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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29-09-2008 to 11-01-2010
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

1
Chemical structure
Reference substance name:
Dimethoxydimethylsilane
EC Number:
214-189-4
EC Name:
Dimethoxydimethylsilane
Cas Number:
1112-39-6
Molecular formula:
C4H12O2Si
IUPAC Name:
dimethoxydimethylsilane
Details on test material:
- Name of test material (as cited in study report): Dimethyldimethoxysilane
- Substance type: Alkoxysilane
- Physical state: Colourless liquid
- Stability under test conditions: stable in absence of water, moisture or humid air.
- Other: Solubility: Soluble in acetone, ethanol

Test animals

Species:
rat
Strain:
other: Crl: CD® (SD) IGS BR VAF/Plus®
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC 27610
- Age at study initiation: 9 weeks at experimental start
- Weight at study initiation: 192 to 239 g at experimental start (Females). 283 to 341 g at experimental start (Males)
- Housing: Animals were individually housed in suspended wire-mesh cages elevated above faecal pans containing Bed-O’ Cobs® litter, during quarantine/ acclimation and during the in-life phase of the study. Animals were given Nylabones® and Cozee Pads for environmental enrichment while in standard housing. Environmental enrichment was removed from the toxicity group male and female animals the afternoon/evening prior to necropsy.
- Diet : Lab diet 5002, Certified Rodent Diet (PMI Nutrition International) was provided ad libitum during the quarantine/acclimation period and throughout the study. Food was removed the afternoon/evening prior to necropsy of toxicology animals
- Water: Municipal water, further purified by reverse osmosis was available ad libitum via automatic watering system.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 -21.7
- Humidity (%): 53-59
- Air changes (per hr): 14.3
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20-10-2008 To: 30-07-2009.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Each dosing solution was prepared individually. Dosing solutions were prepared by adding the appropriate amount of the test article to a tared container and adding the appropriate amount of corn oil to yield the desired dose level. Dosing solutions were administered by oral gavage with a 100 mm, 15 gauge (1.8 mm), plastic feeding tube and syringe. The test article was administered at a dose volume of 4 ml/kg bw and was calculated from the most recent body weight.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based upon the physical and chemical properties of the test article, dried and deacidified corn oil was considered to be the most appropriate vehicle for oral administration.
- Lot/batch no. (if required): 058K0070
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing solution analysis was performed by a GC/FID method to verify concentration, stability, and homogeneity of the test article in carrier. Concentration verification was conducted for the initial and third dose preparations.
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 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:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed daily immediately following exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were determined beginning with randomization into test groups, on the first day of dosing, at least weekly thereafter, and on the day of euthanasia. All animals received a detailed physical examination once before the first dose (to allow for within-subject comparisons), and weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected for hematological evaluations from all animals on the day of scheduled euthanasia as a terminal procedure.
- Anaesthetic used for blood collection: Yes (identity). While the animals were under Isoflurane anesthesia a syringe and needle were used to collect blood samples from the abdominal vena cava and distributed to three sample collection tubes containing either sodium citrate, EDTA, or no anticoagulant.
- Animals fasted: No data
- How many animals: All
- Parameters checked: RBC, MCV, MCH, MCHC, WBC, total and differential, Hgb, Hct and PLT.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected for clinical chemistry evaluations from all animals on the day of scheduled euthanasia as a terminal procedure.
- Animals fasted: Yes
- How many animals: All
- Parameters checked: Alanine aminotransferase, Albumin, Alkaline phosphatase, Aspartate aminotransferase, Calcium, Cholesterol, Chloride, Creatine, Glucose, Phosphorus, Potassium, Sodium, Total Bilirubin, Total protein and Urea nitrogen.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes /
- Time schedule for examinations: Functional Observation Battery (FOB) evaluation were performed on all animals prior to the start of dosing (baseline evaluation). Testing performed during the fourth week of dosing took place prior to daily dosing. FOB and motor activity examinations were performed on all animals since no signs of toxicity to an extent that would significantly interfere with the evaluation were noted.
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity /

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Body weight, changes in body weight, organ weight, organ to body weight ratios, food consumption data, haematology data, serum chemistry and pro-thrombin times were analysed using a one-way Analysis of Variance (ANOVA) if the data satisfied the requirements of normality of the residuals and homogeneity of variance as determined using a statistical test for normality and homogeneity of variance (Wilks-Shapiro and Bartlett’s, respectively; Gad and Chenglis, 1998). If the data did not satisfy the parametric requirements, a Kruskal-Wallis test was used (Kruskal and Wallis, 1952; Kruskal, 1952). In the case of clinical pathology data a log or rank transformation was performed. If the ANOVA or Kruskal-Wallis test was significant (P<0.05), a pair-wise comparisons of the exposed groups to control were made using the Dunnett’s test (Dunnet, 1955, 1964) or a Wilcoxon test, respectively.
For variables with multiple measurements across time (motor activity, body weight and food consumption), a repeated measurement ANOVA was performed to determine if a significant time by treatment group interaction exists. Repeated measurement ANOVAs were done using the time (baseline and post-treatment), interval, sex, and treatment to look for interactions between the treatment, the time and sex to determine if analysis could be done with the sexes combined.
Microscopic findings were also analysed using a Cochran-Armitage trend test to indicate an increasing incidence trend regardless of grade with a Fisher’s exact test used to indicate increased incidence (non-grade specific) over the control. In addition, for those microscopic findings with one or more incidences in the control groups, the graded animals only were compared using a Kruskal-Wallis test to determine if there was an increase in severity grade between the control group and the treated groups. Pair-wise comparisons of the graded-only animals were done using a Wilcoxon test if the overall Kruskal-Wallis test was significant.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
For the males at 1000 mg/kg bw/day, significant abnormal observations were noted and included soiling of the chin and urogenital area. Soiling of the chin, muzzle and urogenital areas were significant abnormal observations in the toxicity group females at 1000 mg/kg bw/day.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant differences between controls and treatment groups in the mean body weights on any of the test groups.

HAEMATOLOGY and CLINICAL CHEMISTRY
Statistically significant changes noted in haematological parameters and pro-thrombin times and clinical chemistry parameters for males and females were within or slightly above/below historical control values and these findings did not correlate with a pathological outcome, therefore, no toxicological significance is given to any of these statistically identified differences

NEUROBEHAVIOUR
No statistically significant differences were found between the control and treatment groups in either sex for all the FOB ranked tests. There were no treatment-related changes associated with dimethyldimethoxysilane administration on rat neurobiological function as evaluated with FOB and motor activity parameters.
ORGAN WEIGHTS
There were statistically significant differences compared to controls noted for the following organ weights in males; adrenal glands (decrease at 1000 mg/kg bw/day), thymus (decrease at 1000 mg/kg bw/day), epididymides (decrease at 1000 mg/kg bw/day), prostate gland (decrease at 1000 mg/kg bw/day) and seminal vesicles (decrease at 1000 mg/kg bw/day). There were statistically significant differences for the mean percentage of organ weights relative to body weights for adrenal glands (decrease at 1000 mg/kg bw/day), liver (increase at 250 and 1000 mg/kg bw/day), thymus (decrease at 1000 mg/kg bw/day) and testes (decrease at 1000 mg/kg bw/day) for the males.
There were statistically significant differences noted for organ weights in females compared to controls; liver (increase at 250 and 1000 mg/kg bw/day) and spleen (decrease at 1000 mg/kg bw/day). There were statistically significant differences for the mean percentage of organ weights relative to body weights for liver (increase at 250 and 1000 mg/kg bw/day) and spleen (decrease a 1000 mg/kg bw/day) in females.

GROSS PATHOLOGY
Effects attributable to test article administration in males occurred in the liver, thyroid gland, adrenal glands, kidneys, testes, and epididymides. In females test material effects were observed in the liver and thyroid glands. There were questionable effects in the spleen of males and the lungs of both sexes. Target tissues examined at 50 and 250 mg/kg bw/day in males were liver, lungs, thyroid glands, adrenal glands, kidneys, testes, epididymides and spleen. In females, the target tissues examined were liver, lungs and thyroid glands.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the liver, there were three primary effects of the test article observed, including panlobular hepatocellular hypertrophy in both sexes at 1000 mg/kg bw/day and centrilobular hypertrophy in females at 250 mg/kg bw/day, increased periportal hepatocellular vacuolation (microvesicular lipidosis, females administered 1000 mg/kg bw/day), and pigment accumulation (males only). The latter was usually accompanied by chronic inflammation and bile duct hyperplasia. Hepatocellular hypertrophy (panlobular or centrilobular) is considered an adaptive change. Hepatic vacuolation, unless severe, is generally considered non-adverse. Hepatic pigment accumulation is considered an adverse effect.
Thyroid follicular cell hypertrophy was observed in the thyroid gland of high –dose rats of both sexes. This is considered an adaptive secondary effect and adverse for the rat, but the mechanism is generally not applicable to species with significant levels of thyroid binding globulin (Capen et al., 2002).
There was minimal adrenal cortical atrophy in half of the male rats dosed at 1000 mg/kg bw/day. The pathogenesis and significance of this finding is not clear.
In the testes there was moderate to marked seminiferous tubule degeneration observed in all 1000 mg/kg bw/day male rats that was characterised by degeneration of spermatocytes. A downstream effect was observed in the epididymides of the same rats. This is an adverse finding.
The increased liver weights in males and females at 1000 mg/kg bw/day correlated with the histopathologic finding of panlobular hypertrophy and centrilobular hypertrophy in females at 250 mg/kg bw/day. In males, adrenal cortical atrophy was accompanied by a decrease in absolute and relative adrenal weights at 1000 mg/kg bw/day, seminiferous tubule degeneration was accompanied by a decrease in absolute and relative testes weights at 1000 mg/kg bw/day, epididymal effects were accompanied by a decrease in absolute and relative decreases in epididymal weights, and kidney nephropathy findings (subtle increase in eosinophilic rhomboidal or needle-shaped crystalline cytoplasmatic inclusions in renal renal proximal tubule epithelial lining cells, and a small increase in degenerating and sloughing cells in this same cell population) were accompanied by an increase in relative kidney weights. However, these findings are consistent with minimal alpha-2-µ-nephropathy, which is a specific finding for male rats. This conclusion is further supported by the fact, that female kidneys were not affected. Alpha-2-µ-globulin deposition is not relevant for humans and therefore not taken into account for setting the effect level for the human hazard assessment.


Effect levels

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on results observed at 1000 mg/kg bw/day in males (hepatic pigment accumulation, adrenal cortical atrophy, testicular seminiferous tubule degeneration with epididymides involvement) and in female rats (periportal vacuolation).

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on results observed at 1000 mg/kg bw/day in males (hepatic protoporphyrin accumulation, adrenal cortical atrophy, kidney protein droplet nephropathy, testicular seminiferous tubule degeneration with epididymides involvement) and in females rats (periportal vacuolation), the NOAEL (No-Observed-Adverse-Effect-Level) for systemic toxicity of dimethyldimethoxysilane is 250 mg/kg bw/day.