Registration Dossier

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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 September 2016 to 07 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
examined within the range of a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 September 2016 to 07 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening
Test” adopted on 29 July 2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: ESD0021912
- Expiration date of the lot/batch: 17.03.2020
- Purity test date: 100 % [w/w]
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility and stability of the test substance in the vehicle: Vehicle used was corn oil and test item
formulation was stable for 24 hours at room temperature
FORM AS APPLIED IN THE TEST: Liquid
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: In-house bred animals
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: Male: 220 to 260 g
Female: 200 to 240 g
- Housing: standard polypropylene cage (size: L 430 x B 285 x H 150 mm)
- Diet (e.g. ad libitum): Teklad Certified (2014SC) Global 14 % Protein Rodent Maintenance Diet -
Pellet (Manufactured by Envigo)
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed
to ultraviolet rays in Aquaguard water filter with purifier
- Acclimation period: 29 September 2016 to 17 October 2016
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 to 22.9oC
- Humidity (%): 48 to 69%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle
IN-LIFE DATES: From: 18 October 2016 To: 24 December 2016
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Test item formulation was prepared daily before administration
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During first week and week 5 of treatment
Duration of treatment / exposure:
Males: 37 days
Females: Approximately 70 days including premating, mating, gestation and lactation periods.
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Main group: 12 males and 12 females per dose
Recovery group: 5 males and 5 females per dose (control and high dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control/ vehicle control recovery, low dose, mid dose and high dose/ high dose recovery groups respectively were selected in consultation with the sponsor based on the results of dose range finding study of test item when administered through oral gavage for a period of 14 consecutive days to Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 1944).
These doses were selected as the test item Hostastat FE 20 LIQ when administered orally to Sprague Dawley rats once daily for 14 consecutive days did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).

- Rationale for animal assignment: The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ± 20%
Positive control:
no
Observations and examinations performed and frequency:
Parental animals: Observations and examinations
CAGE SIDE OBSERVATIONS: Yes
- Once daily throughout the experimental period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly
- Signs noted included, but not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14
and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was measured for main group animals once a week during premating and for main group males during post mating period. Feed consumption was not measured during mating period for both males and females. Thereafter, feed consumption for females was recorded during gestation days 0 to 7, 7 to 14 and 14 to 20 and on lactation days 1 to 4, 4 to 7 and 7 to 13. Feed consumption was measured for recovery group animals once a week throughout the experimental period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmological examination was carried out once before treatment for all animals, during end of the dosing period for males (shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice) of vehicle control and high dose main group animals and during last week for recovery group animals. The examination was not extended to lower dose groups as there were no treatment related changes noted in high dose group animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Dams at termination (lactation day 14), adult males at termination (after completion of 37 days of treatment)
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: five males and five females randomly selected from each main group and from all recovery group animals
- Parameters checked: at least according to guideline

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Dams at termination (lactation day 14), adult males at termination (after completion of 37 days of treatment)
- Animals fasted: Yes
- How many animals: five males and five females randomly selected from each main group and from all recovery group animals
- Parameters checked: at least according to guideline, including T4 levels

URINALYSIS: Yes
- Time schedule for collection of urine: At termination of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Albumin, Leucocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: five males and five females, randomly selected from each group towards the end of the dosing period for males (shortly prior to scheduled sacrifice
- Dose groups that were examined: all groups
- Battery of functions tested: sensory activity / grip strength / motor activity / Home Cage Observations / Handling Observations / Open Field Observations / Neuromuscular Observations / Physiological Observation (Rectal temperature)

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Estrous cyclicity (parental animals)
Oestrus cycles were monitored during the acclimatization to evaluate its normal oestrus cyclicity (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal sm
ears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce
pseudopregnancy. Oestrus cyclicity was also monitored on the day of sacrifice for females. Recovery group females were not evaluated for oestrus cyclicity.

