Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 October 1999 to 29 February 2000
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Solid
Details on test material:
- Name of test material (as cited in study report): Pymordiol
- Physical state: Cream white solid
- Purity of test material: 96 wt%
- Lot/batch No.: DGM096K1A
- Expiration date of the lot/batch: 1 January 2001
- Stability under test conditions: Stable under test conditions and throughout test period.
- Storage condition of test material: At room temperature in the dark.

Test animals

Species:
rat
Strain:
other: Wistar Crl:(WI) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld Germany
- Age at study initiation: Approx. 6 weeks
- Weight at study initiation: +/- 20% of the sex mean.
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors. During activity monitoring, individually housed over night in Macrolon plastic cages with sterilised sawdust (SAWI, Jelu Werk, Rosenberg, Germany).
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet (Carfil Quality BVBA, Oud-Turnhout, Belgium).
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21˚C +/- 3˚
- Humidity (%): 30-70%
- Air changes (per hr): Approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial light/12 hours dark

IN-LIFE DATES: From: 7 October 1999 To: 4 November 1999

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 4 hours prior to dosing. Adjustment was made for specific gravity of vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX and confirmed to be a suitable vehicle for chemical analysis of dose preparations.
- Amount of vehicle (if gavage): 5mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of week 4 formulations were analysed to check stability over 4 hours, homogeneity (highest and lowest concentration) and accuracy of preparations (all concentrations).
Duration of treatment / exposure:
28-Days
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
5, 15, 50 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5 males and 5 females (10 total)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of a 5-day dose range finding study

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to start of treatment and on days 8, 15, 22 and 28

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to post mortem examination
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Animals fasted: Yes
- How many animals: All animals tested
- Parameters checked in table No. 1 (below) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to post mortem examination
- Animals fasted: Yes
- How many animals: All animals tested
- Parameters checked in table No. 2 (below) were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 4 of treatment
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Hearing ability, pupillary reflex, static righting reflex, grip strength, and activity test.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes, Table No. 3 (below) for parameters examined. All animals surviving to the end of the observation period were anaesthetised using ether vapour and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities were recorded. Samples were collected and fixed in 4% formaldehyde solution.
HISTOPATHOLOGY: Yes, Table No. 3 (below) for parameter examined. All organs were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
Slides of all organs and tissues collected at the scheduled sacrifice from all animals of the control and the highest dose group, and all gross lesions of all animals were examined by a pathologist. Liver, kidneys (males only), spleen and lungs were examined for all rats of the low and mid does groups.
Statistics:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled 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 tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Test statistics were calculated on the basis of exact values for means and pooled variances.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
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 examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no clinical signs of toxicity or behavioural changes over the 28-day observation period that were considered to be related to treatment. No mortality occurred during the study period.

BODY WEIGHT AND WEIGHT GAIN: Body weight gain of low dose males and females during the 4-week treatment period was higher when compared to control animals, and achieved levels of statistical significance on days 8 (both sexes) and day 28 (males only). As a result, body weights of low dose males and females were higher than that of controls after 4 weeks of treatment (for males and not statistically significant for females).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Slightly lower food consumption was seen in high dose females over the 4 week study period when compared to controls.
An inverse relation to treatment was found for the food consumption in males. Whereas food consumption in males increased at lower dose levels of the test substance. These effects in food consumption correlated with changes in body weights in animals.

