Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Between 3 March and 14 August 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Recently conducted GLP compliant study using the most recent test methods

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): JKY-214
- Physical state: white solid
- Analytical purity: No information
- Lot/batch No.: Y002E
- Expiration date of the lot/batch: No information
- Stability under test conditions: No information. Assumed stable due to no data to the contrary
- Storage condition of test material: room temperature in the dark
- Other:

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd
- Age at study initiation: six to eight weeks old
- Weight at study initiation: males weighed 216 to 251g, the females weighed 163 to 196g
- Fasting period before study: not reported
- Housing: housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd, Cheshire, UK).
- Diet (e.g. ad libitum): free access to a pelleted diet (Rodent 2014C Teklad Global Certified Diet Harlan Laboratories UK, Oxon, UK)
- Water (e.g. ad libitum): free access to mains drinking water, supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15% respectively
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: Not reported

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis oil
- Concentration in vehicle: 30, 300, 600, 1000 mg/kg/day
- Amount of vehicle (if gavage): 4 mg/kg/day
- Lot/batch no. (if required):
- Purity: 97.5%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The standard and sample solutions were analysed by HPLC using the following conditions:
HPLC: Agilent Technologies 1050 or 1200,
Incorporating autosampler and workstation
Column: Gemini 5p C18 (50 x 4.6 mm id) @ 40°C
Mobile phase: Methanols. 1% formic acid (70:30 v/v)
Flow rate: 1.5 ml/min
UV Detector
Wavelength: 263 nm
Injection volume: 10 μl
Retention time: ~2.1 mins
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
600 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 14 day range finding study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: JMETI/MHLW/ MOE guideline
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random): JMETI/MHLW/ MOE guideline
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before, after and 1 hour after dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before, after and 1 hour after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1 and then at weekly intervals

FOOD Consumption: Yes
- Time schedule: weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Daily

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 for all non recovery anumals and Day 42 for Recover animals
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: No data
- Animals fasted: No data
- How many animals: No data

URINALYSIS: Yes
- Time schedule for collection of urine: No data
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: No data
- Dose groups that were examined: No data


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
Other examinations:
Histopathology Sample Processing
All tissues were despatched to the Test Site (Harlan Laboratories Ltd, Zelgliweg 1, CH- 4452 Itingen, SWITZERLAND) for processing (Principal Investigator K Weber). The tissues shown in bold from all non-recovery control and 1000 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 pm and stained with Haematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed together with the liver and spleen from all 30, 300 and 600 mg/kg/day dose group animals. In addition, sections of testes and epididymides from all Control and 1000 mg/kg/day males were stained with Periodic Acid-Shiff (PAS) stain and examined.
Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE 84 Pathology computerisation system for tabulation and report production.
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
All data were summarised in tabular form. Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett's test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non- parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non- parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (P) are presented as follows:
P < 0.01 ** P < 0.05 *
p > 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
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
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Effect levels

Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related changes observed at the highest xposure level of 1000 mg/kg bw/ day

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Summary forms including tabular data are attached.

Applicant's summary and conclusion

Conclusions:
The NOEL following dosing over a period of 28 days by oral gavage is 1000 mg/kg bw/day.
Executive summary:

The test substance has been dosed to rats at three exposure levels upto a maximum dose of 1000 mg/kg bw/ day over a period of 28 days by oral gavage with a 14 -day recovery period at high and solvent control dose levels. Under the conditions of the test, no toxicologically significant effects, clinically adverse observations and no macroscopic or microscopic observations. The observed No Effect Level is at the maximum tested dose of 1000 mg/kg bw/day