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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Jul 2018 to 1 Nov 2018
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 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals : The male and female rats of the parental (P) generation were treated for at least ten weeks prior to mating
- Basis for dose level selection : The dose levels were selected based on results of a dose range finding study.
- Inclusion of extension of Cohort 1B
- Termination time for F2 : F2 pups were sacrificed on postnatal day (PND) 21
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B
- Exclusion of developmental immunotoxicity Cohort 3
- Route of administration : Oral, through diet
- Other considerations, on choice of species: The rat is the preferred test system because it is a readily available laboratory animal. The rat has been historically shown to be an acceptable animal for reproduction toxicity testing and is recommended by the OECD and other regulatory authorities.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Purity: 98%
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0000091655
- Expiration date of the lot/batch: 31 August 2018, The test item expired during the experimental phase of the study as per the declared expiry date on the CoA (Certificate of Analysis). To establish the stability of the test item, the active ingredient content will be analysed concurrently with the dosing period i.e., within one week before declared expiry and within one week after completion of dosing following the validated analytical method. The CoA and comparison of analysed purity were reported in the final report.
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: As per the instruction received from the Sponsor on storage of the test item, the test item will be stored: Storage Temperature : Room Temperature, Storage Container : In original container as supplied by the Sponsor
- Stability under test conditions: The test item is stable up to 14 days at room temperature based on the results of the method validation study. The test diet will be prepared at least once every 8 days.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The required quantity of the test item was received from the Test Item Control Office (TICO). The test item was mixed with the 10% of total diet or 500 g of food (whichever is higher) in order to prepare the pre-mix. To this pre-mix diet, the remaining amount of diet was added and mixed. This whole amount of diet, thus prepared, was mixed in a blender and served to the animals ad libitum throughout the study. Details of the diet preparation were recorded in the raw data.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Female rats were nulliparous and non-pregnant. At the initiation of acclimatisation, rats were 5-6 weeks old.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jai Research Foundation
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5-6 weeks
- Housing: During the experiment period, rats were housed individually or in groups of 2/3 rats/cage/sex and enrichment material were provided. During the mating period, rats were housed in groups of two rats/cage (one male and one female). Mated female rats were housed individually and nesting material were provided at near parturition. During the study, rats were housed in solid floor polypropylene rat cages (size: 41 cm x 28.2 cm x 18 cm). Each cage was fitted with a stainless steel top grille having provision for a polypropylene water bottle with stainless steel drinking nozzle. Separate food hoppers were attached to the cages for diet. The bottom of the cages was layered with clean sterilised rice (paddy) husk as the bedding material. Cages were arranged in such way that possible effects due to cage placement are minimised. Contaminant analysis of samples of the bedding material were performed at six months interval and most recent results were included in the final report.
- Diet: The experimental rats were fed ad libitum with powdered food. Every food consignment received was accompanied with a certificate of analysis of nutrient content from the food supplier. Quality of food and water is being monitored regularly. The most recent result of microbial analysis were reported in the final report. Sample quantity, approximately 500 g of each batch of the food consignment used in the study were stored at -20 ± 5 ºC, until finalisation of the report.
- Water: unlimited supply of clean and filtered drinking water (filtered through reverse osmosis water filtration system) in polypropylene bottles. The most recent result of microbial and chemical contaminant analysis were reported in the final report.
- Acclimation period: A total of 105 male rats and 105 female rats were selected for acclimatisation. Rats were received into the experimental room and acclimatised for a minimum period of 5 days prior to randomisation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
The test item was mixed with the 10% of total diet or 500 g of food (whichever was higher) in order to prepare the pre-mix. To this pre-mix diet, the remaining amount of diet was added and mixed. This whole amount of diet, thus prepared, was mixed in a blender and served to the animals ad libitum throughout the study.
Details on mating procedure:
- M/F ratio per cage: 1:1 pairing
- Length of cohabitation: until evidence of copulation is observed or 2 weeks have elapsed
-After ten-weeks of pre-mating period, parent male and female rats were mated to obtain F1 generation pups. During the mating period, each female was placed with a randomly selected and unrelated male rat from the same dose group . Each morning, the female rats were examined for the presence of sperm or copulatory plugand the ‘day 0’ of gestation was recorded. ‘Day 0’ of gestation was defined as the day on which sperm was observed in the vaginal smear.
