Registration Dossier

Administrative data

Description of key information

There are several reliable studies that investigated the potential for formation of laryngeal granulomas following inhalation of aerosols of aqueous solution of 3-trimethoxysilylpropyl methacrylate. These studies exposed rats for 28 or 90 days. In addition, there is one nine day study. These studies on the aerosol tested several concentrations, solution strengths and durations, making interpretation of results complex. Following a weight-of-evidence approach it was concluded that the overall NOAEC for granuloma formation is 15 mg/m3 and this is a local effect.

Granulomas were not observed in a nine day vapour inhalation study.

The nine-day studies were assigned reliability 4 since they were of insufficient duration to meet current criteria for repeated dose toxicity testing. The results, however, are useful for weight of evidence.

The only consistently significant finding relating to general systemic toxicity was a reduction in body weight gains. The overall NOAEC for this effect appears to be between 50 and 100 mg/m3 for a 1 or 2% solution. As a worst-case interpretation, it is assumed that the body weight effects are not linked to granuloma formation and the lowest available systemic NOAEC of 50 mg/m3 is used as a starting point for risk characterisation purposes.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
14.08.1989 to 08.09.1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted specifically to identify whether an aerosol of the test substance induced laryngeal granulomas. While this study does not meet standards of the current guidelines in terms of the diversity of the examinations conducted, it is a well conducted study that achieves its objective.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of this study was to confirm the development of laryngeal granulomas in rats following short-term repeated inhalation exposures to 3-trimethoxysilylpropyl methacrylate-hydrolysate aerosol.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY.
- Age at study initiation: 41 days
- Weight at study initiation: Males: 130-131 grams
- Fasting period before study: none
- Housing: One or two per cage 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 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.5-21 °C
- Humidity (%): 45-77 %
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 14.08.1989 To: 08.09.1989
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: distilled water
Remarks on MMAD:
MMAD / GSD: 2.65 microns with standard deviation of 1.74.
Details on inhalation exposure:
The inhalation chambers were constructed from stainless steel and glass windows for animal observations. The volume was approximately 1330 litres, and the airflow was approximately 300 litres/minutes (13.5 air changes/hour). Temperature and relative humidity measurements were recorded at least twelve times per exposure.

A 2% solution of 3-(trimethoxysilyl)propyl methacrylate hydrolysate was metered from a piston pump into an atomiser. The atomiser was inserted into the top of the inhalation chamber turret where the liquid aerosol was dispersed throughout the chambers by filtered chamber supply air.

The particle size distribution was measured using a TSI Aerodynamic Particle Sizer and a 20:1 diluter. These determinations were made once per day for the duration of the study (except on day 3 due to equipment failure).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of 3-(trimethoxysilyl)propyl methacrylate hydrolysate were determined by gravimetric methods. Four samples were obtained each day.
Duration of treatment / exposure:
4 weeks (19 days of exposure over a 4-week period, 18 days of exposure for satellite group)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
143 mg/m³ air
Dose / conc.:
150 mg/m³ air
No. of animals per sex per dose:
15 males rats/treated group; 15 male rats/control group
5 animals from each group were designated to a satellite group for further ultrastructural evaluation of the larynges (this was cancelled)
Control animals:
yes, concurrent no treatment
Details on study design:
A group of 15 male Fischer 344 rats was exposed 6 hours per day for 19 days of exposure (18 days of exposure for the satellite group) over a 4-week period to an aerosol of 3-(trimethoxysilyl)propyl methacrylate hydrolysate (3-(trimethoxysilyl)propyl methacrylate hydrolysate; gamma-methacryloxypropyltrimethoxysilane hydrolysate; CAS: 2530-85-0) at a target concentration of 150 mg/m3. A control group of 15 male rats was exposed with the same exposure regimen to filtered air only. Five animals of the control and 3-(trimethoxysilyl)propyl methacrylate hydrolysate exposure groups were assigned to a satellite group for possible ultrastructural evaluation of the larynges, but not subsequently evaluated. 
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Preceding and following each exposure. On non-exposure days, animals were observed once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the time of body weight collection and just preceding sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on the morning prior to initiation of the first exposure. Then weekly during the four week exposure period and immediately preceding sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes a full necropsy was performed.
HISTOPATHOLOGY: Yes, of gross lesions, larynx, lungs, trachea, nasal turbinates and kidneys.
Statistics:
The data for continuous, parametric variables were intercompared for the exposure and control groups by use of Levene's test for homogeneity of variances and by t-tests.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only exposure-related clinical sign, observed in most of the animals, was perinasal encrustation.
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreases in body weight gain were observed for the 3-(trimethoxysilyl)propyl methacrylate-hydrolysate exposure group during the first two weeks of the study.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Nasal crust around the nares of the 3-(trimethoxysilyl)propyl methacrylate-hydrolysate exposed rats was the only gross lesion noted at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Upon histological examination, changes were noted within the respiratory tracts of the3-(trimethoxysilyl)propyl methacrylate-hydrolysate exposed rats. Cytoplasmic hyalinization was observed within the olfactory mucosa of the nasal cavity. In addition, squamous metaplasia and foci of minimal to mild granulomatous laryngitis (laryngeal granulomas) were present within the larynges of 3-(trimethoxysilyl)propyl methacrylate-hydrolysate exposed rats. These findings concur with microscopic changes observed in previous 3-(trimethoxysilyl)propyl methacrylate-hydrolysate aerosol studies.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
LOAEL
Effect level:
143 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Laryngeal granulomas developed.
Critical effects observed:
not specified
Conclusions:
In a four week study aerosol inhalation study, not conducted according to any guideline but in compliance with GLP, a LOAEL was concluded to be 143 mg/m3 in males, based on the development of laryngeal granulomas.
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 15.09.1986 to 05.06.1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: unknown
Principles of method if other than guideline:
The purpose of this study was to determine a no-observable-effect level for the development of laryngeal granulomas in rats following inhalation exposures to aerosol of hydrolyzed and condensed 3-trimethoxysilylpropyl methacrylate. It was also an additional objective of this study to further characterize the initial development of this lesion and the potential progression of this lesion during the lifetime of the laboratory rodent. However, because no laryngeal granulomas were developed in this study, all rats held for recovery were sacrificed approximately 6 months after the termination of exposures.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Two-hundred male and fifty-three female Fischer 344/CDF rats, 40 days of age, were received on August 25, 1986, from Charles River Breeding Laboratories, Inc. (Kingston, NY). Three male and three female rats were sacrificed on August 26, 1986, and the trachea, lungs, liver, kidneys, heart, spleen, salivary glands, submandibular lymph nodes, and nasal cavities were fixed and examined microscopically. Fecal samples were collected from these animals and examined for intestinal parasites by zinc sulfate flotation, and a Scotch~ tape test was performed on the perineum for examination of pinworms. Prior to sacrifice, blood samples were obtained for possible serologic evaluations. Blood samples were collected from five male and five female rats two weeks later for serologic evaluations. In addition, body weight determinations, clinical observations, and ophthalmic evaluations of all animals were performed prior to the initiation of the 14-week exposure regimen. Physical examination, parasitology, and histopathology indicated these animals were free of infectious disease and were suitable for use on this study.
An additional 42 male Fischer 344/CDF rats, 39 days of age, were received on September IS, 1986, fro~ Charles River Breeding Laboratories, Inc. (Kingston, NY). Three rats were sacrificed for quality control on September 16, 1986, and the same tissues as described above were fixed and examined micro¬scopically. Fecal samples were collected and examined using the zinc sulfate flotation method and these animals were examined for pinworms. Prior to the sacrifice, blood samples were collected for possible serologic evaluation. Blood samples were collected from five animals on September 30, 1986, for serologic evaluations. In addition, body weight determinations and clinical observations were performed on these male rats prior to the initiation of their 7-week exposure regimen (this group of rats started exposure on October 6, 1986, and was exposed for 7 weeks).

Animal Husbandry:
The rats were housed two per cage in stainless steel wire-mesh cages, 23.5 cm x 20 cm x 18 cm high in Room 165 for approximately 16 weeks. The rats were housed individually during the recovery period in the same type of cages as during the 14-week exposure regimen. They were located in Room 165 for approximately one week, then moved to Room 164 Bioclean Unit F for the duration of the recovery period. All animals were separated by test group and sex. Deotized Animal Cage Board. (Shepherd Specialty Papers, Inc., Kalamazoo, HI) was placed under each row of cages to collect excrement. The room temperature and relative humidity were monitored continuously by a Hygrothermograph. Seven-Day Continuous Recorder, Model #8368-00 (Cole-Parmer Instrument Company, Chicago, IL).The animals were kept on a 12-hour photoperiod throughout the study.
The additional male rats received on September 15, 1986, were housed two per cage in stainless steel, wire-mesh cages, 23.5 cm x 20 cm x 18 cm high in Room 164 Bioclean Unit B for 15 days, then transferred to Room 165 for approximately 12 weeks, then moved to Room 164 Bioclean Unit F for the duration of their recovery period. The animal husbandry procedures for these additional male rats were similar to those used for the animals received on July 17, 1986.
During 3-(trimethoxysilyl)propyl methacrylate exposures, the animals were transferred to.9.5 cm x 18 cm x 17.5 cm stainless steel, wire-mesh cages, separated by test group, and housed one per cage. Some of the high concentration group male rats were individually housed in 21 cm x 12.5 cm x 18 cm stainless steel, wire-mesh cages.
During nonexposure periods, water (Municipal Authority of Westmoreland County, Greensburg, PA), supplied by an automatic watering system, and powdered food (Purina Certified Rodent Chow '5002, Ralston Purina Company, St. Louis, MO) were available to the animals ad libitum. Analyses of the food and water showed no contaminants at concentrations high enough to interfere with the outcome of the study. The animal husbandry procedures for the recovery animals were similar to those used during nonexposure periods. At the time of group assignment, only animals with body weights within two standard deviations of the group mean for each sex were used in the study. Any animal in poor health was rejected from group -assignment.

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: distilled water, pH=5
Remarks on MMAD:
MMAD / GSD: Aerosol Generation: Aqueous solution of test substance was metered from a piston pump (Fluid Metering, Inc., Oyster Bay, NY), equipped with a 3/8-inch (50 mg/m ) or lIS-inch (5 and 15 mg/m3) piston. The solution was metered into an atomizer (Spraying Systems Company, -Wheaton, IL) fitted with a No. 1650 liquid nozzle and a No. 64 air nozzle. The atomizer was inserted into the top of the inhalation turret where the liquid aerosol was dispersed throughout the chambers by filtered chamber supply air. The operating pressure of the atomizers used to generate the solution of test substance for each target concentration was 20 psig.
Details on inhalation exposure:
Inhalation Chamber Design and Operation: The chambers were constructed from stainless steel with glass windows for animal observation. The volume of the chambers was approximately 1330 liters and a typical airflow was approximately 300 L/min (13 air changes per hour). Chamber temperature and relative humidity were recorded using a minimum-maximum thermometer (Brooklyn Thermometer Company, Inc., Farmingdale, NY) and an Airguide hunidity indicator (Airguide Instrument Company, Chicago, IL). Temperature and relative humidity measurements were recorded 12 times per exposure.

Target Concentrations and Exposure Regimen: The animals were acclimated to the exposure chambers located in Room 139 (air-only exposure) for two days prior to the initiation of the exposure regimen. Target concentrations of 0 (control), 5, 15, and 50 mg/m3 were selected for this study. Two chambers were required for the 50 mg/m3 target concentration group because of the large number of animals. Equal numbers of animals were placed in each of the 50 mg/m3 group chambers. The rats (60 days of age) were exposed for six hours per day beginning on September 15, 1986. Fourteen male rats (61 days of age) were exposed for one six-hour exposure on September 16, 1986. Eighteen male rats (62 days of age) were exposed for three six-hour exposures beginning on September 17, 1986. Beginning on September 22, 1986, 14 male rats (67 days of age) were exposed for five days. Twenty-two male rats (60 days of age) were exposed for six hours per day for 34 days beginning on October 6, 1986. The balance of the animals were exposed five days per week for 13 weeks, and the males were exposed for two days and the females for three days during the final (14th) week. The six-hour chamber exposure interval was defined as the time when the aerosol generation system was turned on and subsequently turned off.
The cage placement pattern was changed weekly in a predetermined manner within each chamber to compensate for any possible, but undetected, variations in chamber exposure conditions.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber Concentrations. Particle Size Analyses. and Chamber Aerosol Distribution:
Chamber concentrations of the test substance (1% solution) were determined by gravimetric methods. Two samples were obtained from each test substance aerosol exposure chamber each day. The sample flowrates for the 5, 15, 50 (chamber 137-4), and 50 mg/m3 (chamber 137-5) concentrations were 23.20, 11.11, 4.30, and 3.95 L/min, respectively. The sample collection time was 150 minutes for all groups. The glass fiber filters (47 mm, Type A-E, Gelman Instrument Company, Ann Arbor, MI) used to collect the test substance aerosols were connected to a dry gas meter (Rockwell International, Pittsburgh, PA), a critical orifice, and a vacuum pump (Terracon Corp., Waltham, MA). The filters with the collected A-174 were dried for 20 minutes in a drying oven (approximate temperature l20°C) and then cooled for 10 minutes in a desiccator with anhydrous calcium sulfate. The amount of A-174 in the chamber was determined from the change in weight according to the formula:
Equivalent test substance Concentration = Measured Concentration / 0.7217
where the resulting silicone formed on the dried filter paper represents approximately 72.17% of the test substance. The desiccation procedure and adjustment of the gravimetric concentration reflect procedures that have been followed in previous studies (BRRC Report Nos. 46-44, 1983, and 47-78, 1984).
The particle size distribution was measured using a TSI Aerodynamic Particle Sizer (Model APS 33, TSI Incorporated, St. Paul, MN). These determinations were made two times per week for the first 13 weeks and once during the final week of the exposure. The daily dats collected from each test substance exposure chamber were analyzed by probit analysis to obtain the count median aerodynamic diameter (CMAD), mass median aerodynamic diameter (HMAD), and their respective geometric standard deviations (ag).
Prior to the initiation of exposures, the distribution of the aerosol was evaluated for one of the 50 mg/m3 group chambers. The results of the chamber aerosol distribution assessment indicated that there was acceptable uniformity of distribution for the aerosol.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
5 mg/m³ air
Dose / conc.:
15 mg/m³ air
Dose / conc.:
50 mg/m³ air
No. of animals per sex per dose:
See study design.
Control animals:
yes, concurrent no treatment
Details on study design:
Four groups of F-344 rats were exposed for 6 hours/day, 5 days/week, for up to 14 weeks to filtered air (control group) or to aerosols of hydrolyzed and condensed test substance (in a 1% aqueous solution) at target concentrations of 5, 15, or 50 mg/m3. Selected animals were then maintained for approximately six months after the termination of the exposure regimen.  

Tables 1 and 2 provide information on the original study design which is briefly described below.
All male rats of the 5 and 15 mg/m3 groups and all 40 female rats on study started exposures on September 15, 1986. All but 4 male rats of the control group also started the exposure regimen on September 15. Because of the large number of rats assigned to the 50 mg/m3 group, the first day of exposure was staggered for some subgroups: 62 rats started exposure on September 15 and received 13-1/2 weeks (66-67 days) of exposure; 14 rats started exposure on September 16 and received 1 exposure; 14 rats started exposure on September 17 and received 3 exposures (additionally, the only 4 control male rats that did not start the exposure regimen on September 15 started on September 17 and had 3 days of the exposure regimen); 14 rats started exposure on September 22 and received 5 exposures; and 22 rats started exposure on October 6 and had 7 weeks (34 days) of exposure.

Forty-two of the male rats were sacrificed on December 17, 1986, two of whiCh were selected for perfusion fixation of the laryngeal tissue (Tables 1 and 2). All the female rats were sacrificed on December 18, 1986. Since no laryngeal granulomas were induced, the male rats held for recovery (35 rats of the control group and 50 rats of the 50 mg/m3 group) were sacrificed and discarded on June 5, 1987. The male rats exposed for 1, 3, 5, or 34 days' starting on September 16, 17, or 22, 1986, or on October 6, 1986, respectively, and which were involved in perfusion fixation for possible evaluation of the larynx by transmission electron microscopy (TEM), scanning electron microscopy/energy dispersive X-ray diffraction (SEM/EDX), scanning electron microscopy (SEM), or light microscopy techniques were sacrificed on various dates as described in Table 2.
Observations and examinations performed and frequency:
All animals were individually observed for signs of toxic effects except during exposure. During the exposures, observations were recorded on a group basis. Preceding and following each exposure, observations were recorded for animals exhibiting overt clinical signs. The animals were examined for mortality on weekends and holidays. At the time of body weight collection and just preceding sacrifice, detailed observations were performed on all animals. All male animals held for recovery were observed every Monday, Friday, and alternate Wednesdays, except holidays, for overt clinical signs. A mortality check was conducted daily on all animals during the recovery period.

Ophthalmic Examinations: Prior to the first exposure, the anterior chambers and corneas of the eyes of each animal received on August 25, 1986, were examined by a veterinarian using an external light source and a magnifying lens. The animals scheduled for a complete necropsy during the 14th week of the study had an ophthalmic examination performed prior to sacrifice.

Body Weights: All animals were weighed on the morning prior to the initiation of their first exposure. These values were used as the pre exposure reference weights and were subtracted from each subsequent weight to determine the change in body weight. The animals were also weighed weekly during the exposure regimen of the study and prior to sacrifice. The male animals held for recovery were weighed every other week throughout that postexposure period.

Food and Water Consumption: Food and water consumption were measured while the rats were in the metabolism cages (see Urinalysis). The rats were fed powdered food (Certified Rodent Chow #5002, Ralston Purina Company).

Blood Analysis: Serum chemistry and hematology evaluations were performed on blood samples collected from 40 male and 40 female rats (10/group) sacrificed at the end of the 14-week exposure regimen. Blood was obtained from the orbital sinuses of methoxyflurane-anesthetized animals immediately before sacrifice. Food was removed from all cages at the start of the blood collection period, but water was supplied ad libitum.

Urinalysis: Urine was collected from 10 animals/sex/group. The anima~s were housed individually in polycarbonate metabolism cages with stainless steel, wire-mesh bottoms approximately 20 cm in diameter x 11 cm high (Nalge Company, Rochester, NY). Food and water were available ad libitum. Approximately two to three thymol crystals were added to the urine collection tube as a preservative. Urine was collected for approximately 15 hours following 66 exposures for male rats and for approximately 16 hours following 67 exposures for female rats.

Organ Weights: The brain, liver, kidneys, lungs, spleen, heart, adrenal glands, and testes (males) from the 10 rats/s~x/group sacrificed-during the 14th week were weighed at necropsy. Organ weights were statistically compared to those of the control animals both as absolute weights and as a percentage of body weight.
Sacrifice and pathology:
Necropsy and Histopathology: Ten rats per exposure group were subjected to a complete necropsy on December 17, 1986 (males), and December 18, 1986 (females). Except for the rats designated for perfusion fixation, rats were killed by exsanguination via the brachial blood vessels following anesthesia with methoxyflurane. Gross examinations were performed on selected animals (rats sacrificed at the end of the 14-week exposure regimen) and the tissues listed below were saved in 10% neutral buffered formalin (except eyes - preserved in Bouin's solution) for histologic evaluations. Histologic evaluations were performed on selected tissues embedded in paraffin and stained with hematoxylin and eosin from selected animals in the control and high concentration croups. The larnynx was also examined from animals of the low and middle concentration croups. The larynges of selected male rats were perfused for possible examination by SEM/EDX.

In addition, 10 high concentration group male rats (start of exposures on October 6, 1986) were subjected to a minimal necropsy on November 11, 1986, following 34 exposures and 10 high concentration group male rats were subjected to a minimal necropsy on January 8, 1987, following approximately seven weeks of nonexposure. The larynges were evaluated by light microscopy.

The purpose of the recovery group was to determine the regression of laryngeal cranulomas over the lifetime of these laboratory rodents; however, since no laryngeal granulomas were detected macroscopically or microscopically, the Sponsor determined there was no reason to maintain these rats. Therefore, the study was terminated on June 3, 1987, and all surviving male rats (except for one rat sacrificed as moribund on May 29, 1987) were removed from the study on June 5, 1987, killed by carbon dioxide asphyxiation, and discarded.
Statistics:
For statistical purposes, to negate the constant fluctuations in size of the groups, and hence mean body weight values, a core group of animals was chosen for the control and 50 mg/m3 group male rats for conducting statistics on body weight data. The core group for the control males consisted of 45 rats and excluded the 4 rats that received just 3 exposures. The core group for the 50 mg/m3 group males consisted of the 62 rats which received the entire 14 weeks of exposure. The body weights for all males of the 5 and 15 mg/m3 groups and all female rats on study were used for the statistical analyses.

Statistical Procedures: Results of quantitative continuous variables (e.g., body weight change) were intercompared for the three exposure groups and one control group by use of Bartlett's test for homogeneity of variances (Sokal and Rohlf, 1969), analysis of variances (ANOVA) (Sokal and Rohlf, 1969), and Duncan's multiple range test (Snedecor and Cochran, 1967). The latter was used, if F for the !NOVA was significant, to delineate which groups differed from the control. If Bartlett's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances (Welch and Brown and Forsythe, 1974) followed, if necessary, by t-tests. Bonferroni corrections were applied to all t-test comparisons. The fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
of statistical significance
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Actual mean chamber aerosol concentrations were 4.8, 14.7, and 50.1 mg/m3. The mass median aerodynamic diameter for the particle size distribution for the groups ranged from 2.3 to 2.9 microns. The only exposure-related effects were in the serum protein electrophoresis and histologic evaluations. With regard to serum protein electrophoresis, males of the 50 mg/m3 group had a 27% decrease in globulin concentration as determined by protein electrophoresis. There was also a 20% decrease in the globulin concentration for female rats of both the 15 and 50 mg/m3 groups, but the change was not statistically significant. The biological significance of this finding is unknown. However, decreased globulin can be an indication of liver of kidney dysfunction. There were no adverse microscopic findings, therefore this finding is probably not toxicologically significant (additional comment of study reviewer). The principal histologic change in the olfactory mucosa, cytoplasmic hyalinization without any indication of cellular necrosis, was found in all but one of the aerosol-exposed animals.  Additionally, macrophage infiltration was found in the lungs of the 50 mg/m3 group, a normal local biological response to foreign particle deposition in the lungs.  However, the major finding in this study was that laryngeal granulomas were not produced in rats.  

Therefore the NOAEC for laryngeal granulomas and general systemic toxicity was >=50 mg/m3.
Dose descriptor:
NOAEL
Effect level:
50 mg/m³ air
Sex:
male/female
Basis for effect level:
other: Laryngeal granulomas were not produced in rats inhaling aerosols generated from a 1% pH=5 solution of 3-trimethoxysilylpropyl methacrylate at concentrations up to 50 mg/m3.
Critical effects observed:
not specified

Chamber Concentrations. Particle Size Analyses. Temperature, and Relative Humidity:

The following table presents data for the nominal and gravimetric exposure concentrations obtained for the study:

Target Concentration (mg/m3)

Nominal Concentration of test substance solution (mg/m3)

Corrected* Nominal Concentration (mg/m3)

Gravimetric Concentration (mg/m3)

Equivalent(a) test substance Concentration (mg/m3)

5

692

6.92

3.5+/-0.29

4.9

15

1671

16.71

10.6+/-0.66

14.7

50(A)**

5881

58.81

36.1+/-1.27

50.0

50(B)**

5821

58.21

36.2+/-1.65

50.2

*Corrected Nominal Concentration = Nominal Concentration x 0.01 for 1% solutions

**Two chambers had to be used for this group because of the large number of animals.

(a)Equivalent test substance concentration = Gravimetric Concentration + 0.7217

 

The following table summarizes the particle size data collected during the study:

Target Conc. (mg/m3)

CMAD (µ)

sg

MMAS (µ)

sg

5

1.2

1.5

2.3

1.7

15

1.2

1.5

2.6

1.7

50A

1.2

1.5

2.9

1.8

50(B)

1.2

1.8

2.9

1.8

Where

CMAD = Count Median Aerodynamic Diameter

MMAD = Mass Median Aerodynamic Diameter

sg = Geometric Standard Deviation

The means of the daily mean chamber temperature and relative humidity ranged from 21.2 to 23.8°C and from 40-56%,

respectively.

Conclusions:
In a repeated inhalation dose toxicity study, ot conducted according to any guideline but in compliance with GLP, a NOAEC of 50 mg/m3 was concluded. This was based on that laryngeal granulomas were not produced in rats inhaling aerosols generated from a 1% pH=5 solution of test substance at concentrations up to 50 mg/m3.
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
20.05.1985 to 14.06.1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted specifically to identify whether an aerosol of the test substance induced laryngeal granulomas. While this study does not meet standards of the current guidelines in terms of the diversity of the examinations conducted, it is a well conducted study that achieves its objective.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study was to characterize factors relating to the development of laryngeal granulomas in male rats following repeated inhalation exposures (four weeks) of gamma-methacryloxypropyltrimethoxysilane 3-trimethoxysilylpropyl methacrylate aerosol. Because the rate of hydrolysis of the methoxy groups of 3-trimethoxysilylpropyl methacrylate, with resultant polymerization and change from a liquid to a solid state, is increased as the pH of the solution becomes more acidic, there was concern that the methods for preparation of the generation solution (i.e. acidic pH of 3-4 and recirculation of the test material) for studies conducted prior to the present study may have resulted in aerosols of a solid, polymerized material that was not representative of the formulation, usage, and exposure hazard in the industrial setting. Therefore, this study was designed to answer several major questions as follows:
1) Are the laryngeal granulomas reproducible using concentrations and generation parameters employed in previous studies?;
2) Will there be a significant difference in biological response in rats exposed to aerosols derived from a formulation more typical of industrial usage (1%, pH 5, non-recirculated solution) versus that used for previous studies (1%, pH 3-4, recirculated solution)?;
3) Does the recirculation of a 15%, pH 3 solution hasten the polymerization of 3-trimethoxysilylpropyl methacrylate and thereby become an important factor in the production of the laryngeal granulomas?;
4) If differences in the biological response to inhalation of aerosols from a 1%, pH 5, non-recirculated solution versus a 15%, pH 3, non-recirculated solution versus a 15%, pH 3, recirculated solution were found, could those differences be related to different A-174 moieties in the exposure atmosphere and/or the laryngeal tissue?;
5) Does a dose-response relationship exist for inhalation of 3-trimethoxysilylpropyl methacrylate aerosol?;
6) Can the presence of 3-trimethoxysilylpropyl methacrylate be analytically verified in the sites of these granulomas and what form does it assume in these sites?
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY
- Age at study initiation: 42 days
- Weight at study initiation: Approximately 100 grams
- Fasting period before study: None
- Housing: Two per cage in stainless steel wire-mesh cages.
- Diet: Ad libitum (except during exposure)
- Water: Ad libitum (except during exposure)
- Acclimation period: Two days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5-23 °C
- Humidity (%): 28-71 %
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 20.05.1985 To: 14.06.1985
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: 1% solution in distilled water and acetic acid (pH 5) or 15% solution in distilled water and acetic acid (pH 3)
Remarks on MMAD:
MMAD / GSD: Mean 2.92 to 5.2 microns
Details on inhalation exposure:
The inhalation chambers were constructed from stainless steel and glass windows for animal observations. The volume was approximately 1330 litres, and the airflow was approximately 360 litres/minutes (16 air changes/hour). Temperature and relative humidity measurements were recorded at least five times per exposure.

A solution of 3-(trimethoxysilyl)propyl methacrylate was metered from a piston pump directly into an atomiser for the non-recirculating method of aerosol generation. The atomiser was inserted into the top of the inhalation chamber turret where the liquid aerosol was diluted to the desired concentration and dispersed throughout the chambers by filtered chamber supply air. The recirculation system for generation (Group IV only) used one piston pump and atomiser to continuously spray the 3-(trimethoxysilyl)propyl methacrylate solution back into the reservoir, A second piston pump and atomiser were then used in a similar manner to the non-circulated method, with the pump drawing the continuously recirculated fluid from the reservoir.

The particle size distribution was measured daily in each chamber with a TSI Aerodynamic Particle Sizer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of 3-(trimethoxysilyl)propyl methacrylate hydrolysate were determined by gravimetric methods. Samples were obtained twice per day from the 3-(trimethoxysilyl)propyl methacrylate test chamber and once per day from the air-control chamber.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
See methods
No. of animals per sex per dose:
5 males/dose
Control animals:
yes, concurrent no treatment
Details on study design:
Five groups of 20 to 40 male F-344 rats were exposed for 6 hours/day, 5 days/week for up to 4 weeks to filtered air (control group; Group I) or respirable aerosols of 3-(trimethoxysilyl)propyl methacrylate (Groups II - V). The 3-(trimethoxysilyl)propyl methacrylate exposure groups were exposed to target concentrations generated from solutions varying in percent concentration, pH, and method of aerosol generation: Group II - 10 mg/m3, 1% (v/v), pH 5, non-recirculated solution; Group III - 50 mg/m3, 1%, pH 5, non-recirculated solution; Group IV - 50 mg/m3, 15%, pH 3, recirculated solution; and Group V - 50 mg/m3, 15%, pH 3, non-recirculated solution. 
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals observed prior to, during and following each exposure.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on the morning prior to initiation of the first exposure, then weekly and preceding sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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: No

CLINICAL CHEMISTRY: YNo

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Macroscopic observations and microscopic evaluation of the respiratory tract were conducted. Additionally, tissue samples (laryngeal granulomas) were evaluated by SEM/EDX, as well as by transmission electron microscopy.
Other examinations:
Samples from the chamber atmosphere were evaluated by SEM/EDX (scanning electron microscopy/ energy dispersive X-ray analysis) and infrared (IR) spectroscopy.
Statistics:
The data for continuous, parametric variables were intercompared for the exposure and control groups by use of Levene's test for homogeneity of variances and by t-tests.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Actual mean equivalent 3-(trimethoxysilyl)propyl methacrylate concentrations obtained for Groups II through V were 13.5, 68.2, 67.8, and 69.0 mg/m3, respectively.  

Laryngeal granulomas were found in all groups (III, IV, and V) of rats exposed to approximately 70 mg/m3 of 3-(trimethoxysilyl)propyl methacrylate, regardless of the formulation of the generation solution. No granulomas were found in control rats (Group I) or rats exposed to approximately 14 mg/m3 of the aerosol generated from a 1%, pH 5, non-recirculated solution (Group II). At the site of laryngeal granulomas, spherical siliceous particles were observed ultrastructurally, these being present both extracellularly and within the cytoplasm of histiocytes and fibroblasts. Additionally, there was generally an increased incidence of hyaline bodies observed in the nasal mucosa of rats exposed to 3-(trimethoxysilyl)propyl methacrylate when compared to control rats. Transient decreases in body weight gain occurred in rats exposed to approximately 70 mg/m3 of 3-(trimethoxysilyl)propyl methacrylate (Groups III, IV, and V), but no exposure-related clinical signs or macroscopic abnormalities were observed.
Dose descriptor:
NOAEC
Effect level:
13.5 mg/m³ air
Sex:
male
Basis for effect level:
other: Aerosol exposure at 13.5 mg/m3 did not produce laryngeal granulomas to rats under the conditions of this study.
Critical effects observed:
not specified

Discussion of study author regarding formation of laryngeal granuloma

The formulation of generated solution had no biological significance; rats from all three groups exposed to approximately 70 mg/m3 of 3-(trimethoxysilyl)propyl methacrylate developed laryngeal granulomas after 2 and/or 4 weeks of exposure. The granulomas from rats exposed to aerosols of the 1%, pH5 solution (Group III) were smaller than those observed for both groups exposed to aerosols of the 15%, pH3 solution. However, no qualitative difference in the IR spectra was observed in samples obtained from the chamber atmosphere of Groups III and IV to explain these slight differences in granuloma incidence (at 2 weeks) and size (at both 2 and 4 weeks). Similarly, no difference was observed in the appearance of the material in the granuloma, or the morphological and cytological characteristics of the granuloma itself, to account for these changes. The incidence and severity of the lesion was very similar in both groups exposed to the 15%, pH3 solution (Groups IV and V). Therefore, the recirculation of the 15% solution (Group IV) produced no apparent biological differences from the non-recirculated solution (Group V).

The mechanism whereby the laryngeal granuloma is produced remains unknown. The reasons for the unusual location of the lesion (larynx) and the presence of particles (of approximately 2 microns) in the submucosa under the intact epithelium are not apparent. Some factors that could relate to the site of the lesion may be an unusual sensitivity of these cells to 3-(trimethoxysilyl)propyl methacrylate toxicity, or an increased deposition and/or decreased clearance of particles in this area. Possible explanations for the appearance of spherical siliceous particles in the submucosa include the transport of particles across the epithelium or an initial necrosis and ulceration of the epithelium, followed by particulate deposition, with reepithelialization of the ulcerated site(s). However, there was no evidence of focal necrosis, reepithelialization, or a prominent change in cell type was observed in this study. An additional explanation, which gains support from the fact that the material was determined not to be a solid particulate in the chamber atmosphere, is that the 3-(trimethoxysilyl)propyl methacrylate is inhaled as a liquid aerosol. It then somehow crosses the intact epithelium to the submucosa, where it eventually polymerizes into a solid particulate.

