Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Remarks:
Study included to support DNEL risk assessment.
Adequacy of study:
supporting study
Study period:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

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:
Seventy-eight female and seventy-eight male rats were obtained from Charles River Laboratories, Kingston, NY. Animals were 10 weeks old at experimental start.
Animal Receipt and Quarantine/Acclimation: Upon receipt, animal resource personnel inspected each animal. Animals judged to be in good health and suitable as test animals were quarantined/acclimated for a minimum of five days. The attending veterinarian examined all animals before release from quarantine/acclimation and documented the general state of animal health.
Animal Housing: Animals were individually housed in suspended wire-mesh cages during the course of the study. The cages were elevated above Bed-O’Cobs bedding and subjected to routine cleaning consistent with good housekeeping practices. Prior to exposure, animals were acclimated according to the following schedule:
Three days prior to exposure: Animals were placed in the exposure caging for 2.8 – 3.0 hours.
Two days prior to exposure: Animals were placed in the exposure caging for 6.0 hours.
One day prior to exposure: Animals were placed in the exposure caging for 6.0 – 6.1 hours in the inhalation chambers.
Following each exposure, the test article treated animals were housed in a separate animal room from the control animals.

Environmental Enrichment: Animals were given Gnaw PucksTM and Cozee PadsTM in their home cages for environmental enrichment.

Environmental Conditions: Animals were housed in environmentally controlled animal rooms. With the exception of the deviations noted in Table 13 environmental conditions were within 18 – 26°C for temperature, 30 – 70% relative humidity, and 10 – 15 air changes per hour. Lighting was controlled to provide a 12-hour fluorescent-light/dark cycle. Temperature and humidity were recorded approximately every fifteen minutes using a HOBO® data logger (Onset Computer, Bourne, MA; software: BoxCar® Pro 4.3.1.1.). In addition, twice a day on weekdays and once a day during weekends and holidays these values were manually recorded.

Basal Diet: Certified Rodent Diet #5002, PMI Nutritional International Inc., St. Louis, MO, was offered ad libitum except while animals were in the inhalation exposure cages and during the fasting period prior to terminal sacrifice. Manufacturer’s periodic analyses of the certified feed for the presence of heavy metals and pesticides was reviewed by the study director to ensure that none are present in concentrations that would be expected to affect the outcome of the study.

Drinking Water: Municipal water, further purified by reverse osmosis (RO) was available ad libitum except while animals were in the inhalation exposure cages. Water collected from the RO system was monitored on a semi-annual basis to determine compliance with the EPA drinking water standards. The most recent analysis was reviewed by the study director to ensure that there were no contaminants known to be present in water, at levels expected to interfere with the integrity of the study.

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
Vapour Generation: Generation of test article vapor was performed using heated stainless steel J-tubes containing stainless steel beads. Test article was metered from reservoirs into J-tubes using either a Fluid Metering Incorporated (FMI®) pump or a Harvard model syringe pump equipped with Hamilton brand glass syringes. Compressed air flowed through the J-tube at a predetermined controlled rate. The carrier/vapour mixture passed from the J-tube directed to the inlet port at the top of the exposure chamber. Just prior to entering the exposure chamber, the carrier/vapour mixture was combined with chamber supply (dilution) air where it was diluted to the target chamber concentration as it entered the exposure chamber.