HAEMATOLOGY: Treatment related changes were seen in erythrocyte parameters and erythrocyte indices derived thereof and white blood cells in male and female rats.
A dose related increase was seen for the erythrocyte counts (RBC) and decreases in mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH), resulting statistically significant differences in values for RBC in high dose males, for MCV in high dose females and for MCH in high dose males and mid and high dose females. A statistically significant increase in red cell distribution width was seen in both high dose males and high dose females.
An increase number of white blood cells was found in the high dose animals, but achieved a level of statistically significance in females only when compared to controls. Although in both sexes the increase in the number of lymphocytes was mainly responsible for the increase in white blood cells, the increase in number of neutrophils was more pronounced. The increase number of neutrophils in high dose females, both relative and absolute, achieved a level of statistical significance. As a result, the relative number of lymphocytes was statistically significant decreased, despite an increase in absolute number.
In low dose females, statistically significant differences were also found for the increase in relative and absolute number of neutrophils, which was the cause of the statistically significant decrease for relative number of lymphocytes. In the absence of a clear dose related response, the toxicological significance of changes in the differential leucocyte counts in low dose females was doubted.
In the absence of clear changes in the values for haemoglobin and haematocrit, no toxicological significance was attached to the statistically significant changes in mean corpuscular haemoglobin concentration in males of all dose groups and low and mid dose females.
The minor statistically significant decrease in partial thromboplatin time arising between controls and low dose males was considered to have arisen by chance and not to represent a change of biological significance.

CLINICAL CHEMISTRY: In the high dose males and females treated with test substance, increased levels of alanine aminotransferase were reported, reaching levels of statistical significance in the females, when compared to controls for any of the dose levels.
In mid dose males, a statistically significant increase in the level of alkaline phosphatase was seen, whereas a tendency to increased levels was seen in high dose males and females (not statistically significant).
A very slight dose related decrease of total protein, and concurrent decrease of total albumin, was reported, but all values remained well within the historical background data. In high dose females, the values of albumin achieved a level of statistically significance when compared to controls.
The statistical significance for the (increased) levels of sodium in low and mid dose males was considered to have arisen by chance. In the absence of a treatment-related distribution or corroborative findings in the opposite sex, these changes were considered to be of no toxicological significance.

NEUROBEHAVIOUR: No changes were reported in hearing ability, pupillary reflex, static righting reflex and grip strength in the animals treated with test substance, when compared to control animals.
The variation in motor activity between control and treated animals did not indicate a relation with treatment.

ORGAN WEIGHTS: Treatment related changes in liver and spleen weights were found in high dose males and females and in kidneys weights of males of all dose groups.
In males, a statistically significant increase was found for the liver weights and the liver:body weight ratio and in females a level of statistical significance was achieved for the liver:body weight ratio only.
A dose related increase was found for the spleen weights of both males and females. Both absolute and relative spleen weights of high dose animals showed a statistical significance of p<0.01. The relative spleen weights of mid dose females was also statistically significant increase, when compared to controls.
Statistically significant increases were seen for the absolute kidney weights in males of all dose groups. The kidney:body weight ratios were statistically increased in mid and high dose males only.
No toxicological significance was attached to the (statistically significant) difference for heart weights in high dose females, since they were considered to be mainly the result of the changes in (terminal) body weight of the animals compared to that of controls.

GROSS PATHOLOGY: Enlarged size of the spleen was observed in 2/5 males of the high dose group.
The findings of alopecia reported in a few mid and high dose females confirm the clinical signs in these animals and were considered of no toxicological significance.

HISTOPATHOLOGY: NON-NEOPLASTIC: Microscopic findings related to treatment were noted in lungs, liver and spleen as well as in kidneys of male rats only.
Lungs: There was an increase in the incidence and severity of primarily inflammatory alterations in the lungs of mid and high dose rats of both sexes. These consisted of perivascular/peribronchial inflammatory cell foci, alveolar inflammation and foci of alveolar macrophages, which were recorded at minimal to moderate degrees of severity. The level of these findings in the low dose animals were similar to those in the control group.
Liver: In the liver there was an increase in the incidence and severity of multifocal granulomatous inflammation, periportal lymphoid inflammation and single cell necrosis in mid and high dose rats of both sexes. The level of these findings in low dose animals was similar to those in the control group.
Spleen: In the spleen, lymphoid hyperplasia was noted at minimal to moderate degrees of severity in five mid dose and eight high dose rats. This finding was the correlate to the enlarged spleen noted in two high dose males at necropsy.
Kidneys - males: Hyaline droplets in the cortical renal tubules were recorded in all groups of males including controls. However, there was a slight increase in severity - from minimal to slight, and incidence in treated groups.

Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 5 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Excluding kidney effects.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the current test conditions and excluding the kidney effects NOAEL = 5 mg/kg/day for both male and female rats.