Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Decemeber 2018 to 16 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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
- Basis for dose level selection

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Nitika Pharmaceutical Specialties Pvt Ltd / Bx IRST7H780L
- Expiration date of the lot/batch: August 2023
- Purity test date: September 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In original container in dark, cool place.
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle: Based on the results obtained, the test item in the vehicle was stable for 8 days.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
- other information:

Test animals

Species:
rat
Strain:
other: RccHan:WIST
Details on species / strain selection:
The preferred species is the rat. It has been historically shown to be a suitable model for developmental and reproductive toxicity testing and is recommended by the regulatory authorities. The results may be of value in predicting the toxicity of the test item to humans.
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Commercial laboratory animal supplier.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 12-14 wks; (F1) 0 wks
- Weight at study initiation: (P) Males: 381.09 to 386.66 g; Females: 246.89 to 247.94 g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: no
- Housing: Throughout the experiment period, the male and female rats were housed in groups of 2 or 3 rats/sex/cage. Except, during the mating period, rats were housed in groups of 2 rats/cage (one male plus one female) and mated female rat was caged individually. Enrichment material was provided to all animals. Nesting material was provided at near parturition (from gestation day 17). During study, rats were housed in solid floor polypropylene rat cages (size: 41 cm x 28.2 cm x 18 cm). Each cage were fitted with a stainless steel top grille having provision for polypropylene water bottle with stainless steel drinking nozzle. The bottom of cages were layered with clean sterilised rice (paddy) husk as the bedding material. Cages were placed on 5 tier racks. Cages and enrichment material were changed minimum twice a week. Cages were arranged in such a way that possible effects due to cage placement were minimised.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet: Rats were fed ad libitum with standard rodent diet (Teklad Certified Global 16% Protein Rodent Maintenance Diet, Batch N° 2016SC-050818MA procured from Envigo Laboratories, Inc., USA).
- Water: Rats were provided reverse osmosis (RO) water ad libitum (RO water filtration system) in polypropylene bottles.
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23°C
- Humidity (%): 64 to 66%
- Air changes (per hr): 19 to 21
- Photoperiod (hrs dark / hrs light): The photoperiod was 12 hours light and 12 hours darkness, fluorescence light hours being 06.00 - 18.00 hours.
IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% carboxy-methyl-cellulose (CMC) with 1% tween 80
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): after consultation with the Sponsor,
- Concentration in vehicle: 1%
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
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
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: [no / yes (explain)]
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Instrumental parameters for method of analyses:
Instrument : Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES)
Element : Iron (Fe)
Wavelength : 238.204 nm
RF power : 1300 Watts
Pump (Flow rate) : 1.5 mL/minute
Gas Flow Rate
Plasma : 15.0 L/minute
Auxillary : 0.2 L/minute
Nebulizer : 0.8 L/minute
Duration of treatment / exposure:
Male: two weeks prior to mating, during the mating (two weeks) and until approximately 80% of the females have delivered. Total days of treatment was 47 days.
Female: two weeks prior to mating, the variable time to conception, the duration of pregnancy and fourteen days after delivery.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
G1 - Control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
G2 - low dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
G3 - Mid dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
G4 - High dose
No. of animals per sex per dose:
15
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Dose levels have been selected based on the results of the dose range finding (DRF) study (JRF Study N° 410-1-02-21405). In the DRF study, no treatment related effects were observed on body weight, body weight gain, Food consumption and organ weight (absolute and relative) up to 1000 mg/kg b. wt./day. Therefore, the following dose levels were selected for the present study in consultation with the Sponsor: 100, 300 and 1000 mg/kg b. wt./day.
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry: yes
- Other:
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of all male rats was recorded on the first day of dosing and at weekly intervals thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The food consumption was determined by differentiating the weight of food input and leftover.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OTHER:
Oestrous cyclicity (parental animals):
Oestrous cycle length and pattern were evaluated by vaginal smears of individual female rats during the pre-treatment period of two weeks. Vaginal smear was monitored daily from the beginning of the treatment period until evidence of mating. Vaginal smear, from each pregnant animal, was also observed on the day of terminal sacrifice. Care was taken to avoid disturbance to mucosa while obtaining vaginal cells.
Sperm parameters (parental animals):
Parameters examined in P/F1 male parental generations: no
[testis weight, epididymis weight, enumeration of cauda epididymal sperm reserve
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia]

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead]

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals male rats were sacrificed after approximately 80% of females have delivered.
- Maternal animals: All surviving animals Female rats were sacrificed on LD 15

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated were prepared for microscopic examination and weighed, respectively.
Testes
Epididymis
Levator ani plus bulbocavernosus muscle complex (LABC)
Cowper’s glands
Glans penis
Ovary
Thyroid gland
Seminal vesicles with Coagulating gland
Prostate (dorsolateral and ventral)
Uterus with oviducts and cervix
Gross lesions
Vagina
Male mammary gland
Pituitary
Liver
Kidneys
Adrenals
Thymus
Spleen
Brain
(cerebrum, cerebellum, and medulla/pons)
Heart
Spinal cord
(at three levels; cervical, mid-thoracic and lumbar)
Stomach
Small intestine (Duodenum, jejunum, ileum)
Large intestine (Caecum, colon, rectum)
Eye
Trachea
Lungs (preserved by inflation with fixative and then immersion)
Urinary bladder
Lymph nodes (mandibular, mesenteric)
Sciatic nerve
Skeletal muscle and bone
Bone marrow (Femur)
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at post-natal day 4days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Pup blood was pooled by litter. Blood samples were collected in vials without anticoagulant.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated were prepared for microscopic examination and weighed, respectively. Serum thyroid hormone (T4) level was analysed from PND 4 pups.
Reproductive indices:
No data
Offspring viability indices:
No data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined

Details on results (P0)

No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 1000 mg/kg b. wt./day based on no effect on systemic end-points.

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
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Effect levels (P1)

Key result
Remarks on result:
not measured/tested

Target system / organ toxicity (P1)

Key result
Critical effects observed:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed

Details on results (F1)

The NOAEL for developmental toxicity was 1000 mg/kg b. wt./day dose level based on no effect on mortality, clinical sign, and, postnatal growth.

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
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Key result
Remarks on result:
not measured/tested

Target system / organ toxicity (F2)

Key result
Critical effects observed:
not specified

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
Based on results of present study, it is concluded that:
No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 1000 mg/kg b. wt./day based on no effect on systemic end-points.
NOAEL for reproduction and fertility toxicity was 1000 mg/kg b. wt./day based on no effect on fertility and reproduction performance.
The NOAEL for developmental toxicity was 1000 mg/kg b. wt./day dose level based on no effect on mortality, clinical sign, and, postnatal growth.