Sacrifice and pathology:
Postmortem examinations (parental animals)
SACRIFICE
- Male Animals: All Animals after 37 days of treatment
- Female Animals: All animals were sacrificed on Lactation day 14.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera: Yes
HISTOPATHOLOGY / ORGAN WEIGHTS
Control and high dose groups examined, examination range at least according to guideline, with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure
Statistics:
One-way ANOVA with Dunnett’s post test
Kruskal-Wallis followed by the Mann-Whitney test if differences were indicated
Chi-square test/ Fischer's Exact Test
The Parameters include Body weight , Change in body weight, Feed consumption, Copulatory interval, Gestation length, Hematology, Clinical chemistryAbsolute organ weights, Relative organ weights, FOB, Body temperature, Defecation, Urination, Rearing, Grip strength, Litter weights , Pup weight , Preimplantation loss, Pre natal loss, Post natal loss, Total No. of early/late resorptions/dam, Corpora lutea/dam, Implantations/dam, No. of pups/dam, Sex ratio, Mean litter size, Mean pup weight, Pregnancy rate, No. of dams with/without live pups, No. of dams with/without dead pups, No. of litters with/without resorptions.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity observed at any of the tested dose main groups of either sex during premating period and mating period, during post mating period in males, during gestation/lactation period in females and throughout the experimental period in recovery group animals.
The detailed clinical examination of animals did not reveal any treatment related changes at any of the main and recovery group animals of either sex.
Mortality:
no mortality observed
Description (incidence):
There were no mortality/morbidity observed at any of the tested dose main groups of either sex during premating period and mating period, during post mating period in males, during gestation/lactation period in females and throughout the experimental period in recovery group animals.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment related changes in mean body weight and percent change in body weight with respect to day 1 at any of the main and recovery group animals of either sex
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment related changes noted in feed consumption at any of the doses at any of the main and recovery group animals of either sex.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular changes observed in vehicle control and high dose group animals during the ophthalmological examination for males conducted at the end of the dosing period, for females during the lactation period and for recovery group animals at the end of recovery period.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in haematology at any of the tested doses of either sex. However, statistically significant decrease in prothrombin time in low, mid and high dose group females and statistically significant increase in activated prothrombin time in the recovery group females when compared with vehicle control was noted. This change is considered as incidental and not treatment related, as the values are within historical control range and same changes not observed in males.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in clinical chemistry at any of the doses tested in both the sex.

However, in main group males statistically significant decrease in alkaline phosphatase level at low dose and statistically significant increase in sodium level at mid and high dose noted when compared with vehicle control. In females, statistically significant increase in cholesterol at mid dose, statistically significant increase in albumin at high dose and statistically significant increase in calcium at high dose were noted when compared with vehicle control.

In recovery males, statistically significant increase in creatinine, total protein, albumin, and chloride and statistically significant decrease in phosphorous at recovery when compared with vehicle control and in recovery females statistically significant increase in creatinine, cholesterol, albumin and sodium and statistically significant decrease in phosphorous at recovery when compared with vehicle control was noted.

These changes are considered as an incidental and not treatment related, as the values are within historical control range and is due to biological variations.

Statistically significant decrease in T4 levels in high dose males when compared with vehicle control group were noted when compared with vehicle control group.
The variation observed in the T4 levels could not be attributed as treatment related as there were no microscopic observations noted in thyroid gland and no effects were noted in thyroid weight at all the tested dose groups. This significant variation could be due to the increased T4 levels in vehicle control group males (Animal No. Rb8893: 99.593 ng/mL and Animal No. Rb8898: 99.416 ng/mL) and low dose group male (Animal No. Rb8905: 110.605 ng/mL). The T4 levels obtained across the groups are within historical control range. Hence, the variation observed in the T4 levels is considered to be incidental

Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed at any of the dose tested. However, statistically significant decrease in urine volume at mid dose (males), significant decrease in urobilinogen in low, mid and high dose males and significant increase in pH at the end of recovery (females) was noted. This change is considered as an incidental and not treatment related, as the values are within historical control range.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The detailed clinical examination of animals did not reveal any treatment related changes at any of the main and recovery group animals of either sex.
There were no changes observed in the neurological/functional examination conducted for main group males (prior to scheduled sacrifice), for main group females during the lactation period (prior to scheduled sacrifice) and for all recovery group animals at the end of recovery period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in the absolute and relative organ weights at any of the doses tested of both the sex in main and recovery group animals.
However, statistically significant decrease in absolute brain and heart weight high dose males, statistically significant increase in absolute and relative spleen and heart weight in mid dose females and statistically significant increase in absolute PSC weight (Prostate+Seminal vesicles with coagulating glands) in low dose males were noted when compared with vehicle control. This change can be considered as incidental and not treatment related, as there were no gross and microscopic findings were noted in these organs.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes (both external and internal) observed at any of the doses
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment related histopathological findings noticed in high dose group animals.
Testes, which were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all expected associations.
All other observed histopathological lesions in high dose group animals, considered as spontaneous and incidental to Sprague Dawley rats of this particular age as they lack in consistency.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no changes observed in the oestrus cyclicity of females at any of the doses tested during pre-mating treatment, mating treatment and on lactation day 14.
There were no treatment related changes in gestation and lactation body weight, percent change in gestation and lactation body weights, feed consumption during gestation period and lactation period, uteri observations, litter and pup weights, anogenital distance of pups (see in detail in chapter 7.8.1).
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of treatment-related adverse effects
Key result
Critical effects observed:
no

The dose formulation analysis for concentration verification was performed during week 1 and 5 of treatment period and the results were within acceptable limits.

Conclusions:
Based on the results of this study, the NOAEL was 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), and to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 under the experimental conditions employed in this study.
Executive summary:

The test item, was evaluated for possible adverse effects following repeated oral dosing to males for 37 days and to females, two weeks pre-mating period, during mating, pregnancy

(gestation) and up to lactation day 13 to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus,

parturition, and early neonatal development.

A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups. G1 (control), G2 (low dose), G3 (mid dose) and G4 (high dose) groups consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle[Corn oil], the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of100, 300 and 1000 mg/kg body weight for low dose, mid dose and high dose/high dose recovery groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. The main group males were treated for two weeks pre-mating, during mating and up to the day before

sacrifice during post-mating period (total of 37 days of treatment). The main group females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after

which the pups were sacrificed on lactation Day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed). The recovery group males and females were treated until the

first scheduled sacrifice of dams and kept without treatment for a further 16 days observation. The dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.

All animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight and feed consumption. Ophthalmological examination was carried out once before treatment

for all animals, duringend of the dosing period for males (shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice)for vehicle control and high dose

group animals andduring last weekfor recovery group animals.Neurological/Functional examination was performed for five males and five females, randomly selected from each group towards the end of the

dosing period for males (shortly prior to scheduled sacrifice), during lactation period for females (shortly prior to scheduled sacrifice) and from both the recovery group animals towards the end of the recovery

period (shortly prior to scheduled sacrifice).

The clinical pathology (haematological and clinical chemistry) examinations were conducted in five males and five females from each main group (randomly selected) andfor all recovery group animals.

Urinalysis was performed from the five randomly selected males of each main group during the last week of the treatment period and for all recovery animals at termination.

Gross pathology and organ weighing were performed on day 38 for males, on lactation day 14 for dams and on day 68 for recovery group animals. The number of corpora lutea, implantation sites and resorptions for dams were recorded.

The animals did not reveal any clinical signs of toxicity. No mortality or morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight,

feed consumption, ophthalmoscopic examination, neurological/functional examination, haematology, clinical chemistry, urine analysis nor in organ weights (both absolute and relative) were observed in both

the sex of main and recovery group animals. The dams did not reveal any treatment related changes in gestation and lactation body weight, percent change in gestation and lactation body weights, feed consumption during gestation period and lactation period, uteri observations, litter and pup weights, anogenital distance of pups. The animals did not reveal any treatment related gross pathological and histopathological findings.

