnatal exposure on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females, young and adult offspring. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, was expected to identify specific target organs in the offspring. In addition, this study was conducted to provide information about the effects of Strontium chloride hexahydrate on the integrity and performance of the adult male and female reproductive systems. This EOGRT study was also conducted to evaluate gonadal function, the oestrus cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition and lactation and to provide sufficient information on developmental neurotoxicity and developmental immunotoxicity assessments. The data derived from these tests were expected to allow the determination of No-Observed Adverse Effect Levels (NOAEL)/No-Observed Effect Levels (NOEL)/Lowest Observed Adverse Effect Levels (LOAEL)/ Lowest Observed Effect Levels (LOEL).


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 and homogeneity of the test item in dose formulations was established before initiation of the treatment. The dose concentrations were stable up to 48 hours at room temperature with dose concentrations of 10 mg/mL and 100 mg/mL in water. 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%.


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 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 and feed consumption were 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 C1B 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. Oestrus cyclicity was evaluated during pre-mating, cohabitation period and at termination for all C1B females. The body weights (on gestation day 0, 5, 7, 9, 11, 13, 15, 17 and 20; on lactation day 1, 4, 7, 14 and 21) and feed consumption (during gestation day 0 to 7, 7 to 14 and 14 to 20; during lactation day 1 to 4, 4 to 7, 7 to 14 and 14 to 21) were recorded during gestation and lactation periods. 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 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 and feed consumption were 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. 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 noted in any of the high dose animals of both sexes.


All the Cohort 2B 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 neuropathological changes in any of the high dose animals of both sexes.