If there were insufficient male rats, for example due to male death before pairing, then male(s), which have already mated, were paired (1:1) with a second female such that all females were paired. If mating did not occurred after 2 weeks, the rats were separated without further opportunity for mating and were considered as mated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The active ingredient (A.I.) concentration and homogeneity of the test item in diet were analysed once before initiation of treatment and at least once in a month thereafter throughout the treatment period. Duplicate samples of control (one location) and the test item treated diet (from different locations i.e., top, middle, and bottom of container) of each group were taken to determine the homogeneity and concentration of the test item in the test diets. A set of test diet was sent for analysis and a second set was stored at 2-8 °C. Stored samples were used, if re-analysis was required. On each occasion, the mean concentration was determined and compared with the nominal value. The acceptance criteria was ± 20% deviation from the nominal value and %CV < 20. Samples were analysed using a validated analytical method. Following instrumental parameters will be used for analyses: Instrument : HPLC, Column : C-18 [250 mm x 4.6 mm (i.d), 5 μm particle size], Wavelength (nm) : 210, Injection volume (μL) : 20, Flow rate (mL/minute) : 1.0, Mobile phase : Solvent (A) - Acetonitrile (80%), Solvent (B) - 0.1 % Orthophosphoric acid in Milli-Q Water (20%)
Duration of treatment / exposure:
-Parental (P) and F1 (selected for Cohort 1A and 1B) rats were offered the control or test diet continuously on a 7-days/week basis, until the scheduled necropsy. Exposure to the test item to both P sexes were commenced, at least, ten weeks prior to mating and continued during the twoweeks mating period. After mating, P male rats were further exposed, up to, and including the day before scheduled sacrifice (after delivery of P females). P female rats were further exposed during gestation and at least up to weaning of F1 pups. Unless already initiated during the lactation period, direct treatment of the selected F1 male and female pups began at weaning and continue, until the scheduled necropsy.
-Cohort 1B rats was maintained on treatment beyond PND 90 and bred to obtain a F2 generation. Male rats and female rats of the same dose group were mated (avoiding the pairing of siblings) for up to two weeks, beginning on or after PND 90, but not exceeding PND 120. Procedures were similar to those for the P rats. F2 generation pups were evaluated up to PND 21.
Frequency of treatment:
Continuous
Details on study schedule:
- F1 parental animals not mated until Postnatal day 90 after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [...] days of age.
- Age at mating of the mated animals in the study: [...] weeks
Doses / concentrationsopen allclose all
Dose / conc.:
150 ppm (analytical)
Remarks:
P Generation, F1 Generation (Cohort 1A) and F1 Generation (Cohort 1B)
Dose / conc.:
300 ppm (analytical)
Remarks:
P Generation, F1 Generation (Cohort 1A) and F1 Generation (Cohort 1B)
Dose / conc.:
600 ppm (analytical)
Remarks:
P Generation, F1 Generation (Cohort 1A) and F1 Generation (Cohort 1B)
No. of animals per sex per dose:
25 rats/sex/group P Generation, 20 rats/sex/group F1 Generation (Cohort 1A) and 20 rats/sex/group F1 Generation (Cohort 1B)
Control animals:
yes, plain diet
Details on study design:
- Dose Justification: The dose levels were selected based on results of a dose range finding study
- Standardisation of Litter Size: On postnatal day (PND) four, the size of each litter will be adjusted by eliminating extra pups by random selection to yield, as nearly as possible, five male pups and five female pups per litter. Whenever the number of male pups or female pups prevents having five of each sex per litter, partial adjustment (e.g., six male and four female) will be performed. Adjustments will not be done for litters of ten pups or less.
-Selection of Pups for Post-Weaning Evaluations on Post-natal Day: A) Cohort 1A: One male and one female F1 pup per litter (20 pups/sex/group; wherever possible) will be randomly assigned for primary assessment of effects on reproductive systems and general toxicity. , B) Cohort 1B: One male and one female F1 pup per litter (20 pups/sex/group; wherever possible) will be randomly assigned for follow-up assessment of reproductive performance by mating F1 rats.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All visible clinical signs were noted and recorded daily, at least once, throughout the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of P male rats was recorded on the first day of treatment and weekly thereafter. Body weight of P female rats was recorded on the first day of treatment and weekly thereafter during pre-mating and mating period. Body weight of P female rats was also recorded at least on gestation days 0, 7, 14 and 20, and lactation days 0, 4, 7, 14, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The food consumption was determined by weighing of food input and leftover. Food weight of all P male rats was measured weekly during pre-mating and post-mating period. In P female rats, during pre-mating period, food weight was recorded, at least, at weekly intervals. During gestation period, food weight was recorded, at least, on gestation days 0, 7, 14, and 20. During lactation period, food weight was recorded, at least, on lactation days 0, 4, 7, 14, and 21. Additional food was offered as and when required. Food consumption was not measured during mating period. Food spillage/waste was not estimated during the course of the study.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