Based on the results discussed, the No Observed Adverse Effect Level (NOAEL) of the test item was found to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), and to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 under the experimental conditions employed in this study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” adopted on 29 July 2016
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals: 14 days
- Basis for dose level selection: The doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control/vehicle control recovery, low dose, mid dose and high dose/ high dose recovery groups respectively were selected in consultation with the sponsor based on the results of dose range finding study of test item Hostastat FE 20 LIQ when administered through oral gavage for a period of 14 consecutive days to Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 1944).
These doses were selected as the test item Hostastat FE 20 LIQ when administered orally to Sprague Dawley rats once daily for 14 consecutive days did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight).
- Route of administration: Oral

Test material

Constituent 1
Chemical structure
Reference substance name:
Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]
EC Number:
285-089-6
EC Name:
Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]
Cas Number:
85029-63-6
Molecular formula:
not applicable, mixture
IUPAC Name:
Fatty acids, coco, esters with 3,3'-oxybis[1,2-propanediol]
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: ESD0021912
- Expiration date of the lot/batch: 17.03.2020
- Purity test date: 100 % [w/w]

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Solubility and stability of the test substance in the vehicle: Vehicle used was corn oil and test item formulation was stable for 24 hours at room temperature

FORM AS APPLIED IN THE TEST: Liquid

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: In-house bred animals
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: Male: 220 to 260 g
Female: 200 to 240 g
- Housing: standard polypropylene cage (size: L 430 x B 285 x H 150 mm)
- Diet (e.g. ad libitum): Teklad Certified (2014SC) Global 14 % Protein Rodent Maintenance Diet - Pellet (Manufactured by Envigo)
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to ultraviolet rays in Aquaguard water filter with purifier
- Acclimation period: 29 September 2016 to 17 October 2016
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 to 22.9oC
- Humidity (%): 48 to 69%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle
IN-LIFE DATES: From: 18 October 2016 To: 24 December 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Test item formulation was prepared daily before administration
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 21 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): females were housed individually
- Any other deviations from standard protocol: No
Analytical verification of doses or concentrations:
yes
Remarks:
during first week and week 5 of treatment
Details on analytical verification of doses or concentrations:
vehicle control/ vehicle control recovery: 0 mg/mL
Low dose: 10 mg/mL
Mid dose: 30 mg/mL
High dose/ high dose recovery: 100 mg/mL
Duration of treatment / exposure:
Males: 37 days
Females: Approximately 70 days including premating, mating, gestation and lactation periods.
Frequency of treatment:
Once daily
Details on study schedule:
- Age at mating of the mated animals in the study: 13 to 14 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control/vehicle control recovery
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High dose/high dose recovery
No. of animals per sex per dose:
Main group: 12 male and 12 female per dose
Recovery group: 05 male and 05 female per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses of 0, 100, 300 and 1000 mg/kg body weight for vehicle control/vehicle control recovery, low dose, mid dose and high dose/ high dose recovery groups respectively were selected in consultation with the sponsor based on the results of dose range finding study of test item Hostastat FE 20 LIQ when administered through oral gavage for a period of 14 consecutive days to Sprague Dawley Rats (Bioneeds Study No.: BIO-TX 1944).
These doses were selected as the test item Hostastat FE 20 LIQ when administered orally to Sprague Dawley rats once daily for 14 consecutive days did not reveal any treatment related effects at all the tested doses (100, 300 and 1000 mg/kg body weight)

- Rationale for animal assignment: The animals were weighed and arranged in ascending order of their body weights. These body weight stratified animals were distributed to all the groups using Microsoft Excel Spreadsheet, such that body weight variation of animals selected for the study did not exceed ± 20%
Positive control:
Not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Once daily throughout the experimental period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly once

BODY WEIGHT: Yes
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The recovery group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination
Oestrous cyclicity (parental animals):
Oestrus cycles were monitored during the acclimatization to evaluate its normal oestrus cyclicity (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Vaginal smears were monitored daily from the beginning of the treatment period until evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid disturbance of mucosa, which may induce pseudopregnancy. Oestrus cyclicity was also monitored on the day of sacrifice for females. Recovery group females were not evaluated for oestrus cyclicity
Sperm parameters (parental animals):
Not applicable
Litter observations:
The day of littering was considered as lactation day 1. The number of pups born (dead and live) in a litter, sex and external observations were recorded at birth. Individual body weight of live pups on lactation day 1 (within 24 hours of parturition), 4, 7 and 13 was recorded. The anogenital distance of each pup was measured on postnatal day 4 (lactation day 4). The number of nipples/areolae in male pups were counted on postnatal day 13 (lactation day 13)
Postmortem examinations (parental animals):
SACRIFICE
- Male Animals: All Animals
- Female Animals: All animals were sacrificed on Lactation day 14.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera: Yes

HISTOPATHOLOGY / ORGAN WEIGHTS
Control and high dose groups examined, examination range at least according to guideline, with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure
Postmortem examinations (offspring):
The pups were sacrificed on lactation day 13 and the sacrificed pups and dead pups were examined for gross abnormalities and the findings were recorded.
Statistics:
(SPSS software version 22)
One-way ANOVA with Dunnett’s post test
Kruskal-Wallis followed by the Mann-Whitney test if differences were indicated
Chi-square test/ Fischer's Exact Test
The Parameters include Body weight , Change in body weight, Feed consumption, Copulatory interval, Gestation length, Hematology, Clinical chemistryAbsolute organ weights, Relative organ weights, FOB, Body temperature, Defecation, Urination, Rearing, Grip strength, Litter weights , Pup weight , Pre implantation loss, Pre natal loss
Post natal loss, Total No. of early/late resorptions/dam, Corpora lutea/dam, Implantations/dam, No. of pups/dam, Sex ratio, Mean litter size, Mean pup weight, Pregnancy rate, No. of dams with/without live pups, No. of dams with/without dead pups, No. of litters with/without resorptions.
Reproductive indices:
Male Fertility Index (%) = Number of Males Impregnating Females
------------------------------------------------- x 100
Number of Males used for Mating
Female Fertility Index (%) = Number of Females confirmed with Pregnancy
--------------------------------------------------------- x 100
Number of Females used for Mating
Parturition (%) = Number Of Parturitions
------------------------------- x 100
Number Of Pregnancies
Copulatory Interval (Days) = Date of Initiation of Cohabitation - Date of Confirmation of Mating
Gestation Length (days) = Date of Delivery - Date of Confirmation of Mating
Offspring viability indices:
Male/Female Sex Ratio = Number Male Pups Per Dam
-----------------------------------
Number Female Pups Per Dam
Live Birth Index (%) = Number Of Viable Pups Born
------------------------------------ x 100
Number Of Pups Born
Lactation Day 4
Pup Survival Index (%) = Number Of Viable Pups on Lactation Day 4
----------------------------------------------------- x 100
Number Of Pups Born
Lactation Day 7
Pup Survival Index (%) = Number Of Viable Pups on Lactation Day 7
------------------------------------------------------ x 100
Number Of Viable Pups on Lactation Day 4

Lactation Day 13 Pup Survival Index (%) = Number Of Viable Pups on Lactation Day 13
------------------------------------------------------- x 100
Number Of Viable Pups on Lactation Day 7

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 observed at any of the tested dose main groups of either sex during premating period and mating period, during post mating period in males, during gestation/l
actation period in females and throughout the experimental period in recovery group animals.
The detailed clinical examination of animals did not reveal any treatment related changes at any of the main and recovery group animals of either sex.
Dermal irritation (if dermal study):
not specified
Description (incidence and severity):
Not applicable
Mortality:
no mortality observed
Description (incidence):
There were no mortality/morbidity observed at any of the tested dose main groups of either sex during premating period and mating period, during post mating period in males, during gestation/lactation
period in females and throughout the experimental period in recovery group animals.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment related changes in mean body weight and percent change in body weight with respect to day 1 at any of the main and recovery group animals of either sex
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment related changes noted in feed consumption at any of the doses at any of the main and recovery group animals of either sex.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular changes observed in vehicle control and high dose group animals during the ophthalmological examination for males conducted at the end of the dosing period, for females during
the lactation period and for recovery group animals at the end of recovery period.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in haematology at any of the tested doses of either sex. However, statistically significant decrease in prothrombin time in low, mid and high dose group females and statistically significant increase in activated prothrombin time in the recovery group females when compared with vehicle control was noted. This change is considered as incidental and not treatment related, as the values are within historical control range and same changes not observed in males
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed in clinical chemistry at any of the doses tested in both sexes.
However, in main group males statistically significant decrease in alkaline phosphatase level at low dose and statistically significant increase in sodium level at mid and high dose noted when compared with vehicle control. In females, statistically significant increase in cholesterol at mid dose, statistically
significant increase in albumin at high dose and statistically significant increase in calcium at high dose were noted when compared with vehicle control.
In recovery males, statistically significant increase in creatinine, total protein, albumin, and chloride and statistically significant decrease in phosphorous at recovery when compared with vehicle control and in recovery females statistically significant increase in creatinine, cholesterol, albumin and sodium
and statistically significant decrease in phosphorous at recovery when compared with vehicle control was noted.
These changes are considered as an incidental and not treatment related, as the values are within historical control range and is due to biological variations.

