Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-07-13 to 1999-10-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

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:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21. September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy S.r.l., 33049 San Pietro al Natisone (UD), Italy
- Age at study initiation: 27-29 days old
- Weight at study initiation: weight range of 10 g per sex
- Housing: Clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh Iid and floor (Code 1354 G, Techniplast Gazzada S.a.r.l., Buguggiate, V arese ). Each cage tray held absorbent paper which was inspected daily and changed three times a week.
- Diet: Commercially available laboratory rodent diet (Altromin MT pelleted diet, A. Rieper, Bolzano, Italy)
- Water: Drinking water ad libitum
- Acclimation period: 18 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 2 °C
- Humidity: 55 % ± 10 %
- Air changes: 15 to 20 air changes per hour
- Photoperiod: twelve hours each day

IN-LIFE DATES: From: 1999-07-13 To: 1999-10-12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test solutions were prepared daily. The test substance was administered as a solution in corn oil. The formulations were prepared daily ( concentrations of 15, 50 and 200 mg/mL). The dose volume required for daily administration was calculated in advance based on the most recently recorded body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment the proposed formulation procedure was checked by chemical analysis to confirm that the method was acceptable. The stability over a 6 hour period was also checked on this occasion. In addition, samples of the formulations prepared in weeks 1 and 13 were analysed to check the concentration. Results of these analyses, carried out by the Analytical Chemistry Department at RTC. Results ofthese analyses were within the Iimits of acceptance.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily; 7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 75, 250 and 1000 mg/kg/day
Basis:
other: formulation in corn oil
No. of animals per sex per dose:
10 animals per sex per dose;
control and high dose groups included 5 additional animals per sex
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose selection was assessed on the basis of results obtained in a preliminary study (RTC StudyNo. 7115).
- Rationale for animal assignment: randomizing

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily before dosing, immediately after, and approximately I and 2 hours after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food consumed by each cage of rats was recorded weekly following allocation and the group mean daily intake per rat calculated.

OPHTHALMOSCOPIC EXAMINATION: Yes
Both eyes of all animals assigned to the study were examined just prior to the commencement of treatment. The eyes of all animals in all groups were re-examined during week 12 of treatment.
- Parameters examined: anterior chamber, conjunctivae and eyelids, cornea and sclera, iris, jens, posterior segment - vitreous humour, ocular fundus

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Once before the start of treatment and once during weeks 6 and 13.
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- How many animals: all
- Parameters examined: haematocrit, haemoglobin, red blood cell count, reticulocyte count (not performedas no signs of anaemia were present), mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count (neutraphits, lymphocytes, eosinophils, basophil, monocytes, large unstained cells), abnormalities of the blood film, platelets, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Once before the start of treatment and once during weeks 6 and 13.
- How many animals: all
- Parameters examined: alkahne phosphatase, alanine aminotransferase, aspartate aminotransferase, urea, creatinine, glucose, albumin, total bilirubin, total cholesterol, total protein, sodium, potassium, calcium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: Once before the start of treatment and once during weeks 6 and 13, ovemight urine samples
- Parameters examined: appearance, valurne, specific gravity, pH, protein, total reducing substances, glucose, ketones, bilirubin, urobilinogen, blood

Motor activity assessment:
The motor activity of the first 5 males and 5 females was measured once during week 13 of treatment and week 4 of recovery by an automated activity recording device (Mini Opto-Varimex, Columbus International Corp.).

Clinical signs and neurotoxicity assessment:
AlI clinical signs were recorded for individual animals. Examination of individual animals for signs of reaction to treatment was carried out daily before dosing, immediately after, and approximately 1 and 2 hours after dosing.
Animals were examined in an open arena for a period of three minutes.
-Parameter examined: Removal (from cage), handling reactivity, lachrymation, palpebral closure, salivation, piloerection, rearing, mobility impairrnent, arousal (animal activity), vocalisation, stereotypies, unusual respiratory pattern, bizarre behaviour, urination, detecation.


Sacrifice and pathology:
All animals were killed by carbon dioxide narcosis at the end of the scheduled treatment period and were subjected to necropsy supervised by a pathologist.

GROSS PATHOLOGY: Yes
- Number of animals: all
- Organs examined: Aadrenal glands, brain, epididymides, heart, li ver, kidneys, ovaries, spleen, testes, thymus, thyroid and parathyroid glands (after fixation), uterus

HISTOPATHOLOGY: Yes
- Number of animals: all
- Organs examined: adrenal glands, aorta, bone marrow (from sternum), brain, caecum, colon, duodenum, epididymides, eyes, femour, heart, ileum (including Peyer's patches), jejunum, kidneys, larynx, li ver, lungs (including mainstem bronchi), lymph nodes- cervical, lymph nodes- mesenteric, mammary gland, nasopharynx, oesophagus, optic nerves, ovaries, pancreas, parathyroid glands, pituitary gland, prostate gland, rectum, salivary glands, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinalcord, spleen, sternum, stomach, testes, thymus (where present), thyroid gland, trachea, urinary bladder, uterus, vagina


Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst group means was assessed by analysis of variance. Differences between each treated group and the control group was assessed by Dunnett's test using a pooled error variance. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off. Microscopic observations were tested for statistical significance using the non-parametric Kolmogorov-Smimov test.

Results and discussion

Results of examinations

Details on results:
No biologically relevant treatment-related changes were observed. Statistically significant variations in food consumption and clinical chemistry parameters, as weil as those in organs weights, were slight, in many cases not consistent between sexes and always within historical control values. No treatment related changes were observed at histopathological examination.

CLINICAL SIGNS AND MORTALITY
No death occurred during the study. Daily post-dose observations did not show any significant signs. Detailed clinical signs with neurotoxicity assessment did not show any treatment-related etfects.

BODY WEIGHT AND WEIGHT GAIN
No statistically significant differences were observed between control and treated groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No change of toxicological relevance was observed in food consumption.

OPHTHALMOSCOPIC EXAMINATION
No findings were seen at the ophthalmic examination performed at the end ofthe study.

HAEMATOLOGY
No treatment-related changes were observed in haematological parameters.

CLINICAL CHEMISTRY
No treatment-related changes were seen in clinical chemistry parameters. The statistically signiticant changes observed were within historical control values and, therefore, were considered of no toxicological relevance.

NEUROBEHAVIOUR
Neurotaxicity tests and measurements performed at the end of treatment did not show changes attributable to the test substance.

ORGAN WEIGHTS
No treatment-related changes were seen in the organ weights. The statistically significant variations in relative liver weights and other occasional changes in weights of various organs were within the range of our historical control data.

OTHER:
No macroscopic and/or microscopic change was observed that could be considered treatment related.


No treatment-related local changes were observed at the site of treatment (i.e. the mucose of the gastro-intestinal tract). Therefore, the Ievel of local tolerance after repeated contact of the substance (NOAEC) is regarded to be 20 % of the test item in corn oil.
Furthermore, no adverse systemic effects were observed. No changes of toxicological importance were seen at any of the dose Ievels investigated and, therefore, the No Observed Effect Level (NOEL) in this study is considered to be 1000 mg/kg/day/bw.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion