Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
19.11.2003 to 19.05.2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to the appropriate OECD guideline and in compliance with GLP and is therefore considered to be reliability 1. Read-across of the study itself is considered to be reliability 2. Further information on read-across is given in the endpoint summary.
Justification for type of information:
Please refer to the attached justification below and the overall justification for grouping of substances attached in IUCLID Section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

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: USEPA OPPTS 870.3650
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test article, methyltrimethoxysilane, was received from Gelest Inc, Morrisville, Pennsylvania, for Silicones Environmental, Health and Safety Council of North America, as follows:
Methyltrimethoxysilane
Lot No. 9H-7709
CAS No. 1185-55-3
Clear liquid

Characterization analyses were performed by Dow Corning Corporation Health and Environmental Sciences Laboratory. The purity of the test article was 96.74%. The test article was stored in sealed containers (FF) at room temperature and was considered stable under these conditions. A reserve sample of the test article was retained.

The test article, methyltrimethoxysilane (Lot No. 9H-7709) was characterized according to current EPA Good Laboratory Practice Standards. The characterization included physical description, GC-FID for area % purity, and GC-MS for identification of the major component in the test article.

The physical description of the test article was observed to be a clear liquid. The GC-FID analyses of methyltrimethoxysilane afforded a mean area % purity of 96.74 +/- 0.001. The GC-MS data verified the major component in the test article to be methyltrimethoxysilane and the minor components to be methanol, Dimethoxydimethylsilane and dimethoxymethyldisiloxane. Also, the mass spectrum of each component in the test article was consistent with that of reference mass spectra found in library searches.

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR VAF/Plus®
Sex:
male/female
Details on test animals and environmental conditions:
ANIMAL RECEIPT AND ACCLIMATION
Ninety female and forty five male Crl:CD®(SD)IGS BR VAF/Plus® rats were received from Charles River Laboratories, Portage, MI. The animals were 9 weeks old. Upon receipt, each animal was inspected by animal resource personnel and animals judged to be in good health and suitable as test animals were quarantined for six days. During the quarantine period animal resource personnel observed each animal at least once daily. The attending veterinarian examined all animals before release from quarantine and documented the general state of animal health.

ANIMAL HOUSING
Animals were individually housed in suspended wire-mesh cages elevated over Bed-O’Cobs Alf-a’ bedding, during quarantine and throughout the course of the study, with the following exceptions. During cohabitation, reproductive group females were paired 1 to 1 with a male from the same treatment group in suspended wire-mesh cages elevated over Bed-O’Cobs Alf-a’ bedding. The animals were paired in the home cage of the male. On day 0 of gestation, reproductive group females were moved into shoebox cages containing Bed-O’ Cobs Combination bedding and remained there throughout the remainder of the study. The results of the manufacturer’s periodic analysis of the Bed-O’ Cobs Combination bedding were reviewed to ensure that there were no contaminants present at levels that would be expected to affect the outcome of the study. The cages, bedding/fecal pans were routinely cleaned, consistent with good husbandry practices.

DIET, DRINKING WATER AND MAINTENANCE
PMIcertified Rodent Diet #5002, manufactured by PMI Nutritional International, St. Louis, MO, was offered ad libitum except during functional observational battery assessment period. Males and toxicity group females were fasted prior to necropsy by removing food on the evening of the day before necropsy. The results of the manufacturer’s periodic analyses of the certified feed were reviewed to ensure that heavy metals and pesticides were not present in concentrations that would be expected to affect the outcome of the study.

Municipal water, further purified by reverse osmosis was available ad libitum except during performance of functional observational battery testing. The drinking water was monitored on at least a semi-annual basis to determine compliance with the U.S.E.P.A. drinking water standards. The most recent analysis was reviewed and there were no contaminants in the water known to be present at levels expected to interfere with the integrity of the study.