Statistically significant decrease in T4 levels in high dose males when compared with vehicle control group were noted when compared with vehicle control group. The variation observed in the T4 levels could not be attributed as treatment related as there were no microscopic observations noted in thyroid gland and no effects were noted in thyroid weight at all the tested dose groups. This significant variation could be due to the increased T4 levels in vehicle control group males (Animal No. Rb8893: 99.593 ng/mL and Animal No. Rb8898: 99.416 ng/mL) and low dose group male (Animal No. Rb8905: 110.605 ng/mL). The T4 levels obtained across the groups are within historical control range. Hence, the variation observed in the T4 levels is considered to be incidental
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related changes observed at any of the dose tested. However, statistically significant decrease in urine volume at mid dose (males), significant decrease in urobilinogen in low, mid and high dose males and significant increase in pH at the end of recovery (females) was noted. This change is considered as an incidental and not treatment related, as the values are within historical control range.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The detailed clinical examination of animals did not reveal any treatment related changes at any of the main and recovery group animals of either sex.
There were no changes observed in the neurological/functional examination conducted for main group males (prior to scheduled sacrifice), for main group females during the lactation period (prior to scheduled sacrifice) and for all recovery group animals at the end of recovery period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related histopathological findings noticed in high dose group animals.
Testes, which were screened with special emphasis on stages of spermatogenesis and interstitial testicular cell structure, revealed normal progression of the spermatogenic cycle and presence of all expected associations.
All other observed histopathological lesions in high dose group animals, considered as spontaneous and incidental to Sprague Dawley rats of this particular age as they lack in consistency.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no treatment related changes in gestation and lactation body weight, percent change in gestation and lactation body weights, feed consumption during gestation period and lactation period, uteri observations, litter and pup weights, anogenital distance of pups

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no changes observed in the oestrus cyclicity of females at any of the doses tested during pre-mating treatment, mating treatment and on lactation day 14.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no changes observed in number of corpora lutea, implantations, resorptions, pre-implantation loss and pre/post natal losses at any of the tested doses.
There were no treatment related changes in the gestation length,in the gestation length, litter delivered, number of pups delivered, sex ratio, live birth index and observation of litter on lactation day 1 to 13 at any of the tested doses.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of treatment-related adverse effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no changes observed in implantations, resorptions, pre-implantation loss and pre/post natal losses at any of the tested doses.
There were no treatment related changes in number of pups delivered and live birth index and observation of litter on lactation day 1 to 13 at any of the tested doses.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were changes observed in mean pup weight during lactation day 1 to 13 at any of the doses tested. However, statistically significant increase in mean male pup weight on lactation day 7 in the mid dose group and mean female pup weight on lactation day 7 in low and mid dose groups was noted when compared with vehicle control. This can be considered as incidental and not to be treatment related as there were no external observations noted in these pups.
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:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant decrease in T4 levels of pups on lactation day 13 at G2, G3 and G4 groups were noted when compared with vehicle control group.
The variation observed in the T4 levels could not be attributed as treatment related as there were no microscopic observations noted in thyroid gland and no effects were noted in thyroid weight at all the tested dose groups. This significant variation could be due to the increased T4 levels in vehicle control group pups (Animal No. Rb8945/Pup No. 2 & 3: 98.703 ng/mL, Rb8948/Pup No. 2 & 3: 91.128 ng/mL), low dose group pups (Animal No. Rb8957/Pup No. 2 & 3: 90.457 ng/mL). The T4 levels obtained in pups across the groups are within historical control range. Hence, the variation observed in the T4 levels is considered to be incidental.
Urinalysis findings:
not examined
Sexual maturation:
not examined
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 (both external and internal) observed at any of the doses tested in pups of both sexes
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no changes observed in anogenital distance of both male and female pups at any of the tested doses.
There were no occurrences of nipples in male pups at any of the tested doses on lactation day 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of treatment-related adverse effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

The dose formulation analysis for concentration verification was performed during week 1 and 5 of treatment period and the results were within acceptable limits.

