Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 24 AUG 1999 to 19 OCT 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
27 JUL 1995
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
30 SEP 1996
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch: V.N0199.064
Expiry: 09.07.2001

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Recognized by international guidelines as the recommended test system
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Biotechnology & Animal Breeding Division, Füllinsdorf /Switzerland
- Age at delivery: 6 weeks
- Weight at start of acclimatization: males: 120-157 g (mean 136 g), females: 96 - 144 g (mean 116 g)
- Fasting period before study: no data
- Housing: in groups of five in Makrolon type-4 cageswith wire mesh tops and standardized softwood bedding
- Diet: pelleted standard Provimi Kliba 3433 rat maintainace diet, ad libitum.
- Water: Community tap water, ad libitum
- Acclimation period: yes, 7 days

DETAILS OF FOOD AND WATER QUALITY: The feed batch and tap water were analysed for contaminants. None of the contaminants analysed in the water and diet is considered to have been present at a concentration which would have affected the validity of the results.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
PEG300
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article formulations were prepared weekly. Test substance was weighed into a glass beaker an a tared Mettler Balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature (17 - 23°C).
Homogeneity of the test article in the vehicle were maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): PEG 300 was found to yield the most homogenous test article/vehicle mixture
- Concentration in vehicle: 0, 5, 20 or 100 mg/mL
- Amount of vehicle (if gavage): 10 ml/kg bw/d
- Lot/batch no. (if required): 395304/1 30699
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken during acclimatization. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed by RCC Ltd (Environmental Chemistry & Pharmanalytics Division) according to a photometric measurement method supplied by the Sponsor.
Duration of treatment / exposure:
28 days for exposure groups 1-4 (0, 50, 200 and 1000 mg/kg bw/d, consisting of 5 male and 5 female animals respectively)
Two additional exposure groups (0 and 1000 mg/kg bw/d, 5 male and 5 female animals/ exposure group) were exposed for 28 days followed by a recovery period of 14 days.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
0 and 1000 mg/kg bw/d: 10 males and 10 females
50 and 200 mg/kg bw/d: 5 male and 5 females
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale: Based on the results of a non-GLP 5-day dose-range-finding study in which the test substance was administerd by gavage to 2 rats per group and sex. Animals showed only passive effects to the treatment ( yellow colored feces)
- Rationale for animal assignment: computer generated random algorithm

Positive control:
none

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
recorded once during pre-test period, and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after week 4 and after week 6
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes for 18 h
- How many animals: all

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after week 4 and after week 6
- Animals fasted: Yes for 18 h
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: after week 4 and after week 6
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes for 18 h, during this time urine was collected

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 4
- Dose groups that were examined: all animals evaluated
- Battery of functions tested: grip strength and locomotor activity

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Statistics:
The following statistical methods were used to analyze grip strength, locomotor activity, body weights, organ weights and all ratios, as well as clinical laboratory data :
- The Dunnett-test (many to one t-test) based an a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Student's t-test was applied to grip strength and locomotor activity.
- Fisher's exact-test was applied to macroscopic findings.

References :
- C.W. Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
- R.G. Miller: Simultaneous Statistical Inference, Springer Verlag, New York (1981).
- R.A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
GENERAL CAGESIDE OBSERVATIONS (DAILY)
No test article-related clinical signs were evident at any dose level.
Yellow feces was noted in all test article-treated animals. The severity of the discoloration was related to dose and persisted during the first few days of recovery in the remaining animals treated with 1000 mg/kg/day. Discoloration of the feces is considered to be a common passive effect resulting from oral administration of a dyestuff and does not reflect a toxic response.
Slightly soft feces were noted in all animals from treatment day 2 until the last day of treatment. This finding persisted during the first few days of recovery in the remaining animals of the control group and in animals treated with 1000 mg/kg/day. Soft feces is known to be a common effect caused by PEG 300 and is unrelated to the test article.

DETAILED CLINICAL OBSERVATIONS (WEEKLY)
Reduced grooming was noted during treatment week 1 in all females treated with 50 mg/kg/day. The push-off reflex was reduced during week 1 in one female ( treated with 1000 mg/kg/day.
Slight chromodacryorrhea was noted during treatment week 1 in one male rat treated with 1000 mg/kg/day.
These findings were considered to be incidental and unrelated to the treatment with the test article.
All other animals were without clinical signs during pretest and during treatment weeks 1-3.
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled necroscopy.
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test article-related differences to the control values were noted at any dose level. The few statistically significant differences were considered to be incidental.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Plasma uric acid levels were higher in males and females treated with the test article at 1000 mg/kg/day. Although the differences to the control values attained statistical significance (males, p<0.01; females p<0.05), they were within the upper range of the 95% confidence limits of the historical control data and therefore considered to be unrelated to the test article treatment.
The remaining differences to the control values noted in the males treated with 1000 mg/kg/day (higher total bilirubin, sodium, albumin and total protein) were not evident in the females, nor were corresponding findings noted in related parameters. These differences were considered to be incidental.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Slight chromodacryorrhea was noted in one male rat during functional observational battery at treatment week 4. This finding was not seen in the remaining males or females of this group and therefore considered to be incidental.
All other animals were without clinical signs during treatment week 4.

The forelimb grip strength of the test article-treated animals was similar to that of the controls.
The hindlimb grip strength of the males treated with 200 mg/kg/day or 1000 mg/kg/day was higher than that of the controls, and attained statistical significance (p<0.01). These differences were considered to be unrelated to the test article.
The hind limb grip strength of the test article-treated females compared favorably with that of the control females.

Locomotor Activity
No test article-related differences in locomotor activity were noted when compared with the control values.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
After 4 Weeks
No test article-related differences to the control values were noted at any dose level tested.
The statistically significant higher absolute brain weight (p<0.05) noted in the males treated with 1000 mg/kg/day was considered to be incidental. Subsequently, the brain-to-kidney weight ratio noted in these animals was slightly lower. This difference to the control value attained statistical significance (p<0.05).
All other organ weights and ratios compared favorably with the controls.

After 6 Weeks
The absolute mean liver and testes weights of males treated with 1000 mg/kg/day were higher than those of the control males. The differences attained statistical significance (p<0.05), but were considered to be incidental. The absolute mean liver weight of test article-treated females compared favorably with that of the control females.
All other organ weights compared favorably with those of the controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All findings were considered to be within the range of spontaneous background alterations in rats of this strain and age. They consisted of incompletely collapsed lungs, renal pelvis dilation, accentuated lobular pattern of the liver or size reduction of a liver lobe, enlarged adrenal glands, uterus horn dilation and discoloration and/or discolored foci in several organs. In one kidney, a nodule was recorded.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A malignant nephroblastoma was the histological correlate to the nodule noted in the kidney of one control male. This tumor and the rernaining findings were within the range of spontaneous background lesions recorded in rats of this strain and age.
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: no adverse effects up to the highest dose tested (i.e. also limit dose for testing)

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
In this 28-day study according to OECD guideline 407 performed under GLP, male and female Wistar rats were exposed to 0, 50, 200 or 1000 mg test item/kg bw/d in PEG 300 via gavage. Cage side observations, clinical observations, food consumption, body weight development, haematology, urine analysis, macroscopic investigations, histopathology of selected organs and assays concerning the functional observational battery (grip strength and locomotor activity) did not reveal any substance related toxic effects, except for a yellow discoloration of the feces, higher plasma uric acid levels in males and females treated with the test article at 1000 mg/kg/day and a higher absolute brain weight noted in the males treated with 1000 mg/kg/day. However, these effects were considered incidential by the study authors. These effects were minimal and therefore not accounted for as adverse. The NOAEL in this study was 1000 mg/kg bw/d.
Executive summary:

The test item did not reveal substance related adverse effects under the conditions tested (according to OECD TG 407, performed under GLP). Yellow feces was noted in all test article-treated animals. Discoloration of the feces is considered to be a common passive effect resulting from oral administration of a dyestuff and does not reflect a toxic response.

Minimal  effects were observed in the highest dose group, but considered to be incidental. Plasma uric acid levels were higher in males and females treated with the test article at 1000 mg/kg/day. Although the differences to the control values attained statistical significance (males, p<0.01; females p<0.05), they were within the upper range of the 95% confidence limits of the historical control data and therefore considered to be unrelated to the test article treatment. The statistically significant higher absolute brain weight (p<0.05) noted in the males treated with 1000 mg/kg/day was considered to be incidental, too.

Therefore the NOAEL in this subacute oral feeding study was 1000 mg/kg bw/d, the highest dose tested.