Inhalation Chambers: Exposures were conducted in 2000-liter stainless steel and glass Rochester-style inhalation chambers and stainless steel exposure caging (four layers of 20 animal compartments). The animal cage position assignment within each chamber was rotated daily. Chamber temperature, relative humidity, and airflow were monitored and recorded during the acclimation period similar to that anticipated for the exposure periods. Vapor generation systems were not in operation during acclimation. The chambers were targeted for 12 – 15 chamber volume air changes per hour and environmental conditions of 20 ± 3°C and 50 ± 20%RH. Chamber airflow, temperature and relative humidity were monitored continuously and values recorded approximately once every 30 minutes during exposures. Chamber oxygen content was monitored, and manually recorded, once during the first day of exposure to ensure that under applied experimental conditions the oxygen content was above the minimum 19% acceptable limit.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test Atmosphere Monitoring: The test atmosphere from each chamber was sampled by an automated sampling system. The system was designed such that test atmosphere was continuously pulled from the chamber and delivered to the analyzer through individual chamber sample lines and a stream selector valve. The sample lines were continuously purged with fresh chamber atmosphere during the entire exposure period.
Chamber atmosphere was analyzed using a gas chromatograph equipped with a flame ionization detector (GC/FID) to determine the actual chamber concentration of test article. In addition, given the potential hydrolysis of methyltrimethoxysilane in humid air, the methyl alcohol concentration within each chamber was also determined. The concentration of test article in the chamber atmosphere during the exposure period was evaluated a minimum of once every 60 minutes. Methyl alcohol (a known hydrolysis product of methyltrimethoxysilane) concentrations were measured from each exposure chamber a minimum of seven times during each day’s exposure period. A continuously purged sample line was used to transfer chamber atmosphere to the GC/FID for analysis.
The GC/FID methods were established prior to the experimental start date. Standard curves relating test article vapor concentration and methanol concentration to GC/FID response were established before the first exposure and then again as necessary. Preparation of calibration curves involved bag standards at five different levels that bracketed the expected range of test article or methyl alcohol chamber concentrations. The peak area attributed to the test article or methyl alcohol was plotted against the nominal bag standard concentration. Linear regression analysis of these data was performed to define the parameters of the calibration curve. Acceptance criteria for the GC/FID calibration curve included linear regression correlation coefficient of > 0.98 and < 10% difference between the prepared bag standard concentration and the calculated bag standard concentration derived from the linear regression equation of the calibration curve.
Each calibration curve was verified prior to the exposure period by analysis of a bag standard. The bag standard actual concentration derived from the calibration curve needed to be within 10% of the bag standard nominal concentration for the calibration curve to be considered acceptable for use.
The mean daily actual measured methyltrimethoxysilane vapor concentration was compared with the daily-calculated nominal concentration as a quality control mechanism to evaluate exposure system performance. A difference of ≤15% was considered acceptable for the 25 ppm targeted exposure level, and ≤10% at all other target levels.
Homogeneity of test atmosphere within each chamber was evaluated once prior to initiation of animal exposures. Acceptance criteria required the mean values for each chamber zone not exceed 10% difference from the reference zone (approximate sampling location used during animal exposures).
The stability of methyltrimethoxysilane vapor in a gas bag and sample line loss was evaluated using prepared bag standards. Stability was considered acceptable over the time the measured concentration did not exceed 10% difference from the original concentration measured immediately following preparation.
Exposure chambers were leak tested prior to the first exposure to ensure proper operation. Acceptance criteria required that the mean chamber airflow measured at the inlet not be more than 10% different from the airflow measured on the exhaust side.
Duration of treatment / exposure:
Exposure levels and treatment regimen: Test article was administered by whole-body vapour inhalation. Animals were positioned in the chambers and the exposure period was defined as a 6-hr/day exposure at the target concentration. Animals were removed from the chambers after a second T99 had expired. The T99 period was considered as the time required for the chamber to reach 99% of the target concentration or clearance of 99% of the achieved concentration following generation of test article. Based on chamber flow rate and size, the T99 period was calculated to be 23 minutes for all chambers. For practical purposes, the T99 2 minutes was used for this study.
The target exposure levels were: 0, 25, 100, 400 and 1600 ppm trimethoxy(methyl)silane in air. These exposure levels were determined based on results from a previously conducted 14-day range finding study (Tobin, 2007). Exposures were conducted at approximately the same time each day, five days per week over the course of thirteen weeks.
Frequency of treatment:
6 hours/day, 5 days/week at target concentrations
Doses / concentrations
Remarks:
Doses / Concentrations:
25, 100, 400, and 1600 ppm (ca. 0.14, 0.56, 2.2 and 8.9 mg/L)
Basis:

No. of animals per sex per dose:
10/sex/dose level plus and additional 10/sex/control and high dose groups for a 28-day recovery period
Control animals:
yes, concurrent vehicle
Details on study design:
This study was conducted following the general principals of the OECD Guidelines for Testing Chemicals, “Subchronic Inhalation Toxicity 90-Day Study”, No. 413, adopted May 12, 1981. The study was conducted to evaluate the toxic effects of, and subsequent recovery from, whole-body vapor inhalation exposure of methyltrimethoxysilane. Five (5) groups of 10 male and 10 female Sprague-Dawley rats were exposed to target methyltrimethoxysilane exposure concentrations of 0 (control), 25, 100, 400 and 1600 ppm, 5 days per week for thirteen weeks. Additional satellite groups of 10 males and 10 females were included in the 0 and 1600 ppm target groups for evaluation of a 28-day post exposure recovery period. Exposures terminated on study 90 with non-recovery animals euthanized on study day 91. Recovery group animals remained for 28-days post exposure and were euthanized on study day 119.

Examinations

Observations and examinations performed and frequency:
Mortality/Morbidity/Moribundity: All animals were observed in their cages for mortality, morbidity, and moribundity at least twice daily on weekdays, and once daily during weekends and holidays.
Clinical Observations: General clinical observations were made at least once a day, beginning on the first day of exposure, at approximately the same time each day. The health condition of the animals was recorded. Clinical observations included, but were not limited to, changes in the skin, fur, eyes, and mucous membranes, respiratory system, circulatory system, autonomic and central nervous systems, motor activity, and behavior patterns. General clinical observations were performed on all animals on the day of, and prior to, their scheduled necropsy.

Parameters Measured
Individual Body Weights: Individual body weights were recorded for randomization, weekly throughout the duration of the study, then again prior to sacrifice on the day of scheduled termination.
Individual Food Consumption: Feeder weights were recorded weekly throughout the duration of the study.
Ophthalmic Examinations: Examinations were performed on all animals prior to group assignment. Animals with findings noted during this initial examination were not used on study. Additional examinations were conducted during the final week of exposure prior to 90-day terminal sacrifice, and during the final week of the 28-day post exposure recovery period. An indirect ophthalmoscope (following dilation using mydriatic eye drops) was used for all examinations.
Sacrifice and pathology:
Clinical Pathology: Food was removed from all animals on the evening prior to scheduled termination. Clinical pathology assessments were made on all surviving animals from which adequate samples were collected.
Blood samples were collected for hematological and clinical chemistry evaluations from all animals on the day of scheduled euthanasia as a terminal procedure. While 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.

Hematology: Blood samples for the following hematology tests were collected into test tubes containing EDTA. Hematology samples were analyzed within 24 hours of collection. Analysis was performed using the Cell-Dyn 3700TM (Abbott Diagnostics, Dallas, TX).
Erythrocyte count Hemoglobin
Erythrocyte Indices (MCV, MCH, MCHC) Hematocrit
Leukocyte counts (total and differential) Platelet count
Blood samples for coagulation assessment (prothrombin time) were placed into test tubes containing sodium citrate, centrifuged within two hours of collection and the plasma separated for testing. Samples were maintained at room temperature (18-24 oC) or refrigerated (2-8 oC) prior to analysis. Analysis was conducted within 24 hours of collection. Analysis was preformed using the ACL 100TM (Beckman Coulter, Fullerton, CA).

The following serum chemistries were determined:
Alanine aminotransferase Glucose
Albumin Phosphorus
Alkaline phosphate Potassium
Aspartate aminotransferase Sodium
Calcium Total bilirubin
Cholesterol Total protein
Chloride Urea nitrogen
Creatinine
Blood samples were placed into test tubes without anticoagulant, allowed to clot and centrifuged within two hours of collection and the serum separated for testing. For bilirubin and total protein analysis, the serum samples were frozen at  -20C, until analysis. Samples were analyzed using the Cobas Integra 400 plusTM (Roche Diagnostics, Indianapolis, IN).

Gross Pathology: All animals were subjected to a full gross necropsy which included examination of the external body surface, all orifices, as well as the cranial, thoracic and abdominal cavities including contents.

Histomorphology: Eyes were preserved in Davidson’s solution until processing with the testes and epididymides preserved in Bouin’s solution for 24 – 36 hours then transferred into 70% ethanol until processing. All other tissues were placed in 10% Neutral Buffered Formalin for preservation. Tissue processing included routine procedures including embedding in paraffin, sectioning and staining with hematoxylin and eosin for evaluation.
Histomorphological examination was conducted on all tissues collected from both the recovery and non-recovery animals in groups 1 and 5. Following initial review, additional selected tissues including urinary bladder, kidney, liver, lung and prostate were evaluated for groups 2, 3, and 4.
Other examinations:
The following organs were weighted: adrenals, kidneys, liver, lung, testes, ovaries, brain, epididymides, seminal vesicles and prostate.
Statistics:
Daily mean inhalation exposure concentrations and environmental conditions, along with standard deviations, were calculated using Microsoft Office Excel. Mean and standard deviation were calculated for body weights, changes in body weights, food consumption, organ weights, hematology and clinical chemistry values, using ProvantisTM, version 6.5. Environmental conditions of animal rooms were monitored and recorded using a HOBO® data logger (Onset Computer, Bourne, MA; software: BoxCar® Pro 4.3.1.1.)
All data analysis was carried out using SAS version 9.1.3. Statistically significant probabilities were reported for p-values of < 0.05, <0.02, and < 0.01.
Data from the recovery groups was analyzed separately from the 90 day groups. Body weight, changes in body weight, food consumption data, organ weight, organ to body weight ratios, hematology data, clinical chemistry and prothrombin times were analyzed 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 the Shapiro-Wilk test for normality and Levene’s test for 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, pair-wise comparisons of the exposed groups to control were made using the Dunnett’s Test or a Wilcoxon test, respectively.
For variables with multiple measurements across time (body weight and food consumption), a repeated measurements ANOVA was performed to determine if there was a time by exposure group interaction.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry 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
Details on results:
Average measured methyltrimethoxysilane exposure concentrations included below the limit of quantitation (BLQ), 25 +/- 0.8, 99 +/- 3.2, 398 +/- 12.8, and 1612 +/- 35.6 ppm for groups 1 through 5, respectively. Calculated nominal concentrations included 23 +/- 0.4, 94 +/- 1.6, 383 +/- 10.4, and 1570 +/- 48.9 ppm for groups 2 through 5, respectively. Mean measured methyl alcohol concentrations were below the limit of calibration (8.7 ppm) for for all exposure groups except group 5 which was 19 +/- 3.7 ppm.
Clinical observations were recorded for each animal daily with body weights and food consumption measured on a weekly basis throughout the exposure and recovery periods. Two animals were found dead (one group 4 male on study day 25; one group 5 male on study day 72) and one animal (control group male on study day 65) was sacrificed moribund prior to terminal sacrifice. There were no abnormal clinical or gross pathological findings for the group 4 male. Test article-related clinical signs for the group 5 male included decreased activity, soiling around muzzle, abdomen and urogenital regions with gross pathological findings including dilation of kidneys and urinary bladder with calculus in bladder. Test article-related clinical signs reported for all surviving animals were limited to groups 4 and 5 and primarily included soiling of the urogenital and abdominal regions of both sexes.
Mean body weights trended lower than controls over the exposure period for group 5 males (~6%) and groups 4 and 5 females (~5%). Statistically significant decreases were noted during week one for group 3 and week two for group 5 only. A statistically significant decrease in mean body weight was measured in the group 5 recovery group females beginning exposure week four. This difference persisted through the completion of exposures and into week one of the post exposure recovery period. Although not statistically significant, body weights remained decreased from controls for males (~4%) and females (~6%) during the recovery period. There were no differences in food consumption for either sex, in any of the 90-day exposure groups. Weekly comparison of recovery group food consumption yielded statistical differences during weeks 6, 7, 8 and 10 for group 5 males and weeks 4 and 5 for group 5 females. Food consumption was similar to controls for both sexes in group 5 during each week of the 28-day post exposure period. There were no test article-related ophthalmic finding at the end of the 90-day exposure period.
Test article-related gross necropsy findings were primarily limited to the group 4 and 5 animals and included moderate dilation of the kidney, decreased soft testes, and calculi in the urinary bladder. Histomorphologic changes included minimal to moderate urinary bladder hyperplasia and inflammation in all group 5 males and 9/10 females. Kidney changes were characterized by hyperplasia of the pelvic epithelium and/or granulomatous inflammation. The one group 5 male animal found dead on study day 72, demonstrated an apparent urinary obstruction possibly leading to acute uremia, with calcification of the aorta and pulmonary hemorrhagic edema as secondary effects. Additional changes included prostatic inflammation in moderate or severe degrees in two 1600 ppm exposure group 5 rats.
Following the 28-day recovery period, calculi were observed in the group 5 males only. Minimal to moderate hyperplasia of urinary bladder epithelium persisted in most rats, and exposure-related urinary bladder calculi were observed in several. Chronic or granulomatous inflammation in the renal pelvis was observed in several female rats. In male rats, there was no histomorphological evidence of a residual effect on the kidneys after the recovery period. In females, the incidence of pelvic epithelial hyperplasia and inflammation was modestly increased over controls. There were no indications of a residual effect on the prostate gland following the recovery period. No animals had more than mild inflammation of the prostate gland, and the incidence of inflammation was higher in control animals.
In females, absolute adrenal gland weights were statistically increased in group 4 (18%) and group 5 (25%). Relative to body weight, female adrenal glands were statistically increased (27%) for group 5. There was no histological correlate and the finding was not present in males or in recovery group females. In males, group 4 kidney weight was increased, but a comparable effect was not seen in group 5.