BLOOD AND URINE COLLECTION
-At the time of terminal sacrifice, blood (maximum 3.5 mL) will be collected from P and F1 (cohort 1A) rats (except female rats sacrificed between GD 25 - 26) under anaesthesia (isoflurane) by orbital plexus puncture. Rats will be deprived of food overnight (water provided) prior to blood collection. Blood samples will be collected for haematology (in vials containing 4% EDTA), coagulation parameters PT and APTT (in vials containing 3.2% sodium citrate), clinical chemistry and thyroid hormone analysis (in vials without anticoagulant). Blood will also be collected from F1 surplus pups on PND 4 and weanlings pups on PND 22 (at least from 10 litters – 1 pup/sex), through decapitation and orbital plexus puncture, respectively for serum thyroid hormone analysis. If required, serum samples of pups will be pooled by litter. Serum will be preserved at -70 ± 10 °C, until analysis. Any residual/retained serum samples will be discarded after confirmation for finalisation of the study report. Urine samples will be collected, overnight, at least 10 rats/sex/group of P and F1 (cohort 1A) in rat metabolic cages using graduated collecting tubes.
Oestrous cyclicity (parental animals):
- Oestrous cycle length and pattern was evaluated by vaginal smears for all P female rats for, atleast, two weeks period prior to mating and throughout cohabitation/mating period. It was ensured to avoid disturbance of mucosa while obtaining vaginal smear.
- Vaginal smear will be taken for F1 (Cohort 1A) female pups from the day of vaginal opening, until the first oestrus phase is observed. Oestrous cycle length and pattern will be evaluated for, at least, two weeks period prior to terminal sacrifice by vaginal smears for all F1 female rats in cohort 1A. Vaginal smear will also be evaluated in cohort 1B from the time of pairing until mating evidence is detected. At the time of necropsy, vaginal smear will be examined (from P and F1 rats) to determine the stage of oestrous cycle.
Sperm parameters (parental animals):
Initially, P male rats of control and high dose groups were evaluated for sperm parameters. If treatment related changes were observed, the rats of the low and mid dose groups were also evaluated. Sperm from the cauda epididymis (or vas deferens) was collected for evaluation of sperm motility and sperm morphology. An evaluation of epididymis (or vas deferens) sperm motility was performed, immediately after sacrifice from all rats. A morphological evaluation of an epididymal (or vas deferens) sperm sample was performed. Sperm (at least 200 per sample) was examined as fixed, wet preparations and classified as either normal or abnormal. From all P male rats, sperm from one (right) testis and one (right) epididymis (after recording the weight) were stored for enumeration of homogenisation - resistant spermatids and cauda epididymal sperm reserves, respectively. The total number of homogenisation - resistant testicular spermatids and cauda epididymal sperm were enumerated. This one testis and cauda epididymis were stored in frozen condition until the analysis.
Same parameters were followed for the F1(cohort 1A) male parents.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes. to yield, as nearly as possible, five male pups and five female pups per litter. Adjustments will not be done for litters of ten pups or less.
- If yes, maximum of 10 pups/litter ([/sex/litter as nearly as possible); whenever the number of male pups or female pups prevents having five of each sex per litter, partial adjustment (e.g., six male and four female) was performed. Excess pups were killed and discarded. The size of each litter was adjusted by eliminating extra pups by random selection.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, gross abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups. In addition, the first clinical examination of the pups included a qualitative assessment of body temperature, state of activity, and reaction to handling. 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:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead. Each pup will also be observed for presence of milk band in the stomach from PND 0 to 4. If possible, pups found dead on the day of littering were examined for possible defects and cause of death and preserved after discretion with the Study Director/Pathologist. Pups which died/sacrificed during moribund condition in study were weighed and subjected to post-mortem examination.
Postmortem examinations (parental animals):
SACRIFICE
- Surviving rats (including weanling pups) were sacrificed, by using an over dose of carbon dioxide. Culled pups on PND 4 (not subjected for hormone analysis) were sacrificed, through intraperitoneal administration of thiopentone sodium.
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]

