Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation: August 8, 2012; Study completion: January 18, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD guideline 421 (Reproduction / Developmental Toxicity Screenin Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): KS-235
- Purity test date: 99.9% (Refer Certificate of Analysis in Appendix 21 of the attached report)
- Lot/batch No.: #20411
- Expiration date of the lot/batch: April 11, 2014
- Storage condition of test material: (at JRF): As per the instruction received from the Sponsor on storage of the test item, the test item was stored in its original container as supplied by the Sponsor at cool, dry, well ventilated area in the Test Item Control Office (TICO). The stability of test item in storage is the responsibility of the Sponsor. The test item formulations were stored at the experimental room conditions during preparation and dosing. Data related to the characterization of the test item were documented in a Certificate of analysis provided by the Sponsor and are included as an appendix in the study report. The test item information and analytical identification provided by the Sponsor were considered as an adequate description of the characterisation, purity and stability of the test item and will be responsibility of the Sponsor.
- Other:
- Manufactured by SANKO CO. LTD.
- Date of Manufacture: April 11, 2012
- Appearance: White crystalline powder

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
- Source: Animal Breeding Facility, Jai Research Foundation
- Age at study initiation: At initiation of acclimatization rats were 9 to 12 weeks old.
- Housing: Animals were housed individually in solid floor polypropylene rat cages (size: 41.0 cm x 28.2 cm x 18.0 cm) except during mating period where rats were housed in group of 2 rats/cage (one male and one female) Each cage as fitted with a stainless steel top gull having provision for keeping rat pellet feed and a polypropylene water bottle with stainless steel drinking nozzle. Cages were placed on 5 tier racks. Cages were changed twice a week. The cages and water bottles were cleaned, dried and then sterilized. After sterilization, they were stored in the UV room inside the Barrier Maintained Rodent Facility till used. The bottom of the cages was layered with clean sterilised paddy husk as a bedding material. Paddy husk is being routinely analyzed once in six month interval for commonly found contaminants and the most recent result are incorporated in the final report (Refer to Appendices 17 and 18 of the attached report).
- Diet (e.g. ad libitum): The experimental animals were fed ad libitum with standard rodent pellet diet (obtained from Teklad Certified Global 16% Protein Rodent diet manufactured by Harlan, Madison) with an unlimited supply of clean and filtered drinking water (filtered through Reverse Osmosis water filtration system) in autoclave sterilised polypropylene bottles. Quality of feed, water and bedding material (microbial and chemical contaminant analysis) is being checked and monitored at every six months interval in JRF. Every feed consignment is subject to microbial examination in JRF. The most recent analysis reports are incorporated in the final report (Refer to Appendices 17, 18 and 19 of the attached report).
- Water (e.g. ad libitum): The experimental animals were fed ad libitum with standard rodent pellet diet (obtained from Teklad Certified Global 16% Protein Rodent diet manufactured by Harlan, Madison) with an unlimited supply of clean and filtered drinking water (filtered through Reverse Osmosis water filtration system) in autoclave sterilised polypropylene bottles. Quality of feed, water and bedding material (microbial and chemical contaminant analysis) is being checked and monitored at every six months interval in JRF. Every feed consignment is subject to microbial examination in JRF. The most recent analysis reports are incorporated in the final report (Refer to Appendices 17, 18 and 19 of the attached report).
- Acclimation period: Acclimatization: A total of 52 male and 52 female rats were selected for acclimatization. The rats were received into the experimental room and acclimatized for a period of 7 days prior to treatment. After randomization 48 male and 48 female rats were selected for the study. During this period, rats were observed daily for clinical signs and also for mortality and morbidity.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 65 - 67 (Relative Humidity)
- Air changes (per hr): 17 - 21
- Photoperiod (hrs dark / hrs light): 12 hours fluorescence lighting and 12 hours darkness

Other details:
- Healthy, young adult rats (Rattus norvegicus).
- Animals which were not selected for the study after randomization were returned to Animal Breeding Facility, JRF.
- Female rats were nulliparous and non-pregnant.

- Environmental Conditions: Rats were maintained in Barrier Maintained Rodent Facility in an environment controlled room (BMR Room No. 8). Light hours were 06.00 - 18.00 hours, approximately. Environmental condition during study period was as given in table below:

Environmental Parameters Temperature(C) Relative Humidity (%) Air Change (per hour) Mean Light Intensity (LUX)
Maximum Minimum Maximum Minimum
August 2012 23 20 67 65 21 346
September 2012 23 20 67 65 17 192
October 2012 23 20 66 65 17 300
(The experimental room was cleaned and mopped daily with 2.5% Dettol surface disinfectant).

- Grouping: Prior to randomization, the rats were weighed. Only healthy rats were allocated to the study. Rats were randomly allocated to different groups using an in-house developed validated computer software program (Gad and Well, 1994). Animals were divided into four groups [G1 (control), G2 (low dose), G3 (mid dose), G4 (high dose)]. At the start of the treatment, the body weight variations among the animals were within & ±20% of the mean body weight for each sex.

- Animal Identification: During acclimatization, a temporary animal number were marked with indelible non - toxic marker pen on the dorsal surface of the tail. After grouping, individual animal was identified with individual number tattooed at the base of tail and each cage was labelled with a colored cage card showing Study No., Test Item Code, Group No. & Sex, Dose (mg/kg b. wt./day), Study Code, Cage No. and Animal No. Pups were marked with indelible non-toxic marker pen.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- Route and Mode of Test Item Administration: The oral route of administration has been selected for this study since it is an exaggerated model of the reasonably foreseeable exposure in human.

PREPARATION OF DOSING SOLUTIONS:
- Dose Level and Justification: After consultation with the Sponsor, the following dose levels were selected: 0, 62.5, 250 and 1000 mg/kg b. wt./day in corn oil. These dose levels were selected based on the results of “Repeated Dose 14-Day Oral Toxicity Study of KS-235 in Wistar Rats (Dose Range Finding Study); JRF Study No. 410-1-04-4694”. In this study, dose levels tested were 0, 10, 100 and 1000 mg/kg b. wt./day. No mortality and clinical signs were observed at each dose level tested.
- Test Solution Preparation: The test item KS-235 was mixed with corn oil to obtain desired concentrations. The test formulations were prepared every day prior to dosing and used within 4 hours. Prior to dosing, test formulation was thoroughly mixed using magnetic stirrer and during the administration, they were mixed with glass rod/spatula continuously in order to maintain homogeneity.
- Stability of the Test Item in the Vehicle: The stability of active ingredient (a.i.) in corn oil was determined prior to initiation of the study after validation of the analytical method (JRF Study No. 228-2-13-4616). The stability of test item was determined at 0 and 4 hours at room temperature.
- Homogeneity and Active Ingredient Concentration of the Test Item in the Vehicle: The active ingredient (a.i.) concentration and homogeneity of KS-235 in samples of control and each of the three test formulation prepared for dosing were analyzed at weeks 1, 4 and 6. Duplicate samples were taken for analysis. On each occasion, the mean concentration were determined and compared to nominal value. The acceptance criteria was ± 10% deviation from nominal value and CV% < 10%. The samples were analyzed by JRF after validation of the analytical method provided by the Sponsor (JRF Study No. 228-2-13-4616).
- Dosing: Test solution and corn oil were administered once daily, by oral gavage, using intubation cannula attached to a graduated syringe for seven days a week at approximately same time each day. The fix dose volume of 10 mL/kg body weight was used to calculate the dose volume. Individual close volume was adjusted according to the most recently recorded body weight. The control group animals were dosed with the corn oil alone.

VEHICLE:
- Vehicle and Justification for Choice: The corn oil was selected as a vehicle based on solubility check performed at JRF during JRF Study No. 410-1-04-4694 and after consultation with the sponsor.
- Corn oil (manufactured by Sigma Aldrich, Batch No. #MKBH4894V, Manufactured date: August 17, 2012 and Expiry date: August 16, 2017).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concenration and homogeneity of each concentration and control were analyzed in weeks 1, 4 and 6. Duplicate samples were taken for analysis. On each occasion, the mean concentration was determined and compared to nominal value. The acceptance citeria was +/- 10% deviation from nominal value and CV < 10%. All analysed concentations were within 3% of nominal.
Details on mating procedure:
Mating Procedure: During mating period, the male and female rats were mated in a 1:1 mating ratio to procure the F1 generation. Each morning, the females were examined for the presence of sperm and the ‘day 0’ of pregnancy was recorded. After evidence of copulation, mated female rats were caged individually. For each mating, each female were placed with a single male from the same dose level (1:1 mating) until copulation occurs.
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
Once daily
Duration of test:
6 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 62.5, 250, 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Parental animals: Observations and examinations
DETAILED CLINICAL OBSERVATIONS: Yes
- Clinical Signs: All visible clinical signs, such as changes in skin, fur, eye, mucous membranes as well as behaviour pattern were noted and recorded twice a day, every day throughout the period of the experiment.

BODY WEIGHT: Yes
- Body Weights: Individual body weight of male and female rats were recorded on the first day of dosing and at weekly interval thereafter. During gestation period, females were weighed on gestation days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum), and post-partum 4 (i.e lactation day 4). Parturition ‘day 0’ is defined as the day on which the female littered. At the time of sacrifice body weight of all the rats were recorded.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Feed Consumption: For female, during pre-mating and gestation periods food consumption were measured weekly, and during lactation periods feed consumption were measured during lactation day 0 to 4. For males, feed consumption was measured weekly during pre mating arid post - mating period. Feed consumption was not measured during cohabitation period.

OTHER:
-Mortality and Morbidity: Rats were observed twice a day for mortality and morbidity. Rat and Pup’s, which died during study were weighed and subjected to post-mortem examination.
Litter observations
Pups: Each litter were examined after delivery to establish the number of pups, sex of pups, stillbirths, dead pups and the presence of gross anomalies.
- Pup Body Weight: Individual pup body weight was recorded on lactation days 0 arid 4.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No - animals killed day 4 post partum
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
Statistics:
The test parameters were analyzed using appropriate statistical techniques using Bartlett's test, ANOVA and Dunnett's "t"-test, Chi-square test and Test of significance for difference of proportions.
Indices:
Pre-implantation loss, post-implantation loss, mating index, fertility index, gestation index, live births, pup viability to day 4 post-partum

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
There were no adverse effects on reproduction and development up to the dose level of 1000 mg/kg bw/day when administered by oral gavage to rats prior to mating, during mating, post-mating, gestation period and until post-partum day 3.
Executive summary:

The effects of KS-235 on reproduction and/or development when administered through oral gavage to male and female Wistar rats prior to mating, during mating and gestation periods, and until post-parturn day 3 assessed according to OECD TG421. Groups of 12 male and 12 female Wistar rats were dosed at 0 (corn oil), 62.5, 250 and 1000 mg/kg bw/day. Dose formulation analysis was performed during 1st, 4th and 6th week of treatment period. All rats were observed twice a day for clinical signs and mortality and morbidity throughout the study period. Body weight of individual male animal were determined weekly throughout treatment period. Feed consumption of individual male animal as determined weekly during pre mating and post mating period. Body weight and feed consumption of female animals were determined weekly prior to mating, on gestation days 0, 7, 14 and 20. Dam and pup’s body weight and feed consumption were recorded on lactation day 0 and 4. At the end of the treatment period, male and female rats were sacrificed by carbon dioxide asphyxiation and subjected to gross pathological examination and the organs/tissues defined in study plan were removed, weighed and processed for histopathological evaluation. Pups were sacrificed by i.p injection of sodium thiopentone. Absolute organ weights were recorded and relative organ weight was calculated for the organs defined in study plan. Number of corpora lutea and implantation sites was recorded in dams. Histopathological slides obtained from animals from control and high dose groups were examined microscopically

Results: Dose formulations at each dose concentration was within the acceptance level of ± 10.0% of nominal concentration and CV% < 10% which indicates that the dose formulation prepared were homogeneous. No mortality was observed in parental rats. No treatment related clinical sign and mortality was observed in pups. Transient salivation (approximately 15 to 20 minutes) was observed in post dosing parental rats treated with KS-235 at 250 and 1000 mg/kg bw/day dose groups. Body weight of parental rats was statistically significantly lower during experimental period at 1000 mg/kg bw/day dose group when compared with the control group. The percent body weight change, feed consumption and fertility data were comparable between control and all KS-235 treated groups. No treatment related macroscopic/microscopic finding was observed across all the groups.