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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to an appropriate national standard method and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: EU Method B.26 (Sub-Chronic Oral Toxicity Test; 90-day repeated oral dose using rodent species)
Qualifier:
according to
Guideline:
other: OECD Guideline 408 (Subchronic Oral Toxicity - Rodent: 90-day study)
Qualifier:
according to
Guideline:
other: EPA 798.2650 (Health Effects Testing Guidelines;Oral Toxicity)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Silane, triethoxy[2-(7-oxabicyclo[4.1.0]hept-3yl)ethyl]
- Physical state: Clear, pale liquid
- Analytical purity: 99.3%
- Lot/batch No.: 17036-86
- Expiration date of the lot/batch: 1999-08-08
- Storage condition of test material: At room temperature in the dark, under Nitrogen
- Other: Stable under storage conditions; Density: 1; Stability in vehicle Polyethylene glycol 400: at least 24h

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, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation:
- Housing: group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors until start. Single housing of rats in Macrolon MIII cages with sterilised sawdust (B.M.I. Helmond, The Netherlands) provided as bedding from start of the pretest onwards.
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet (from Carfil Quality BVBA, Oud-Turnhout, Belgium)
- Water (e.g. ad libitum): Free access to tap-water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Approximately 21°C
- Humidity (%): Approximately 50%
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours dark per day

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Polyethylene glycol 400
Details on oral exposure:
TEST SUBSTANCE FORMULATION:
Method: Formulations (w/w) were prepared daily immediately prior to dosing, with the exception of two occasions on which formulations were prepared within 24 hours prior to use.
Storage conditions: At ambient temperature

TREATMENT
Frequency: Once daily for at least 90 days, approximately the same time each day
Dose volume: 5ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate samples of week 1 formulations were analysed to check homogeneity (highest and lowest concentration: samples taken at the top, middle and bottom of container) and duplicate samples of week 1, 4 and 13 were analysed to check accuracy of preparation (all concentrations).
Duration of treatment / exposure:
Test duration: 90 days
Frequency of treatment:
Dosing regime: 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 500 and 1000 mg/kg/day
Basis:
other: nominal oral gavage
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 28-day oral toxicity study
Positive control:
None

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least twice daily at the time of dosing as well as 1-2 hours post-dosing from day 1 onwards (on day 15 the second observation for clinical signs was performed approximately 4 hours post-dosing).

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly, beginning 1 week prior to test substance administration and just prior to scheduled necropsy.

FOOD CONSUMPTION:
- Time schedule: Weekly, beginning 1 week prior to test substance administration.

WATER CONSUMPTION:
- Time schedule: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: At pretest and week 13. Both eyes were examined following instillation of tropicamide solution (5 mg/ml)
- Dose groups that were examined: All animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to treatment, after approx. 30 days of treatment and prior to sacrifice.
- Anaesthetic used for blood collection: Yes, light ether anaesthesia
- Animals fasted: Yes, overnight but water was provided.
- How many animals: all animals


CLINICAL CHEMISTRY: Yes

OTHER:
- Mortality/Viability: Twice daily
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals surviving to the end of the observation period were deeply anaesthetised using ether vapour subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution: Adrenal glands, Aorta, Brain, Caecum, Cervix, (Clitoral gland), Colon, Duodenum, Epididymides, Eyes with optic nerve and Harderian gland, Female mammary gland area, Femur including knee joint, Heart, Ileum, Jejunum, Kidneys, (Larynx), Lacrimal gland, exorbital), Liver, Lung, infused with formalin, Lymph nodes - mandibular mesenteric, (Nasopharynx), Oesophagus, Ovaries, Pancreas, Pituitary gland, (Preputial gland), Prostate gland, Rectum, Salivary glands - mandibular sublingual, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord - cervical midthoracic lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions, tissue masses and tumours.
Following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy: Adrenal glands, Brain, Heart, Kidneys, Liver, Lungs, Ovaries, Spleen, Testes.

HISTOPATHOLOGY: Yes
All organ and tissues samples were processed, embedded and cut at a thickness of 2 - 4 micrometers and stained with haematoxylin and eosin.
Slides of all tissues collected at the scheduled sacrifice from all animals of the control and the highest dose group, as well as from all animals of all dose groups which died spontaneously, all gross lesions, lungs, livers and kidneys of all animals (all dose groups) were examined by a pathologist.
Tissues mentioned between brackets (see GROSS PATHOLOGY) were only examined if indicated by signs of toxicity or target organ involvement. An attempt was made to correlate gross observations with microscopic findings.
Statistics:
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 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.
The exact Fisher-test was applied to the ophthalmoscopic observations.
All tests were two-sided and in all 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).
Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs of toxicity. Mortality was seen but not related to treatment with the test substance.
Mortality:
mortality observed, treatment-related
Description (incidence):
No clinical signs of toxicity. Mortality was seen but not related to treatment with the test substance.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
But no clear sign of toxicity in the absence of a dose-response relationship.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
But no dose-dependent relationship
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS
There were no clinical signs of toxicity or behavioural changes over 90-day observation period that were considered to be related to treatment.

MORTALITY
One control male died after dosing on day 3 and another control male died prior to dosing on the same day. One male receiving 500 mg/kg/day died prior to dosing on day 5. The animal which died after dosing did not exhibit any clinical signs prior to death. At necropsy, haemorrhagic fluid was noted in the thoracic cavity. The male which died on day 3 showed hunched posture and piloerection and at necropsy finding were noted in the pancreas, kidneys, urinary bladder, epididymides, prostate, spleen and thymus. The male which died on day 5 showed labored respiration prior to death, but no macroscopic findings were noted at necropsy.

BODY WEIGHTS
No statistically significant differences were noted in body weights and body gain of treated animals versus controls. Body weight and body weight gain of treated animals remained in the same range as controls over the 90-day study period.

FOOD CONSUMPTION
There were no differences in food consumption before or after allowance for body weight between treated and control animals.


OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related ophthalmology findings at pretest and week 13.

HAEMATOLOGY
At pretest no statistically significant changes were noted between animals assigned to the control and treatment groups.
After 30 days of treatment red blood cell count, haemoglobin, haematocrit and mean corpuscular haemoglobin concentration values were slightly decreased in females of the 1000 mg/kg dose group. Mean corpuscular haemoglobin concentration was also decreased in females receiving 100 and 500 mg/kg/day. In addition total white blood cell count was decreased in females receiving 1000 mg/kg/day. However, none of these parameters showed a clear dose-related response, and the control values were slightly high in comparison with the historical data. Therefore, the toxicological relevance of these decreases is doubted.
After 13 weeks of treatment red blood cell count and haematocrit values were slightly decreased in females of the 1000 mg/kg/ dose group and mean corpuscular haemoglobin concentration was increased in females of this dose group. Partial thromboplastin time was slightly increased in females receiving 500 and 1000 mg/kg/day.
Other minor statistically significant differences arising between control and treated animals after 30 day and 13 weeks of treatment were considered to have arisen by chance and in the absence of a dose-response relationship considered not to represent a change of biological significance.

CLINICAL CHEMISTRY
At pretest sodium, chloride and calcium were slightly lower and urea slightly higher in males assigned to the treatment groups in comparison with animals of the control group. In females of the treatment groups, triglycerides and calcium were lower and sodium higher compared to control values. The values were considered to have resulted from slightly high or low control values but remained within the range of historical data that can be expected for untreated animals of this age and strain.
After 30 days of treatment there were no differences noted between control and treated animals that were related to treatment with the test substance. Statistically significant changes noted in all dose groups did not exhibit a clear dose-dependent relationship and/or were considered to have resulted from slightly high (calcium, aspartate aminotransferase activity) or low control values (glucose).
After 13 weeks of treatment urea values were increased in males receiving 1000 mg/kg/day. Other values in treated animals after 13 weeks achieving a level of statistical significance when compared to controls were considered to have arisen as a result of slightly high (total bilirubin, cholesterol, aspartate aminotransferase activity, total protein, calcium) or low control values (creatinine, sodium) and in the absence of a treatment-related distribution or corroborative findings in the opposite sex, considered to be of no toxicological significance.

ORGAN WEIGHTS
Organ weights and relative organ weights of treated animals were considered to be similar to those of control animals.
Decreased absolute lung weights recorded in males receiving 1000 mg/kg/day were considered to have resulted form slightly high control values and not to represent a sign of toxicity.

MACROSCOPIC EXAMINATION
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as result of treatment. The few macroscopic findings noted were unremarkable and did not distinguish treated animals from the controls. A subcutaneous nodule in the genital region was seen in a single animal of the 100 mg/kg dose group. All findings were considered to be within the range of biological variation for rats of this age and strain and not to represent a change of toxicological significance.

MICROSCOPIC EXAMINATION
There were no microscopic findings noted that were considered to be treatment-related.
Mammary adenocarcinoma was the microscopic correlate to the subcutaneous nodule noted at necropsy in one animal of the 100 mg/kg dose group. This was an early occurrence of a common spontaneous neoplasm and was not related to treatment with the test article. From this tissues examined, a definitive cause of death for the three unscheduled decedents could not be determined. All microscopic findings recorded were considered to be spontaneous in nature and within the range of background morphological alterations encountered in Wistar rats of this age and strain. They occurred at similar incidences and severity in both control and treated groups.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Analytic:

Analysis of the accuracy of dose preparations revealed overall mean values of 102% of nominal.

Results:

100 mg/kg/day: No treatment-related findings noted

500 mg/kg/day: After 13 weeks, partial thromboplastin time was slightly increased in females.

1000 mg/kg/day: After 13 weeks, red blood cell count and haematocrit were slightly decreased, and mean corpuscular haemoglobin concentration and partial thromboplastin time were slightly increased in females. After 13 weeks, urea was slightly increased in males.

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with Silane, triethoxy[2-(7-oxabicyclo[4.1.0]hept-3yl)ethyl]- for at least 90 days produced only some minimal changes which were not accompanied by any evidence of organ dysfunction. Based on these results, a NOAEL of 1000 mg/kg/day was concluded.
Executive summary:

This study was designed to evaluate the oral toxicity of Silane, triethoxy[2-(7-oxabicyclo[4.1.0]hept-3yl)ethyl]- when administered once daily by oral gavage to rats for at least 90 consecutive days. The substance was administered in polyethylene glycol (PEG 400) at 100, 500 and 1000 mg/kg/day. Control animals received PEG 400 at dose volumes equivalent to those administered to treatment group animals.

Mortality was seen in the control group (2 males) and the 500 mg/kg dose group (1 male). During pretest, one of the control males showed already a slightly reduced health status based on low body weight gain and food consumption. In addition, no mortality occurred at the highest dose tested and no definitive cause of dead could be determined from microscopic examination. Therefore, these deaths were considered to have occurred by chance or due to technical reasons (e.g. gavage related injury) and not related to treatment with the test substance.

Changes in red blood cell count and associated parameters were noted in females of the 1000 mg/kg dose group after 30 days and 13 weeks of treatment. The changes seen after 30 days (including white blood cell count) are thought to have resulted from slightly high control values and not to be a clear sign of toxicity. This is in accordance with the absence of treatment-related effects at 1000 mg/kg after 28 days in a previous study (NOTOX Project 194996, registered in IUCLID for Silane, triethoxy[2-(7-oxabicyclo[4.1.0]hept-3yl)ethyl]- on 7.5.1 Repeated dose toxicity: oral 28 days). All haematological changes noted after 13 weeks (including partial thromboplastin time) were only slight in nature and not accompanied by any evidence of organ dysfunction at macroscopic or microscopic level.

Slightly increased serum urea levels were recorded in males of the 1000 mg/kg dose group at pretest and after 13 weeks of treatment. Levels at the end of treatment were only slightly higher than pretest levels, and therefore the effect of treatment is considered minimal. This is supported by the absence of any evidence of organ impairment.

The only change noted below a dose of 1000 mg/kg was an increase in partial thromboplastin time at 500 mg/kg, but in the absence of other relevant findings, a connection with treatment is considered unlikely.

In conclusion, treatment with Silane, triethoxy[2-(7-oxabicyclo[4.1.0]hept-3yl)ethyl]- for at least 90 days produced only some minimal changes which were no accompanied by any evidence of organ dysfunction. Based on these results, a NOAEL of 1000 mg/kg/day was concluded.