GROSS NECROPSY
- Gross necropsy consisted of external and internal abnormalities including the cervical, thoracic, and abdominal viscera. Special attention was paid to the organss of the reproductive systems during gross examination of all rats. The uteri of all cohabited female rats were examined for the presence and number of implantation sites. Number of corpora lutea were recorded from those female rats in which implants were observed on gestation day 25.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- Surviving rats (including weanling pups) were sacrificed, by using an over dose of carbon dioxide. Culled pups on PND 4 (not subjected for hormone analysis) were sacrificed, through intraperitoneal administration of thiopentone sodium.
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Pups which were found dead on day of delivery (i.e., PND 0) were subjected for gross examination and a portion of the lung was immersed in water for confirmation of the status (stillbirth or dead). Pups which were found dead were observed for presence of milk band. Pups, which died or were euthanised before scheduled sacrifice (during PND 0 to 21) were examined, for gross lesions and the cause of death or condition, as soon as possible, after the observation is made. In case of a pup with gross lesions (PND 0 to 21), the whole pup was kept in Bouin’s fluid. Pups without gross lesions were discarded after examination.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Statistics:
Raw data were processed to get group mean and standard deviation with significance between the control and the treated groups of 95 or 99% confidence levels, using validated statistical software. Numerical results were evaluated by an appropriate and accepted statistical methods. The parametric data (body weight, body weight gain/body weight change, food consumption, food efficiency, sperm parameter, oestrous cycle parameter, physical developmental landmark, motor activity, litter size, organ weight, organ weight ratio, etc.) will be analysed by using statistical methods like Bartlett’s test, t-test and ANOVA with Dunnet’s test. The non-parametric data (mortality rate, pregnancy rate, male fertility index, female fertility index, live birth index, survival index, gestation index, lactation index, etc.) were analysed using Chi-square test. Any other appropriate statistical analyses test can be used based on the available data and the details will be included in the study report. Significance between control and treatment groups was calculated with statistical significance of 95 or 99% confidence levels. Non-pregnant rats were not be subjected to statistical analysis.

Results and discussion

Results: P0 (first parental animals)

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks on result:
not measured/tested
Remarks:
Results will be available in November 2019.

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks on result:
not measured/tested
Remarks:
Results will be available in November 2019.

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion