Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 January 1998 to 28 May 1998
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
not specified
GLP compliance:
yes

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): Epyrron
- Physical state: Off white powder
- Analytical purity: 55%
- Composition of test material, percentage of components: Main component - 55%; Other 22%, 18%
- Lot/batch No.: GV-1458 K1
- Expiration date of the lot/batch: 24 September 1998
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: All animals were within +/- 20% of the sex mean
- Housing: Stainless steel suspended cages with wire mesh floors, group housing of 5 animals per sex per cage.
- Diet (e.g. ad libitum): Standard pelleted laboratory animal diet (from Carfil Quality BVBA, Oud-Turnhour, Belgium)
- Water (e.g. ad libitum): Tap-water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ˚C
- Humidity (%): 50%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 light/12 dark

IN-LIFE DATES: From: 5 December 1998 To: 13 February 1998

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily immediately prior to dosing. Adjustment was made for specific gravity of vehicle. Formulations were placed on a magnetic stirrer during dosing. Exposure via oral gavage, using a stainless steel stomach tube.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX
- Concentration in vehicle: 0 (vehicle), 50, 200 and 1000 mg/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of pretreatment were anlaysed to check stability and samples of week 2 formulations were analysed to check stability, homogeneity (highest and lowest concentration) and accuracy of preparations (all concentrations).

Analysis of the accuracy of dose preparations revealed values within the range of 95% to 103% of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.

Formulations were considered to be stable for at least 4 hours, in the 28-day study and group 2 during pretest, although the data of group 2 in the 28-day study were slightly outside the acceptable ranges.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days per week.
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 200, 1000
Basis:
no data
No. of animals per sex per dose:
10 (5 males, 5 females)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on 5-day dose range finding study
- Rationale for animal assignment (if not random): Random

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, detailed clinical observations were completed once prior to start of treatment and on days 8, 15, 22 and 28. All symptoms were graded according to fixed scales (see Table 5 below).

BODY WEIGHT: Yes
- Time schedule for examinations: On days 1, 8, 15, 22 and 28

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, overnight prior to collection
- How many animals: All animals tested
- Parameters checked in table [No. 1] were examined; see below.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Immediately prior to scheduled post mortem examination.
- Animals fasted: Yes, overnight prior to collection
- How many animals: All animals tested
- Parameters checked in table [No. 2] were examined; see below.

URINALYSIS: Yes
- Time schedule for collection of urine: Immediately prior to scheduled post mortem examination.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes, overnight prior to collection
- Parameters checked in table [No. 2] were examined, see below

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all tested animals were necropsied and descriptions of all macroscopic abnormalities were recorded. Samples of the following tissues and organs listed in tables No. 3-4 below were collected and fixed in neutral phosphate buffered 4% formaldehyde solutions.
HISTOPATHOLOGY: Yes, slides of all organs and tissues collected at sacrifice from control and high dose group animals, and all animals which died spontaneously and all gross lesions of all animals were examined by a pathologist.
Based on treatment related morphologic changes, testes, epididymides, seminal vesicles, spleen and thymus were also examined from al rats of the intermediate dose groups.
Tables No. 3-4 below contains the tissues and organs that were examined (tissues in brackets were not examined as there were no signs of toxicity or target organ involvement).
Other examinations:
Histotechnology: All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
Statistics:
The following statistical methods were used to analyse the data:

Univariate one-way analysis of variance was used to assess the significance of intergroup differences.

If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a polled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.

The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.

All test were two-sided and inall cases p<0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: No mortality occurred during the study period. At 1000 mg/kg/day test group reported hunched posture, swelling of the abdomen and alopecia.

A slight decrease in motor activity was reported in animals receiving 1000 mg/kg/day of test substance.

BODY WEIGHT AND WEIGHT GAIN: Males within the dose group of 1000 mg/kg/day reported a statistically significant decrease in body weights and body weight gain.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Males in 1000 mg/kg/day does group reported a decrease in food consumption during weeks 1, 2, and 3 however after allowance for body weight only a decreased over the first week and was increased over the 4th week.

HAEMATOLOGY: Haemoglobin levels and mean corpuscular haemoglobin concentrations were decreased and red cell distribution width was increased in males within the 1000 mg/kg/day. In both males and females total leucocyte count was slightly low.

CLINICAL CHEMISTRY: Statistical significant increases in alanine - and aspartate aminotransferase, glucose and alkaline phosphatase level were noted in females receiving 1000 mg/kg/day. Glucose level and alanine aminotransferase was slightly increased in ales of this dose level.

Cholesterol levels were decreased in males and females receiving 1000 mg/kg/day.

ORGAN WEIGHTS: The thymus, spleen, testes and epidiymides organ weight and/or organ weight ratios were decreased at 1000 mg/kg/day dose level. The liver weights and liver: body weight ratios were increased at this same dose level.

A reported decrease in thymus weights and thymus: body weight ratio was observed in females at the 200 mg/kg/day dose level.

Statistical significant differences were observed in the following weights in males: absolute heart and kidney weights and organ: body weight ratios for brain, heart and adrenals. These organ weights were indirectly affected as a result of lower body weights. No toxicological significance was reported for these findings.

GROSS PATHOLOGY: A reduction in size of the epididymides, prostate, seminal vesicles and/or testes was observed in males. A small size of the thymus was noted in all females and alopecia was observed in all animals of the 1000 mg/kg/day does level.

The following describes the observations reported in the 1000 mg/kg/day dose group. The testes a severe degree of seminiferous tubular atrophy was seen in two rats and was accompanied by epidermal oligospermia. Seminal vesicles were recorded as slightly or moderately atrophic in four rats.

In the thymus, minimal to moderate atrophy was noted in three males and all five females treated at 1000 mg/kg/day and were associated with an increase in incidence and severity of lymphocytolysis. In the spleen of females there was a decrease in the incidence and degree of hemopoiesis which was associated in two cases with lymphoid atrophy.

Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
It can be concluded that under the conditions of this test that a NOAEL of 200 mg/kg/day is appropriate.