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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14-02-2008 to 27-05-2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: OPPTS 870.3650
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Triethoxyoctylsilane
- Substance type: Alkoxysilane
- Physical state: Colourless liquid
- Stability under test conditions: Stable, will react with moisture
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc, Portage.
- Age at study initiation: 9 weeks minimum
- Weight at study initiation: Males: 210.1 to 254.6 g ; Females: 159.6 to 201.7 g
- Fasting period before study: No
- Housing: Individually in suspended wire-mesh cages (except during cohabitation when female was put into male home cage, and pregnant animals were moved into shoebox cages).
- Diet: Ad libitum except during night prior to dosing, FOB and motor activity assessment.
- Water: Ad libitum.
- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 21.3
- Humidity (%): 39 - 60 %
- Air changes (per hr): 13.5
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14.04.2008 To: 07.06.2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance and vehicle were administered to each dose group at an equal dose volume (2ml/kg bw) based on the most recent body weight. Dosing solutions were individually prepared. Based on stability data, dosing solutions were prepared twice during the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the physical and chemical properties of the test substance, dried and deacidified peanut oil was considered to be the most appropriate vehicle for oral administration.
- Concentration in vehicle: 50, 150 and 500 mg/mL
- Lot/batch no. (if required): 080408 and 090408
- Purity: No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing solution analyses were performed by a GC/FID method to verify concentration, stability, and homogeneity of the test substance in the vehicle. Dosing solutions were analysed prior to study inititation to ensure stability and homogeneity. Stability was determined on study days 0, 4, 15, 21 and 32. Dosing solutions were also analysed for concentration verification following each dose solution preparation.
Duration of treatment / exposure:
Males: 28 days
Toxicity phase females: 29 days
Reproductive phase females: two weeks prior to mating, during mating, then through pregnancy, and to post-partum day 3.
Frequency of treatment:
Daily, seven days per week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Ten
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on results of a range-finding study (HES 10708-102)
- Rationale for selecting satellite groups: Toxicity group females used to assess repeated dose toxicity without the influence of pregnancy.
- Post-exposure recovery period in satellite groups: None
Positive control:
N/A

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily for mortality, morbidity and moribundity (once per day during weekends and holidays). General clinical observations were made at least once per day. Clinical observations were not performed on the day of the detailed clinical observations.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals received a detailed physical examination once before the first dose and weekly thereafter. Examinations included but were not limited to changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies, or bizarre behaviour were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were determined beginning with randomisation into test groups, on the first day of dosing, at least weekly thereafter and the day of necropsy. During gestation the pregnant animals were weighed on gestation days 0, 7, 14 and 20, within 24 hours after parturition and on day 4 post-partum or upon terminal euthanasia of the dam. Litter weights were measured within 24 hours of parturition and on day 4 post-partum or upon terminal euthanasia of the dams.

FOOD CONSUMPTION:
Individual animal food consumption was recorded at least weekly on an individual animal basis for the periods listed below.
Male rats: Two week pre-mating only. Feeder weights were taken on days 1, 8 and 15.
Female toxicity phase: Day 1 of dosing to necropsy. Feeder weights were taken on days 1, 8, 15, 22 and the day prior to necropsy.
Reproductive phase females: Two week pre-mating period, gestation and post-partum. Feeder weights were taken on days 1, 8, 15 and on gestation days 0, 7, 14, 20 and on day 0 and 4 post-partum.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On day of scheduled euthanasia.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: All surviving males and toxicity group females.
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On day of scheduled euthanasia.
- Animals fasted: Yes
- How many animals: All surviving males and toxicity group females.
- Parameters checked in table 1 were examined.

URINALYSIS: No

FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Time schedule for examinations: All adult males and all toxicity group females prior to the start of dosing. Testing in the fourth week of dosing took place prior to daily dosing.
- Battery of functions tested: cage side observations (abnormal muscle movements, abnormal behaviour, posture and resistance to removal from the cage), hand-held observations (palpebral closure, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response and reactivity to handling), open field observations (a one-minute evaluation of level of activity, responsiveness to sharp noise, touch and tail pinch, gait evaluation, quantity of urine and fecal pellets voided), categorical observations (behaviour, skin/fur/mucous membrane, respiration, muscle, eyes, urine or feces, soiling, posture and other abnormalities), measurements/counts (rectal temperature, hindlimb/forelimb grip strength, landing foot splay), and motor activity (locomotor activity in 80-minute test lengths).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see table 2. All males and toxicity phase females had a complete gross necropsy that included examination of the external surface and all orifices of the body, the cranial, thoracic and abdominal cavities and their contents. Based on clinical findings during the study, all reproductive females were also subjected to a complete gross necropsy as per the males and toxicity phase females. In addition, the number of corpora lutea and the number of implantation sites were recorded. The uteri of females with positive evidence of mating that failed to produce a litter were stained to enable counting of possible reabsorbed implant sites. Pups found dead were examined for external gross abnormalities only.

At necropsy, a number of organs from males and toxicity group females weighed (see Table 2).

HISTOPATHOLOGY: Yes (see table 2). Tissues and organs required for histopathological examination from the males and toxicity group females were collected from all animals in all treatment groups. Based on various clinical signs in the reproductive phase females, the tissues and organs from all treatment groups of animals were also collected.
Other examinations:
None reported
Statistics:
Body weights, body weight changes, food consumption, haematology and serum chemistry, prothrombin times and organ weights and organ to body weight ratios were analysed using one-way Analysis of Variance (ANOVA) if the data satisfied the requirements of normality of the residuals and homogeneity of variance. If the data did not satisfy the parametric requirements, a Kruskal-Wallis test was used. If the ANOVA or Kruskal-Wallis test was significant (p<0.05), pairwise comparisons of the exposed groups to control were made using the Dunnett's test or the Wilcoxon test, respectively. Reproductive parameters with the exception of litter size were analysed using an ANCOVA with litter size as the covariate. Litter size was analysed using an ANOVA. Measured continuous FOB data were analysed using a combination of ANCOVA with baseline evaluations used as the covariate and repeated measured ANOVA. Categorical FOB data were analysed using the Jonckheere-Terpstra test. Microscopic findings were also analysed using Cochran-Armitage trend test to indicate an increasing incidence trend regardless of grade with a Fisher's Exact test used to indicate increased incidence over the control. The clinical signs data were analysed using Mixed Modeling repeated measures.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All toxicity group males and females and reproductive group females of groups 1 to 3 survived until scheduled necropsy. Clinical signs were mainly restricted to Group 4. There was a dose-related increase in soiling around the nose and chin and generalised soiling around the muzzle in the Group 3 and 4 males and toxicity group females. These clinical findings were statistically significant (p<0.01) in groups 4 males and females.

There were statistically significant changes in the incidence of neurological clinical signs only in Group 4 reproductive group females compared to controls. Hind limb dragging (5/10) and uncoordinated gait (5/10) occurred in Group 4 reproductive group females. None of these neurological signs were noted until study day 32, with the exception of one animal that exhibited uncoordinated gait on day 21 and dragging hind limbs by day 25. These effects were also associated with a generalised decrease in overall activity (p<0.01) in 5/9 affected females. Rapid respiration occurred in 3/10 animals (p<0.05) beginning on the day prior to and/or on the day these animals were euthanised in extremis.
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no significant differences in weekly mean body weights and body weight gains in all male groups compared to controls, but there was a 6.8% decrease in mean overall body weight gain in the Group 4 compared to controls (p<0.05). Weekly mean body weights but not body weight gains were decreased on day 22 (8.9%) and 28 (9.3%) for the Group 4 toxicity group females compared to controls (p<0.05). Mean overall weight gains in Group 4 were also decreased (26.9%) compared to controls (p<0.05).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Average daily food consumption during week 3 in Group 2 (p<0.05) and 4 (p<0.02) toxicity group females were statistically decreased compared to controls, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related, toxicologically significant changes in haematology parameters.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no treatment-related, toxicologically significant changes in haematologyclinical chemistry parameters.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no statistically significant differences across dose groups in either the baseline or treated FOB categorical and motor activity data for the males and toxicity group females.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A dose-related increase in mean absolute (p<0.05) and relative (p<0.01) liver weights occurred in Groups 3 and 4 males, resulting in an 11.7% and 17.3% increase in relative liver weights at these dose levels compared to controls.