ENVIRONMENTAL CONDITIONS
Animals were housed in an environmentally controlled animal room (12-hour fluorescent light/dark cycle, 68.3-72.5 oF, 36.0-62.0% relative humidity, 10-15 air changes per hour) throughout the in-life phase of the study. Temperature and humidity were continuously monitored. The most recent air change verification was reviewed by the study director.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The vehicle and test article formulations were administered orally by gavage, via a 3-4 inch, 15-18 gauge, animal feeding needle and syringe once daily. Volume administrations did not exceed 3 mL/kg of body weight except for a few instances. The volume administered was based upon the most recent body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of methyltrimethoxysilane (MTMS) in corn oil dosing solutions was determined prior to the beginning of the definitive study.

Preparation of Solvent Standards: Stock solvent standards of MTMS in toluene were prepared under a nitrogen atmosphere by weighing an amount of MTMS and mixing with a known volume of toluene. Aliquots of the stock solution were then further diluted with toluene to cover the standard range from approximately 100 µg MTMS/ml toluene to approximately 1000 µg/ml. New solvent standards were prepared with each dosing solution analysis. An aliquot of each solvent standard was placed in an autosampler vial and analyzed.

Preparation of Dosing Solutions for Analysis of Concentration Verification: Aliquots of dosing formulations were volumetrically measured into volumetric flasks, diluted to volume with toluene and mixed well. Aliquots of the diluted dosing solutions were placed in autosampler vials and were analyzed with the solvent standards for determination of the concentration of MTMS.

Gas Chromatography Analysis: A set of solvent standards comprising of at least five concentrations was used to establish a calibration curve. Trendlines and their mathematical equations were generated by linear regression analysis of the peak areas resulting from the analysis of the solvent standards using Microsoft Excel 2000 (Version 9.0). The dosing solution verifications were performed by comparing the target concentration of the dose solutions to the mean observed test substance concentration found by refitting the peak areas resulting from the dosing solution dilution samples to the standard curve. The limit of quantitation was set at the level of the lowest standard analyzed and was equal to 5 mg MTMS/ml dosing solution. The conditions used were as follows:
Injection: 1 µl, split 50:1
Injector Temp: 160 oC
Carrier Gas: Helium
Column Flow: 1.3 ml/min
Hydrogen Flow: 30 ml/min
Air Flow: 400 ml/min
Combined Flow: 25 ml/min (constant column + make-up flow)
Column: HP-5MS, 30 m x 0.25 mm, 0.25 µm film thickness
Oven Temp: 70 oC for 3 min to 210 oC at 17 oC/min
Detector: Flame Ionization Detector (FID)
Detector Temp: 300 oC

Homogeneity and Stability: Homogeneity and stability of MTMS in corn oil dosing solutions was performed as part of the pilot study with MTMS. The dosing solutions were found to be homogeneous and stable up to 15 days if aliquoted into separate vials for daily usage.

Concentration Verification: Verification of each dosing concentration was conducted following each new preparation. New dosing solutions were prepared at least every 15 days in order to stay within the stability timeframe established in the pilot study.

Results: Dose solution analysis for concentration determined that each batch of 50, 250 and 1000 mg/kg/day dose solution was within 95-07%, 95-97% and 99-100% of the target concentration, respectively.
Duration of treatment / exposure:
Test substance was administered once daily by oral gavage, seven days per week at approximately the same time each day. Dose levels of methyltrimethoxysilane (MTMS) were 0 (control), 50, 250 and 1000 mg MTMS/kg/day. MTMS, dissolved in corn oil, was administered by oral gavage once each day for up to 51 consecutive days. Females in each dose level were divided into a toxicity (10 animals/group) and a reproductive group (10 animals/group). A single group of males (10 animals/group) were used for both the toxicity and reproductive phases of the study. Toxicity group females and males were treated for 28 and 29 days, respectively. Reproductive group females were treated for 14 days prior to the mating period, during the mating period, and then up to and including post partum day 3, for a total of up to 51 days.
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (corn oil), 50, 250, and 1000 mg/kg bw/day
Basis:

No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Males and toxicity group females were sacrificed after they had been  treated for 28 days.  Reproductive group females and pups were sacrificed  
on day 4 postpartum.  A functional observational battery (FOB) evaluation  for signs of neurobehavioral effects was performed on all adult males 
and  all toxicity group females prior to the start of dosing and during the  last week of dosing.  Body weights and food consumption were recorded  
weekly.  From all males and all toxicity group females, blood samples  were obtained on the day of scheduled necropsy for hematology and  
clinical chemistry parameters evaluations.  Animals were subjected to a  complete gross necropsy.  Many tissues and organs were collected for  
histological examination and were weighed.  
Positive control:
Not required

Examinations

Observations and examinations performed and frequency:
Mortality/Morbidity: Animals were observed at least twice daily in their cages for moribundity and mortality throughout the in-life phase of the study.
Clinical observations:
Daily Observations: General clinical examinations were made at lease once a day and were conducted immediately after dosing. The examinations included, but were not limited to, changes in the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system functions, motor activity and behavior patterns. Findings were recorded for individual animals. General clinical examinations were not performed on days when detailed physical examinations were performed.

Detailed Physical Examinations: All animals received a detailed physical examination once before the first dose administration (to allow for within-subject comparisons), and weekly thereafter. Examinations were made outside the home cage in a standard arena at approximately the same time each day. Observations were detailed and carefully recorded. 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 movement, stereotypies, difficult or prolonged parturition or bizarre behavior were recorded. The presence or absence of findings was recorded for individual animals.

Body weights and food consumption were recorded weekly. Additional body weights for reproductive group females were obtained on gestational day 0, 7, 14, and 20, and within 24 hours of parturition, and on postnatal day 4. Individual food consumption was determined for each group following group specific schedules. In addition, detailed clinical observations (functional observational battery [FOB] conducted out of the home cage) and locomotor activity were evaluated for all adult male and toxicity phase females once prior to the start of test article administration (baseline evaluations) and again during the last week of the test article administration. Blood samples were collected from males and toxicity group females on the day of scheduled termination for analysis of hematology and serum chemistry parameters.
Sacrifice and pathology:
Complete necropsies were performed on the males and the toxicity group females and selected organs were weighed (adrenals, brain, heart, kidneys, liver, lungs, spleen, thymus, uterus, ovaries, testes, prostate, seminal vesicles, and epididymides). Microscopic examination was performed on protocol-specified tissues from all animals in the control and high dose group males and toxicity group females. Microscopic examination of the liver, thyroid, jejunum and duodenum was extended to males and toxicity group females in the 50 and 250 mg/kg dose groups. In addition, the adrenal gland from these middle dose groups was evaluated for the toxicity group females.
Statistics:
All data analyses were carried out using SAS version 8.2. Statistically significant probabilities were reported for /-values of <0.05, <0.02 and <0.01.

Body weight, body weight changes, organ weight, organ to body weight ratios, food consumption, 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 dosed groups to control were made using the Dunnett’s Test or a Wilcoxon test, respectively.

The red blood morphology findings in the hematology data that were reported by severity grade; polychromasia, anisocytes, poikilocytosis, and ananthocytes were analyzed using a Cochran-Armitage trend test to indicate an increasing incidence trend regardless of grade with Fisher’s Exact tests used to indicate increased incidence (non-grade specific) over the control. To determine shifts in severity, analysis using the Kruskal-Wallis test was performed on the graded data only if there was at least once incidence of severity grade seen in the controls group. If the overall Kruskall-Wallis test was significant, pair wise comparisons of the graded animals between the control and treated groups was done using the Kruskal-Wallis.

