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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Study initiation: August 8, 2012; Study completion: January 18, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: - Study performed according to GLP - Study performed according to the following guideline: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening 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
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- 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).
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.
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.
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
Once daily
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

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.
Postmortem examinations (parental animals):
GROSS NECROPSY
- Necropsy: All the surviving rats were sacrificed by using over dose of CO2 and pups were euthanized by intraperitoneal injection (i.p.) of sodium thiopentone. Male rats were sacrificed after approx 80% of females have delivered. Female rat along with the pups were sacrificed on post-partum day 4. Females which have not delivered on day 25 post-coitum were sacrificed on day 25 post-coitum. At the time of sacrifice or death during the study, all pups were examined macroscopically for any structural abnormalities or pathological changes. Special attention paid to the organs of the reproductive system. During sacrifice, numbers of corpora lutea and implantation sites were recorded in dams.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Organ Weight: At the time of sacrifice, the testes and epididymis of all male adult rats were weighed.
- Preservation of Organs/Tissues: At the time of sacrifice, following organs/ tissues of rats were fixed and stored in a suitable medium;
• Vagina, uterus with oviducts, cervix, seminal vesicles, prostate and coagulating gland (preserved in 10% Neutral Buffered formalin)
• Ovaries, testes and epididymis (preserved in Bouin’s fixative)
• Organs with gross lesion (preserved in 10% Neutral Buffered formalin)
- Histopathology: Detailed histological examination was performed on the ovaries, testes and epididymides of the rats from the highest dose group and the control group. Detailed testicular histopathological examination (e.g., using Bouin’s fixative, paraffin embedding and transverse sections of 4 - 5 µm thickness) were conducted with special emphasis on stages of spermatogenesis and histopathology interstitial testicular cell structure. The evaluation should identify treatment related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells or sloughing of spermatogenic cells into the lumen. Examination of the intact epididymis includes the caput, corpus, and cauda, which can be accomplished by evaluation of a longitudinal section. The epididymis were evaluated for leukocyte infiltration, change in prevalence of cell types, aberrant cell types, and phagocytosis of sperm. PAS and hemnatoxylm staining were used for examination of the male reproductive organs. Histopathological examination of the ovary should detect qualitative depletion of the primordial follicle population.
Postmortem examinations (offspring):
GROSS NECROPSY
- Necropsy: All pups were euthanized by intraperitoneal injection (i.p.) of sodium thiopentone. At the time of sacrifice or death during the study, all pups were examined macroscopically for any structural abnormalities or pathological changes. Special attention paid to the organs of the reproductive system.
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.
Reproductive indices:
- Indices: Pre-implantation loss, post-implantation loss, mating index, fertility index, gestation index, live births, . See attached report for details of indices calculations.
Offspring viability indices:
Viability index on day 4 post-partum: (number of surviving pups on day 4 post-partum/number of live born pups) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
body weight was lower at higher doses
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
body weight was lower at higher doses
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: lowere after 36 days for higher doses

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
- Mortality: No mortality was observed in rats treated with KS-235 or given corn oil only (Table 1; Appendices 1 and 2 of the attached report).

- Clinical Observations: No clinical signs were observed in parental rats given corn oil and treated with KS-235 at 62.5 mg/kg b. wt./day. Post dosing, transient salivation was observed (for approximately 15 to 20 minutes) in male and female rats treated with KS-235 at 250 and 1000 mg/kg b. wt/day dose groups (Tables 2 and 3; Appendices 1 and 2 of the attached report).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
- Body Weight (g) and Body Weight Change (%): Male mean body weight was statistically significantly lower during treatment day 29 to 43 at 1000 mg/kg b. wt./day dose group when compared with the control group. The mean male percent body weight change was also significantly lowest during treatment days 8 to 15 at 62.5 mg/kg b. wt./day dose group and during treatment days 1-8 and 22-29 at 1000mg/kg b. wt/day dose group when compared with the control group. Female mean body weight was statistically significantly lower during gestation days 7 and 14 at 1000 mg/kg b. wt./day. The mean female percent body weight change was significantly lower during treatment days 8 to 15 at 1000 mg/kg b. wt./day dose group. The mean percent body weight change was also statistically significantly lower during gestation days 7 to 14 at 62.5 mg/kg b. wt./day dose group, and during gestation period 0 to 7 and 7 to 14 at 1000 mg/kg b. wt./day dose group when compared with control group (Tables 4, 5, 6 and 7; Appendices 3, 4, 5 and 6 of the attached report). Variation observed in the mean percent body weight change at 62.5 mg/kg b. wt./day dose group was considered as incidental variation because no treatment related changes were observed in body weight and feed consumption and no clinical symptom observed.
- Feed Consumption: The mean feed consumption of rats from the various treatment groups was comparable with the concurrent control group. Except male feed consumption was statistically lower during treatment days 36 to 43 at 62.5 mg/kg b. wt./day dose group when compared with the control group. Changes observed at 62.5 mg/kg b. wt./day dose group was considered as incidental and not related to treatment as same change was not observed at higher dose group (Tables 8 and 9; Appendices 7 and 8 of the attached report).

OTHER FINDINGS (PARENTAL ANIMALS)
- Fertility Data: Male fertility index, female fertility index and gestation index were comparable between control and KS-235 treated groups. Pregnancy rate and parturition index were unaffected by the treatment. The duration of the gestation period and pre-coital interval was comparable among all the dose groups. No treatment related difference was observed in pre-implantation, post- implantation and post-natal loss in KS-235 treated groups as compared to control group (Table 12; Appendices 9 and 14 of the attached report).

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks:
reproduction, systemic toxicity
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant adverse effects at highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

CLINICAL SIGNS (OFFSPRING):
- Clinical Sign: No clinical sign was observed in pups belonging to all test groups and control (Appendix 10 of the attached report).

BODY WEIGHT (OFFSPRING):
- Body Weight: No significant difference in body weight was observed in male and female pups of KS 235 treated groups as compared to control group (Table 11; Appendices 12 and 13 of the attached report).

OTHER FINDINGS (OFFSPRING):
- Mortality: No significant difference in pup mortality during the lactation period for male, female and total (male + female) pup was observed in all KS-235 treated groups when compared to the control group (Table 12 of the attached report).

- Live Birth and Survival Index: Mean number of live pups, pups survival index, lactation index and postnatal index were comparable between all treatment groups and the control group. Statistically significantly difference in live birth index was observed at 62.5 mg/kg b. wt./day dose group when compared with the control group. Variation observed at 62.5 mg/kg b. wt/day dose group was considered as biological variation and not related to treatment because it lacks dose dependent effect at subsequent doses (Table 12 of the attached report).

- Litter Size and Sex Ratio: Mean male, female and total (male + female) litter size and sex ratio were not affected in any of the dose groups (Table 10; Appendix 11 of the attached report).

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

- Homogeneity and Active Ingredient Concentration of the Test Item in the Vehicle: The mean percentage recovery obtained during treatment period of KS-235 in corn oil was;

 

During treatment period

Dose (mg/kg b.wt./day

62.5

%RSD

250

%RSD

1000

%RSD

1stweek

98.24%

0.34

98.16%

0.37

98.69%

0.21

4thweek

97.72%

0.21

98.35%

0.22

97.74%

0.56

6thweek

97.68%

0.12

97.37%

0.41

97.46%

0.18

 

The analysis result revealed that the mean calculated concentration was within the acceptance level of ±10% of nominal and CV% < 10% which indicates that the dose formulation of KS-235 in corn oil was homogeneous and the concentration was within the acceptance level (Appendix 16).

 

- Pathology Report (Appendix 14 of the attached report):

- Organ Weight: The terminal body weight and organ weight (both absolute and relative weight of testes and epididyinides) in male rats belonging to treatment groups were comparable to the control group.

- Macroscopic Findings: External and Internal examination of the terminally sacrificed parental rats and pups of all the groups did not reveal any lesion of pathological significance.

- Microscopic Findings: Detailed histological examinations of ovaries, testes and epididymides from the control and high dose groups did not show any lesion of pathological significance.

Applicant's summary and conclusion

Conclusions:
Transient salivation and body weight reduction was seen at 1000 mg/kg bw/day KS-235, but 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-pat-turn day 3 was evaluated 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. Hlistopathological 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 the treated groups. No treatment related macroscopic/microscopic finding was observed across all the groups.