Also in males, the weights of testes and epididymides were statistically decreased in recovery group rats exposed to 1600 ppm. This finding correlated histologically with two recovery group males showing marked testicular seminiferous tubule degeneration and corresponding epididymal oligospermia (one unilateral, one bilateral). In regular study (90-day) rats, seminiferous tubule degeneration was observed only in one control and one low-exposure (25 ppm) rats. These findings were considered common spontaneous findings in young Sprague-Dawley rats and not test article-related. There were no test article-related changes in clinical pathology or serum chemistry.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
0.56 other: mg/l
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
gross pathology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Dose descriptor:
LOAEL
Effect level:
2.2 other: mg/l
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the increased incidence of grossly observed urinary bladder calculi along with the kidney dilation at the 400 ppm exposure level.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Toxic Response/Effects by Dose Level:
25 ppm (ca. 0.14 mg/L):
No findings attributed to test article 

100 ppm (ca. 0.56 mg/L):
No findings attributed to test article 

400 ppm (ca. 2.2 mg/L): 
Clinical signs:
- Increased incidence of abdominal and urogenital soiling
Gross pathology:
- Calculi in the urinary bladder (2 males)
Histomorphologic changes: 
- Urinary bladder epithelial hyperplasia in males and females 
Organ weights: 
Female adrenal weight increase (absolute):  18%* 

1600  ppm (ca. 8.9 mg/L):
Clinical signs:
- Increased incidence of abdominal and urogenital soiling
Gross pathology:
- Calculi in urinary bladder of 4 males and 1 female, persisting through  28-day recovery
- Kidney dilation 

Organ weights:
Female adrenal weight increase (absolute/relative):  
Absolute:  25%*         Relative:  27%*

Female kidney weight increase (relative):
Relative:  12%* 

Absolute adrenal weights were significantly increased compared to  controls for group 4 (18%) and 5 (25%) females.  Adrenal and kidney to  

body weight ratios were also significantly increased compared to controls  for group 5 (27% and 12%, respectively) females.

Histomorphologic changes:
- Kidney:  hyperplasia of the pelvic epithelium and granulomatous  inflammation for males
- Urinary bladder:  epithelial hyperplasia in males and females
- Prostate:  slight increase in severity of inflammation 
* Statistically significant finding

"        Body weight:  absolute and percent gain was within normal limits for  all test groups
"        Food/water consumption:  within normal limits for all test groups
"        Description, severity, time of onset and duration of clinical signs:   The increased incidence of abdominal and urogenital soiling at the 400  

and 1600 ppm exposure levels was observed beginning on exposure day 11 and persisting on and off throughout the completion entire 90-day  

exposure duration..
"        Ophthalmologic findings incidence and severity:  No test article  attributed findings
"        Hematological findings incidence and severity:  No test article  attributed findings
"        Clinical biochemistry findings incidence and severity:  No test article  attributed findings
"        Mortality and time to death:  One (1) 400 ppm group 4 male on study day  25, One (1) 1600 ppm group 5 male on study day 72.
"        Gross pathology incidence and severity:  increased incidence of urinary  bladder calculi and dilation of the kidneys as described above.
"        Organ weight changes:  Kidney and adrenal glands related to treatment  as specified above.
"        Histopathology incidence and severity:  Urinary bladder epithelial  hyperplasia in both sexes at 400 and 1600 ppm and hyperplasia of the  

pelvic epithelium along with granulomatous inflammation for 1600 ppm male  kidneys.  Prostatic inflammation seen in all groups with a slight  

increase in severity at the 1600 ppm level.

Applicant's summary and conclusion

Conclusions:
Based on the increased incidence of grossly observed urinary bladder calculi along with the kidney dilation at the 400 ppm exposure level, the No Observable Adverse Effect Level (NOAEL) for methyltrimethoxysilane vapor administered six hours per day, five days per week for a 90-day interval via whole-body inhalation exposure to male and female Sprague-Dawley rats, was 100 ppm.