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Repeated dose toxicity: inhalation

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sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in accordance with an appropriate OECD test guideline and in compliance with GLP.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
equivalent or similar to
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
not specified
GLP compliance:
Limit test:

Test material

Details on test material:
- Name of test material (as cited in study report): Tetramethoxysilane
- Physical state: liquid

Test animals

Details on test animals and environmental conditions:
- Source: Charles River Breeding Laboratories, Michigan
- Age at study initiation: Not stated.
- Weight at study initiation: Not stated.
- Fasting period before study: Not stated.
- Housing: Individual in stainless steel, wire mesh cages.
- Diet (e.g. ad libitum): Ad libitum except during exposure
- Water (e.g. ad libitum): Ad libitum except during exposure
- Acclimation period: Two weeks

- Temperature (°C): 20-23
- Humidity (%): 35-45
- Air changes (per hr): 18-20.
- Photoperiod (hrs dark / hrs light): Not stated.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
other: Air
Details on inhalation exposure:
- Exposure apparatus: Five, 30 inch cube (450 litre volume) stainless steel, glass whole body exposure chambers were used in each phase of the study. The chambers were operated under dynamic condictions.
- Source and rate of air: The chamber air was supplied by a heating, ventilation and air conditioning system which was separate from the general laboratory system. The intake air was filtered and drawn through the chambers by means of a low pressure vacuum pump. Flow rate through the chambers was monitored continuously in the inlet line of each chamber by measuring the pressure differential across an orifice plate with a Manehelic gauge. Gauge readings were recorded hourly during the exposure period. Flow rate through each inhalation chamber was set to maintain internal condictions at acceptable levels. Temperature and relative humidity was measured hourly during the exposure periods.
- Method of conditioning air: Filtered using Hepa and charcoal filters.
- Treatment of exhaust air: Exhaust air was passed through a Hepa filter, charcoal filter and water scrubber, and then exited the building through the roof.

- Brief description of analytical method used: Test substance was metered into the J-shaped glass generating tube by a flow FMI laboratory pump. The air/vapour mixture passed directly into the inlet port at the top of the chamber.
- Samples taken from breathing zone: Not stated.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Actual chamber concentrations were measured once per hour of exposure by a varian 4600 gas chromatograph and periodically by an infrared gas analyser.
Duration of treatment / exposure:
Six hours
Frequency of treatment:
All groups were treated concurrently five days per week for four weeks, except for Group IV (60 ppm) in Phase II. Exposure of this group was discontinued after a single 6-hour exposure due to extreme toxicity in the test animals.
Doses / concentrationsopen allclose all
Doses / Concentrations:
1, 5, 10 ppm
other: Nominal conc. Phase 1
Doses / Concentrations:
15. 30, 45 ppm
other: Nominal conc. Phase II
No. of animals per sex per dose:
Control animals:
Details on study design:
- Dose selection rationale: The study comprised two phases. Since an effect level was not achieved in Phase I, a Phase II was conducted with higher exposure concentrations.
- Rationale for animal assignment (if not random): Random
Positive control:


Observations and examinations performed and frequency:
- Time schedule: Frequently (no further information) during exposures and daily during the post-exposure period for overt signs of toxicity and/or mortality. Each animal was examined daily during weekdays for evidence of any treatment-related effects.
- Cage side observations checked in table [No.1] were included.

- Time schedule for examinations: Each animal was weighed at the initiation of the study and then twice weekly throughout the treatment period.

- Food consumption for each animal determined every week throughout the treatment period.


- Time schedule for examinations: Daily on weekdays.
- Dose groups that were examined: All

- Time schedule for collection of blood: At scheduled termination prior to necropsy.
- Anaesthetic used for blood collection: Yes (Rompun and Vetalar mixture)
- Animals fasted: No data
- How many animals: All
- Parameters checked in table [No.2] were examined.

- Time schedule for collection of blood: At scheduled termination prior to necropsy.
- Animals fasted: No data
- How many animals: All
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes, on five randomly selected rats of each sex from the control, 1 and 10 ppm groups in Phase I and the control, 15 and 30 ppm groups from Phase II.
- Time schedule for collection of urine: For 16 hours overnight (no further information).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked in table [No.2] were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3) At completion of the exposure period, all rats from the control, 1, 5, and 10ppm groups from Phase I, and the control, 15, and 30 ppm groups from Phase II were necropsied after sacrifice by exsanguination from the abdominal aorta following clinical pathology blood collection. At necropsy an examination was made of the external body surface and body orifices.
HISTOPATHOLOGY: Yes (see table 3) Where available, tissues and organs were prepared from all rats from the control and 10 ppm groups of Phase I and from the control and 45 ppm groups of Phase II (see table 3, column H marked with #). In the Phase II 45 ppm group some tissues/organs appeared to be target organs or tissues, so they were examined microscopically in the 30 ppm group (see table 3, column H marked @). Of these tissues/organs some were further investigated in the Phase II 15 ppm group because they were target organs or tissues at 30 ppm (see table 3, column H marked X).
Statistical comparisons between control and test groups were conducted where applicable. Body weights, food consumption values, hemograms, myelograms, blood chemistry analyses, and absolute and relative organ weights were analysed by a one-way analysis of variance. Group means were compared to control values using Dunnett multiple t-test. The 95% (P<0.05) confidence level was chosen as the criteria of significance.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: Clinical signs of toxicity generally exhibited by rats exposed to 30 or 45 ppm test substance included lethargy, rough coats, dyspnea, eye squinting, salivation and nasal discharge. All rats in the 45 ppm group either died or were sacrificed in a moribund condition during the course of the study. In most cases, no apparent severe abnormalities were noted during the removal of the animals from the exposure chamber and effects were observed the next day. In many cases deaths were sudden and unexpected in this group. There were no deaths in any other groups of Phase I or II.

BODY WEIGHT AND WEIGHT GAIN: There was a statistically significant, dose-related reduction in mean body weights of males and females exposed to 30 and 45 ppm test substance compared with controls (see table 4).

FOOD CONSUMPTION: There was a dose-dependent decrease in food consumption in the 15, 30 and 45 ppm exposure groups compared with the controls of Phase II. This difference was statistically significant for the 30 and 45 ppm groups. There was no significant difference between treated and control animals for Phase I.


HAEMATOLOGY: No statistically significant or exposure-related effects were observed in the haematologic parameters evaluated in any of the exposure groups. All haematologic parameter values were within the normal ranges for rats of the age used in the test, and there were no trends suggestive of a test substance effect. The authors noted that respiratory tract tissue inflammatory changes were not evident as elevated leucocyte counts.

CLINICAL CHEMISTRY: There were several clinical biochemical parameters in Phase I that showed statistically significant differences when compared with the control values. These differences appeared to occur with a random distribution, were inconsistent between sexes and did not show a dose-response. The authors of the study concluded them to reflect normal biological variation and not to be toxicologically significant. In Phase II there were a number of parameters that showed statistically significant differences when compared with the controls. These differences were consistent between the sexes and had a dose response. Total serum protein and serum albumin values were significantly decreased in both sexes at 30 ppm. There was also a significant decrease in total serum protein in the 15 ppm group males. It was noted that the levels of albumin reported in males and females at all levels appeared to be slightly high for rats of the age used, complicating the interpretation of the data. Decreases in total protein and albumin commonly have a nutritional origin or indicate liver damage. However, the food consumption decrease at 30 ppm was only 13.2% in males and 12.6% in females during the fourth week of the study. The authors of the study concluded that a possible link between serum protein and albumin was unlikely. Nor was there any morphological evidence of liver damage. Serum lactic dehydrogenase values were statistically significantly decreased in males and females at 30 ppm. The significance of this change is unknown. There was also a statistically significant decrease in uric acid in males and females of the 30 ppm group. However, this finding is of doubtful toxicological significance as uric acid is the result of a different metabolic process in rats compared to humans. There was also a significant increase in serum sodium in males from the 15 and 30 ppm groups, and in serum creatinine in males from the 15 ppm group, and a decrease in serum phosphorus in males in the 30 ppm group. The values for these parameters, although different from the controls, were within the normal range for rats, and were not considered to be of toxicological significance. Serum glucose values were elevated in all groups, probably as a result of stress induced by the terminal bleeding procedure. There were considerable elevations over normal rat values for serum alkaline phosphatase in all control and experimental groups in both phases. In addition, serum glutamic oxalacetic transaminase and urea nitrogen values in all groups of Phase II were slightly elevated over normal values. The reasons for these elevations could not be explained.

URINALYSIS: Urinanalysis values for all treated animals were considered to be normal, and there did not appear to be any meaningful differences between values for the control and experimental groups.

ORGAN WEIGHTS: There was a statistically significant decrease in absolute and relative (organ:body weight ratio) adrenal weight in 1 ppm males, decreased relative testes weight in 1 and 5 ppm males, decreased absolute and relative ovary weight in 5 and 10 ppm females, decreased absolute ovary weight in 1 ppm females, increased absolute and relative liver weight in 5ppm females, increased absolute heart weight in 10 ppm females and increased absolute and relative spleen weight in 10 ppm males. These differences do not show a dose-response, were not correlated with morphological changes or consistent between sexes, and were consequently not considered to be related to treatment. In Phase II decreases in absolute liver and heart weight in 30 ppm males, and increases in spleen:body weight ratio in 30 ppm females were a reflection of decreases in body weight in this group and were not considered to be adverse effects caused by exposure to the test substance.

GROSS PATHOLOGY: There were no exposure-related macroscopic lesions in any of the rats from the 1, 5, 10 (Phase I), 15 or 30 (Phase II) ppm groups. In rats from the 45 ppm group, which all died or were sacrificed in extremis during the study period, nasal discharge was a consistent observation and was considered related to treatment. The nasal discharge correlated with lesions of severe nasal irritation observed microscopically. Other lesions in the 45 ppm group were lung congestion and apparent intestinal irritation. These findings were considered to be agonal rather than specific, direct test substance effects.

HISTOPATHOLOGY: No treatment-related microscopic changes were found for the Phase I groups. In Phase II, microscopic pathologic changes to the respiratory tract tissues and eyes occurred as a direct result of exposure to the test substance at all doses in both males and females. At 45 ppm the changes at these sites were severe, present in all animals and were consistent with the severe clinical signs of toxicity that caused the death and morbidity in this group. Changes at 30 ppm, while occurring in all rats were much less severe than those at 45 ppm. At 15 ppm the changes were minimal (probably completely reversible) and only occurred in three males and five females. The fact that all of the affected sites are moist epithelial surfaces suggests that the toxic effects observed occur through hydrolysis on the affected tissue. Specific details on the observed effects are as follows:
Nasal tissue: Exposure-related changes in nasal tissues included ulceration with associated acute inflammation of non-ulcerated epithelium and suppurative exudate in the nasal cavity. At 45 ppm, ulcers occurred in all 17 rats in which nasal tissues were examined, while at the 30 ppm exposure concentration, ulcers occurred in 18 of 20 rats. The nasal exudate at 45 ppm was suppurative in all animals, reflecting the severity of the inflammation at this level. At 30 ppm the exudate was suppurative in 9 and fibrinous in 11, indicating a less severe reaction in some animals. Two rats from the 30 ppm group also had a mild squamous metaplasia of the nasal mucosa. At 15 ppm there was no ulceration, and the epithelium was completely intact in all animals. The only evidence of irritation in this group was minimal acute inflammation of the nasal mucosa in two rats. In one rat with acute inflammation of the nasal mucosa there was associated fibrinous exudate in the nasal cavity. Another 15 ppm rat had fibrinous exudate in the nasal cavity and a second had suppurative nasal exudate, both without evidence of inflammation of the mucosa.
Larynx: Changes at 45 ppm consisted of acute or ulcerative inflammation of the laryngeal epithelium with associated fibrinous or suppurative inflammatory exudate on the epithelial surface or within the lumen. These changes occurred singly or in combination, in 18 of 20 rats at this exposure level. At 30 ppm there was acute or chronic inflammation of the laryngeal mucosa without ulceration in 14 of 20 rats. Exudate was not noted in the lumen or upon the mucosal surface at this level. There was minimal or mild squamous metaplasia of the laryngeal epithelium in two rats from the 30 ppm exposure level. Minimal acute inflammtion of the mucosa in one female was the only exposure-related laryngeal change observed at 15 ppm.
Pharynx: Chnages to the pharynx were only observed at 45 and 30 ppm. They were characterised by acute and ulcerative inflammation at 45 ppm, acute and chronic inflammation at 30 and 45 ppm, fibrinous exudate at 45 ppm, suppurative exudate at 30 ppm and hyperkeratosis of the oropharynx in one rat at 45 ppm.
Trachea: Exposure-related tracheal changes were observed in all rats of the 30 and 45 ppm groups. At 45 ppm these changes were acute or chronic active inflammation with associated fibrinous or suppurative exudate on the epithelial surface or within the tracheal lumen, minimum to severe squamous hyperplasia of the tracheal epithelium and epithelial necrosis. At 30 ppm changes were generally of a lesser severity and were less acute: acute and chronic inflammation, exudate, ulceration and squamous metaplasia.
Lung: Exposure-related effects on the lung occured in all animals in the 45 ppm group and in 18 of 20 animals in the 30 ppm group. No effects were observed in any of the other exposure groups. Adverse effects in the 45 and 30 ppm groups primarily involved the primary and secondary bronchi and did not extend below the bronchioles. Lung parenchyma at the level of the respiratory duct and below was free of lesions related to treatment. Adverse changes noted in the bronchi and bronchioles were chronic bronchitis and squamous metaplasia of the bronchial epithelium. Changes at the higher exposure concentration were much more severe than those at the lower concentration. In addition, oedema and congestion were observed at 45 ppm, but these were considered to be agonal changes.
Eye: Exposure-related ocular changes confined to the cornea occurred in the eyes of all the rats examined from the 45 and 30 ppm groups, and 4 of 20 rats from the 15 ppm group. At 45 and 30 ppm there was acute or chronic keratitis that was usually bilateral and often included vascularisation of the deep corneal stroma and sloughing of the central corneal epithelium. There did not appear to be a dose-response with respect to the severity between 30 and 45 ppm. At 15 ppm, four rats had minimal acute keratitis that was manifested as the presence of a few acute inflammatory cells within or immediately beneath the corneal epithelium with no sloughing of the epithelium.
A number of additional changes in rats exposed to 45 ppm of test substance were considered possibly exposure related and prompted examination of the same tissue in the 30 ppm group animals. These lesions were dilatation of the kidney tubules, hepatocyte vacuolation and hyaline droplet formation in the liver, lymphoid hypoplasia and necrosis in the spleen and hemorrhage, hypoplasia and necrosis of the thymus. However, none of these findings occurred at 30 ppm. The study authors concluded that these effects at 45 ppm could have been direct or indirect test substance effects or agonal. Other microscopic lesions in the rats were typical of mild spontaneous lesions of clinically normal rats of the age tested, and were concluded to be of no toxicological significance.

Effect levels

open allclose all
Dose descriptor:
other: Overall study NOAEC
Effect level:
10 ppm
Based on:
test mat.
Basis for effect level:
other: Based on effects on the respiratory tract.
Dose descriptor:
Effect level:
>= 10 ppm
Based on:
test mat.
Dose descriptor:
Effect level:
>= 15 ppm
Based on:
test mat.
Basis for effect level:
other: Based on only minimal reversible effects at this level.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 4: Average body weights and body weight gains during 28 days of treatment

Analytical concentration (ppm)

Body Weights (g±s.d)

Total Weight Gain (g)

Week 1

Week 2

Week 3

Week 4



278 ±7 

310 ±9

 330 ±11

 348  ±16




274 ±11 

 305 ±14

 327 ±18

345 ±23 




251 ±7** 

277 ±11** 

 298 ±18**

 316 ±19**




243 ±12** 







208 ±13 

218 ±15 

224 ±15 

229 ±22 




205 ±10 

211 ±14 

218 ±16 

222 ±16 




 197 ±11

203 ±10* 

209 ±13 

215 ±14 




204 ±16 



aData obtained from tables XIX and XX  in the study report.

*  Significantly different (p <0.05) from the control, ** Significantly different (p <0.01) from the control.

Applicant's summary and conclusion

In a study comparable to OECD test guidelines and to GLP the NOAEC for local effects of tetramethoxysilane in Charles River CD rats was determined to be 10 ppm when exposed by the inhalation route five days per week for four consecutive weeks. The test substance was found to have a steep dose response curve with no effects at 10 ppm, very minimal effects at 15 ppm, moderate to severe effects at 30 ppm and severe effects and lethality at 45 ppm. Exposure-related morphological changes were typical of the local effects of a corrosive agent, and were limited to moist surfaces of the eye (cornea), and respiratory tract (nasal tissues, pharynx, larynx, trachea, bronchi and bronchioles). As all observed significant toxicological effects appeared to be secondary to the local effects the systemic NOAEC is considered to be 45 ppm. However, the overall NOAEC for the study is 10 ppm, based on local effects on the respiratory tract.