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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
1997-09-04 through 1997-12-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Good Laboratory Practice guideline study

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 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Qualifier:
according to
Guideline:
other: EPA 40 CFR, Part 798 "Health Effects Testing Guidelines:, 1989-07-01, Section 798.2650 Oral Toxicity
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark, under Nitrogen

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: 6 weeks
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors from the day of arrival until start of the pretest. Single housing of rats in Macrolon MIII cages with sterilised sawdust provided as bedding from start of the pretest onwards.
- Diet (e.g. ad libitum): ad libitum standard pelleted laboratory animal dies (Carfil Quality BVBA, Oud-Turnhout, Belgium)
- Water (e.g. ad libitum): ad libitum tap water
- Acclimation period: At least 5 dats before start of treatment under laboratory conditions.

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
Oral gavage, using a stainless steel stomach tube or rubber catheter. Formulations were places on a magnetic stirrer during dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate sampels of Week 1 formulations were alaysed to check homogeneity (highest and lowest concentrations; samples taken at the top, middle and bottom of the container) and accuracy of preparation (all concentrations). Also, duplicate samples of formulations prepared during Weeis 4 and 13 were analysed to check accuracy of preparation (all concentrations). Total number of samples was 32. Test substance formulations in polyethylene glycol formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations of Groups 2, 3 and 4 revealed overall mean values of 102, 102 and 102% of nominal when values of peaks 1 and 2 were combined. This was considered to represent an acceptable level of accuracy for formulations of this type.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily, 7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
other: Vehicle Control
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
other: Nominal by gavage
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
other: Nominal by gavage
Remarks:
Doses / Concentrations:
1,000 mg/kg
Basis:
other: Nominal by gavage
No. of animals per sex per dose:
10 males/10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Dose levels were based on the results of a prior 28-day oral toxicity study in the rat.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
Mortality/Viability were examined twice daily.
Clinical signs were observed at least twice dail at the time of dosing as well as 1-2 hours post-dosing from Day 1 onwards. The time of onset, degree and duration were recorded. Observations included, but were not limited to, changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern. All symptoms were recorded according to fixed scales. Only the absence/presence of signs were recorded.

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

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

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

OPHTHALMOSCOPIC EXAMINATION: Both eyes were examined following installation of tropicamide solution (5 mg/ml0 for all animals at pretest and all animals at Week 13.

CLINICAL LABORATORY INVESTIGATIONS: Blood samples were collected under light ether anaesthesia immediately prior to post mortem examination. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.6 mL), with citrate for clotting tests (1 mL) and untreated tubes for clinical biochemistry parameters (>2 mL) Blood sampling was conducted prior to treatment, at 30 days of treatment and prior to sacrifice at 90 days of treatment.

HAEMATOLOGY: The following haematology parameters were determined from blood prepared with EDTA as an anti-coagulant: RBC count; HB; HCT; MCV; MCH; MCHC; platelet count; RDW; WBC; and differential leucocyte count SEG, EO, BASO, LYMPH & MONO. PT and PTT were determined from blood prepared with citrate as the anti-coagulant.

CLINICAL CHEMISTRY: Clinical biochemistry parameters determined from serum included ALAT/GPT; ASAT/GOT; BILI T.; CHOLEST. T., TRIGL.; Creatinine; Glucose; Urea; PROTEIN T.; ALBUMIN; GLOBULIN; A/G RATIO; ALP; Sodium; Potassium; Chloride; Calcium; and INORG. PHOSPH.
Sacrifice and pathology:
GROSS PATHOLOGY: Examinations included: adrenal glands; aorta; brain; caecum; cervix; colon; duodenum; epididymides; eyes with optic nerve and Harderian gland; female mammary gland area; femur including knee joint; heart; ileum; jejunum; kidneys; liver; lung; lymph nodes' oesophagus; ovaries; pancreas; pituitary gland; prostate gland; rectum; salivary glands; sciatic nerve; seminal vesicles; skeletal muscle; skin; spinal cord; spleensternum with bone marrow; stomach; testes; thyroic including parathyroid; trachea; urinary bladder; uterus; vagina; and all gross lesions, tissue masses and tumors.

ORGAN WEIGHTS: Organ weights were recorded for adrenal glands; brain; heart; kidneys; liver; lungs; ovaries; spleen; and testes.

HISTOPATHOLOGY: SLides of all tissues collected at the scheduled sacrigice 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.
Statistics:
Univariate one-way ANOVA 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) in the summary tables.
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

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

CLINICAL SIGNS:
There were no clinical signs of toxicity or behavioural changes over the 90-day observation period that were considered to be relevant to treatment.

Excessive salivation was observed in control and treated animals in a dose-related manner immediately after dosing. In the majority of animals, this finding was not observed 1-2 hours post-dosing. Excessive salivation is often noted in rats of this age and strain following oral gavage and considered to be related to multiple intra-oesophageal intubation and/or the taste of the test substance. Therefore, this finding was considered not to be a sign of systemic toxicity.

Other findings that were noted did not show a dose-response relationship and included piloerection, rales, brown staining of the forelegs, head and shoulders, red staining around the eye, nose and periorbital region, diarrhoea, blinking eye and black discoloration of the skin. These findings were considered to be within the normal range of biological variation for rats of this age and strain and are not test material related.

BODY WEIGHTS:
No statistically significant differences were noted in body weights and body weight gain of treated animals versus controls. Body weights and body weight gain of treated animals remained in the same range as controls over the 90-day study period. Slightly low values for body weight and body weight gain were recorded for control and treated males on Day 29. This was due to overnight fasting of these animals prior to blood sampling.

FOOD CONSUMPTION:
There were no differences in food consumption before or after allowance for body weight between treated and control animals. Slightly low values for food consumption were recorded for control and treated males over Days 22-29. This was also due to overnight fasting of these animals prior to blood sampling.

OPHTHALMOSCOPIC EXAMINATION:
There were no treatment-related ophthalmology findings at pretest and Week 13. Anterior synechia was noted in one male of the 100 mg/kg/day group at pretest, and corneal opacity with vascularization was seen in one male of the 500 mg/kg/day group in Week 13. Both findings were considered to be within the normal range of variation for rats of this age and strain and no toxicological relevance was attached to these observations.

CLINICAL LABORATORY INVESTIGATIONS:

HAEMATOLOGY:
At pretest no statistically significant differences were noted between animals assigned to the control and treatment groups.

After 30 days of treatment, red blood count, haemoglobin, haematocrit and mean corpuscular haemoglobin concentration values were slightly decreased in females in the 1,000 mg/kg/day 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 1,000 mg/kg/day. However, none of these parameters showed a clear dose-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 1,000 mg/kg/day 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 1,000 mg/kg/day.

Other minor statistically significant differences arising between control and treated animals after 30 days 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 in biological significance.

CLINICAL BIOCHEMISTRY:
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 1,000 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.

PATHOLOGY:

MACROSCOPIC EXAMINATION:
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. The few macroscopic findings noted were unremarkable and did not distinguis treated animals from the controls. A subcutaneous nodule i nthe genital region was seen in a single animal of the 100 mg/kg/day dose group and its histopathological correlate is discussed below. 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 in 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 amales receiving 1,000 mg/kg/day were considered to have resulted from slightly high control values and not to represent a sign of toxicity.

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/day dose group. This was an early occurrence of a common spontaneous neoplasm and was not related to treatment with the test article. From the 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 (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:
In this CoR 1 study, conducted on male and femal Wistar rats using appropriate test guidance and under GLP, treatment with Silane, Triethoxy [2-(7-OxaBicyclo[4.1.0]Hept-3yl)Ethyl]- for 90 days produced only minimal changes that were not accompanied by any evidence of organ dysfunction. Based on these results, a No Observed Adverse Effect Level (NOAEL) of 1,000 mg/kg/day was concluded for repated dose oral exposure to Silane, Triethoxy [2-(7-OxaBicyclo[4.1.0]Hept-3yl)Ethyl]-.
Executive summary:

In this CoR 1 study, conducted on male and femal Wistar rats using appropriate test guidance and under GLP, treatment with Silane, Triethoxy [2-(7-OxaBicyclo[4.1.0]Hept-3yl)Ethyl]- for 90 days produced only minimal changes that were not accompanied by any evidence of organ dysfunction. Based on these results, a No Observed Adverse Effect Level (NOAEL) of 1,000 mg/kg/day was concluded for repated dose oral exposure to Silane, Triethoxy [2-(7-OxaBicyclo[4.1.0]Hept-3yl)Ethyl]-.