A 14.6% increase (p<0.01) in mean relative kidney weights occurred in Group 4 toxicity group females compared to controls. A dose-related increase in mean absolute liver weights also occurred in Group 3 and 4 toxicity group females that attained statistical significance only in the Group 4 toxicity group females compared to controls (p<0.01). These increases in absolute liver weights were associated with an increase in mean relative liver weights. Mean relative liver weights were increased by 4.7% (p<0.05) and 27.8% (p<0.01) in the Group 3 and 4 toxicity group females, respectively. Absolute, but not relative, ovarian weights were also statistically significantly decreased (p<0.05) in the Group 3 and 4 toxicity group females.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related gross findings in the male or female toxicity group animals.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological findings in the liver were associated with an increase in mean absolute and relative liver weights in both the Group 4 male and female toxicity groups and are consistent with common adaptive changes that occur in the liver.

There was a statistically significant increase in the incidence (7/10) of minimal to mild diffuse epithelial hyperplasia of the urinary bladder of Group 4 males compared to controls (p<0.01). One male was affected in Group 2; no males had these findings in Group 3. There was no significant effect in toxicity group females. Epithelial hyperplasia of the urinary bladder was observed; however, in 7/10 Group 4 reproductive group females and not in controls. Although two reproductive group females in Group 2 had this finding in the urinary bladder, there was no dose-related effect as this finding did not occur in reproductive group females in Group 3.

The main finding in the central nervous system was white matter degeneration of the brain and spinal cord in Group 4 toxicity group and reproductive group females, with an increased incidence in the reproductive group females compared to the toxicity group females. The cerebellum and medulla appeared to be the most prominently affected areas of the brain. There was an increase in the incidence of minimal white matter degeneration (vacuolation) of the brain in 4/10 Group 4 toxicity group females and one control. In the Group 4 reproductive group females this effect attained statistical significance, with 8/10 Group 4 animals exhibiting mild to marked vacuolation in the brain and no effects in the controls (p<0.01). The spinal cord of the toxicity group females also exhibited diffuse white matter vacuolation in both the sensory and motor nerve roots in 5/10 Group 4 and no control toxicity group females (p<0.01). Minimal to marked demyelination of the spinal cord also occurred in 9/10 Group 4 reproductive group females and no controls (p<0.01). Although the incidence of the effects in the brain were less prominent and did not attain statistical significance in the toxicity group females, there was evidence of minimal white matter vacuolation of the brain in 4/10 Group 4, 1/10 Group 3 and 1/10 control females. there were no effects on the central nervous system in males.

In the peripheral nerves examined, the sciatic and tibial nerves, there was a statistically significant increase in the incidence of minimal to severe demyelination/degeneration in 8/10 (sciatic) (p<0.01) and 9/9 (tibial) (p<0.01) Group 4 reproductive group females and not in the controls. Affected animals tended to show the change in multiple sites unless only the spinal cord was involved. Minimal to mild nerve degeneration also occurred in 3/10 Group 4 toxicity group females and not in controls, but did not attain statistical significance. The only neurological effect in the males was minimal sciatic nerve degeneration in 2/10 Group 4 males, which also occurred in 1/10 controls.

Based on clinical signs in the Group 4 reproductive group females, two skeletal muscle sections of the hindlimb were evaluated. There was a statistically significant increase in Group 4 reproductive Group female adductor muscle degeneration (10/10) compared with controls (4/10) (p<0.01). There was a trend toward significance in the incidence of degeneration of the gastrocnemius muscle in Group 4 reproductive group females (9/10) compared to controls (5/10), and statistically significant increase in the severity of degeneration compared to controls. Animals affected with neurological findings also showed gross muscle atrophy, diffuse decreased muscle fibre size, fibre fragmentation, increased density of myofibre nuclei, and focal areas of inflammation around necrotic fibres. The animals with these skeletal muscle findings also had notable decreases in hind leg skeletal muscle mass. These findings are consistent with denervation atrophy and considered secondary to the nerve changes. These data indicate that the severity of the clinical and microscopic tissue neuromuscular effects is a likely reflection of duration of exposure rather than an effect specific to the reproductive status of the animal as the toxicity group females were only dosed for 29 days compared up to 45 days for the reproductive females.

These neurological findings did not occur at any of the lower doses or in males.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on bladder epithelial hyperplasia in males and the neuromuscular findings in the toxicity and reproductive phase females at 1000 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
no

Applicant's summary and conclusion

Conclusions:
In an oral combined repeated dose toxicity study with the reproduction / developmental toxicity screening test conducted to OECD 422 and to GLP (reliability score 1) the NOAEL for triethoxyoctylsilane for general systemic toxicity was 300 mg/kg bw/day, based on bladder epithelial hyperplasia in males and the neuromuscular findings in the toxicity and reproductive phase females at 1000 mg/kg bw/day.