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
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
There were two unscheduled deaths (one female each at 0 and 50 mg/kg  bw/day); both were related to dosing errors.  Clinical signs included  
transient inactivity or salivation following dosing.  Statistically  significant decreases in body weight gain and food consumption were noted  
for 1000 mg/kg bw/day group males.  Increased liver weight was observed  for male and female animals at 250 and 1000 mg/kg bw/day.  
Exposure to  MTMS was associated with organ weight and/or histomorphological changes  in males (liver, thymus, thyroid, duodenum,
 jejunum, and red blood cell)  and females (liver, thyroid, duodenum, jejunum, and adrenal gland) at  dose levels at or above 250 mg/kg bw/day.  
A marked increase in  prothrombin time was observed for males at 250 and 1000 mg/kg bw/day  whereas females were unaffected.  Exposure 
was also associated with  increased blood platelet concentration for males and females at 1000  mg/kg bw/day.  The NOAEL for systemic 
toxicity was 50 mg/kg bw/day.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Organ weight and/or histomorphological changes  in males (liver, thymus, thyroid, duodenum,  jejunum)  and females (liver, thyroid, duodenum, jejunum, and adrenal gland) at  dose levels at or above 250 mg/kg bw/day.  

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Toxic Response/Effects by Dose Level:

50 mg/kg bw/day Methyltrimethoxysilane:
Males: None
Females:  None

250 mg/kg bw/day Methyltrimethoxysilane:
Males: 
Increased liver weight (absolute(relative): 19%* (25%*)
Thyroid gland follicular cell hyperplasia/hypertrophy: (incidence 6*/10)
Decreased thymus weight (absolute(relative): 28%*(24%*)
Increased prothrombin time: 2-fold*

Females:
Increased liver weight (absolute(relative): 37%* (41%*)
Centrilobular hepatocellular hypertrophy (incidence 5*/10)
Periportal vacuolation (incidence 10*/10)
Thyroid gland follicular cell hyperplasia/hypertrophy:
(incidence 10*/10)

1000 mg/kg bw/day Methyltrimethoxysilane:
Males:  
Increased liver weight (absolute(relative): 33%* (47%*) 
Diffuse hepatocellular hypertrophy (incidence 10*/10)
Thyroid gland follicular cell hyperplasia/hypertrophy: (incidence 10*/10)
Mucosal lipidosis: duodenum  (incidence 4*/10)
Mucosal lipidosis: jejunum (incidence 7*/10)
Decreased thymus weight (absolute(relative): 35%*(29%*)
Ancanthocytosis (incidence 5*/10)
Increased red blood cell concentration: 16%*
Increased prothrombin time: 2-fold*
Increased serum alanine aminotransferase (48%*)
Increased blood platelet concentration: 16*

Females (Toxicity Phase):
Increased liver weight (absolute(relative)): 197%* (200%*)
Centrilobular hepatocellular hypertrophy (incidence 10*/10)
Periportal vacuolation (incidence 8*/10)
Thyroid gland follicular cell hyperplasia/hypertrophy:(incidence 10*/10)
Adrenal gland hyperplasia/hypertrophy (incidence 10*/10)
Adrenal gland apoptosis (incidence 9*/10)
Adrenal gland lymphocytic infiltration: zona reticularis:(incidence 5*/10)
Mucosal lipidosis: duodenum  (incidence 5*/10)
Mucosal lipidosis: jejunum (incidence 5*/10)
Increased blood platelet concentration: 21%*

Where "*" denotes statistically different from control
(p<0.05)

Applicant's summary and conclusion

Conclusions:
Exposure to methyltrimethoxysilane was associated with organ weight and/or histomorphological changes in males (liver,
thymus, thyroid, duodenum, jejunum, and red blood cell) and females (liver, thyroid, duodenum, jejunum, and adrenal
gland) at dose levels at or above 250 mg/kg bw/day. A marked increase in prothrombin time was observed for males at 250
and 1000 mg/kg bw/day whereas females were unaffected. Exposure was also associated with increased blood platelet
concentration for males and females at 1000 mg/kg bw/day. These data support a NOAEL for the toxicity phase of the
study of 50 mg/kg bw/day.