Applicant's summary and conclusion

Conclusions:
Based on the results discussed, the No Observed Adverse Effect Level (NOAEL) of the test item was found to be 1000 mg/kg body weight when administered to the males for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days), and to the females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13 under the experimental conditions employed in this study.
Executive summary:

The test item, was evaluated for possible adverse effects following repeated oral dosing to males for 37 days and to females, two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 to evaluate effects of test item on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus,

parturition, and early neonatal development. A total of 116 (58 males + 58 females) Sprague Dawley rats were distributed to four main groups and two recovery groups. G1 (control), G2 (low dose), G3 (mid dose) and G4 (high dose) groups consisted of 12 males and 12 females and each recovery group (G1R and G4R) consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle[Corn oil], the animals in G2, G3 and G4/ G4R groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg body weight for low dose, mid dose and high dose/high dose recovery groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. The main group males were treated for two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period (total of 37 days of treatment). The main group females were treated for two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 after which the pups were sacrificed on lactation Day 13 and females (dams) were sacrificed on lactation day 14 after overnight fasting (water allowed). The recovery group males and females were treated until the first scheduled sacrifice of dams and kept without treatment for a further 16 days observation. The dose formulation analysis for homogeneity and concentration verification was performed during weeks 1 and 5 of the treatment period and the results were within acceptable limits.

All animals were observed for clinical signs, mortality and morbidity, detailed clinical examination, body weight and feed consumption. Ophthalmological examination was carried out once before treatment for all animals, duringend of the dosing period for males (shortly prior to scheduled sacrifice) and during lactation period for females (shortly prior to scheduled sacrifice) for vehicle control and high dose group animals andduring last weekfor recovery group animals.Neurological/Functional examination was performed for five males and five females, randomly selected from each group towards the end of the dosing period for males (shortly prior to scheduled sacrifice), during lactation period for females (shortly prior to scheduled sacrifice) and from both the recovery group animals towards the end of the recovery period (shortly prior to scheduled sacrifice.

The clinical pathology (haematological and clinical chemistry) examinations were conducted in five males and five females from each main group (randomly selected) andfor all recovery group animals. Urinalysis was performed from the five randomly selected males of each main group during the last week of the treatment period and for all recovery animals at termination.

Each litter was examined after delivery (lactation day 1) and the number and sex of pups (litter size), stillbirths (dead pups born on day 1) and live births were recorded.

Body weights of male and female pups were recorded separately on lactation days 1, 4, 7 and 13.The anogenital distance of each pup was measured on lactation day 4. The number of nipples/areolae in male pups were counted on lactation day 13.

Gross pathology and organ weighing were performed on day 38 for males, on lactation day 13 for pups, lactation day 14 for dams and on day 68 for recovery group animals. The number of corpora lutea, implantation sites and resorptions for dams were recorded.

The animals did not reveal any clinical signs of toxicity. No mortality or morbidity was observed throughout the experimental period. No treatment related changes in body weight, percent change in body weight, feed consumption, ophthalmoscopic examination, neurological/functional examination, haematology, clinical chemistry, urine analysis nor in organ weights (both absolute and relative) were observed in both the sex of main and recovery group animals.

The dams did not reveal any treatment related changes in gestation and lactation body weight, percent change in gestation and lactation body weights, feed consumption during gestation period and lactation period, uteri observations, litter and pup weights, anogenital distance of pups.

There were no treatment related changes observed in mean pup weight during lactation day 1 to 13 at any of the doses tested.

There were no changes observed in anogenital distance of both male and female pups at any of the tested doses.

There were no occurrences of nipples in male pups at any of the tested doses on lactation day 13.

Statistically significant decrease in T4 levels of pups on lactation day 13 at G2, G3 and G4 groups were noted, but considered to be incidental.

The animals did not reveal any treatment related gross pathological and histopathological findings