Conclusions:
In a four-week inhalation study, not conducted to any guideline but in compliance with GLP (reliability score 2) an aerosol concentration of approximately 70 mg/m3 of A-174 caused laryngeal granulomas. Altering the percent concentration, pH, or method of aerosol generation did not significantly alter the production of laryngeal granulomas at 70 mg/m3. However, no granulomas were observed at a concentration of 15 mg/m3 (pH 5; 1% solution). At the site of laryngeal granulomas, spherical siliceous particles were observed ultrastructurally, these being present both extracellularly and within the cytoplasm of histiocytes and fibroblasts. The only sytemic effect was on body weight, which has resolved in all but only in the 50 mg/m3 (pH3, 5% solution). The NOAEC for laryngeal granulomas and general toxicity (reduced body weights) was 13.5 mg/m3.
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
11.04.1983 to 14.10.1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
some restrictions in the range of examinations performed.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
unknown
Principles of method if other than guideline:
This study was designed to determine the toxic effects, if any, in rats resulting from 14 weeks of repeated inhalation of 3-trimethoxysilylpropyl methacrylate aerosol.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hilltop Lab Animals, Inc., Scottdale, PA.
- Age at study initiation: No data
- Weight at study initiation: No data
- Fasting period before study: No
- Housing: Individually housed 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 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 11.04.1983 To: 14.10.1983
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: distilled water and acetic acid (pH 4)
Remarks on MMAD:
MMAD / GSD: 1.6 microns
Details on inhalation exposure:
Inhalation chambers were constructed from stainless steel with glass windows for animal observation. The volume was approximately 900 litres and the airflow was approximately 200-260 l/min (13-17 air changes per hour).

The test substance aerosol was generated using a Laskin nebuliser. A double aspirator tube was used to generate the 250 mg/m3 test substance target aerosol concentration. single aspirator tubes were used for generation of the 100 and 50 mg/m3 test substance aerosol target concentrations. A predetermined amount of test solution was added once or twice to each flask for the 6-hour exposure. Compressed air supplied to each nebuliser created a negative pressure such that the liquid solution was aspirated into the tube where it was then dispersed as a fine liquid aerosol. The liquid aerosol was then introduced into the top of the exposure chamber where it was diluted to the desired concentration and dispersed through the chamber by filtered chamber supply air.

Particle size distribution was measured daily in each chamber with a TSI Aerodynamic Particle Sizer.

The mean daily chamber temperature was 23-24oC for each chamber. The mean chamber relative humidity increased as the concentration increased. The mean for the control chamber was 45% with a daily mean range of 32-58%.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of test substance were analysed by gravimetric determination approximately once per hour from the test substance chambers and twice per day from the air control chamber.
Duration of treatment / exposure:
14 weeks exposure followed by 91 day recovery/observation period for 8 animals/sex/group.
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
244 mg/m³ air (analytical)
Dose / conc.:
100 mg/m³ air (analytical)
Dose / conc.:
50 mg/m³ air (analytical)
Dose / conc.:
250 mg/m³ air (nominal)
Dose / conc.:
100 mg/m³ air (nominal)
Dose / conc.:
50 mg/m³ air (nominal)
No. of animals per sex per dose:
18
Control animals:
yes, concurrent no treatment
Details on study design:
Groups of 18 male and 18 female F-344 rats were exposed to gamma-methacryloxypropyltrimethoxysllane (3-trimethoxysilylpropyl methacrylate) aerosol, six hours per day, five days per week, for 14 weeks. A control group was exposed to chamber air only.  
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed prior to, during and following each exposure. During the recovery period, all animals were observed at least once per day.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on the morning prior to initiation of the first exposure. Then weekly during the exposure and preceding sacrifice. Animals in the recovery phase were weighed weekly for the first month and biweekly thereafter, and just prior to sacrifice.

FOOD AND WATER CONSUMPTION: Ten rats (per sex/group) were housed in metabolism cages. Food and water consumption were measured for approximately 16 hours following 66 exposures for the female rats and following 67 exposures for the male rats. During the recovery period all animals were housed in metabolism cages and food/water consumption measured for approximately 18 hours following 88 postexposure days for the females and 89 postexposure days for the males.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to first exposure and at sacrifice.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Just prior to sacrifice
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: No
- How many animals: 10/sex/group after exposure period and 8/sex/group after recovery period.
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Just prior to sacrifice
- Animals fasted: No
- How many animals: 10/sex/group after exposure period and 8/sex/group after recovery period.
- Parameters checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: While in metabolism cages for food/water consumption.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Full necropsy and histopathologic examination of tissues were assessed to determined the toxicity of the test material.
Statistics:
The data for continuous, parametric variables were intercompared for the exposure and control groups by use of analysis of variance, Bartlett's homogeneity of variance and Duncan's multiple range.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
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:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
The mean (+/- standard deviation) of the measured exposure concentrations were 244 (+/- 9), 100 (+/- 6), and 50 (+/- 2) mg/m3. The mass median aerodynamic diameter was 1.6 micrometers with a geometric standard deviation in the range of 0.43 to 0.65 for the three concentrations. The mean (± standard deviation) methanol concentrations were 353 (+/- 70), 232 (+/- 40), and 130 (+/- 17) ppm for the three exposure groups.

Body weight gain was reduced by the fourth exposure at the highest concentration in males; following termination of exposures, the body weights returned to 
those seen for the controls.  No other in-life signs of adverse effects were noted in any exposure group.  There were no biologically significant alterations in  clinical pathology evaluations, including serum chemistry, protein electrophoresis, haematology or urinanalysis noted at either sacrifice interval.  
Statistically significant differences for liver weights of the male rats in the highest exposure group were noted.  There were no significant histologic findings for  the liver, but there were treatment-related upper respiratory tract lesions observed in all three exposure groups.  The most biologically significant lesion was granulomatous laryngitis with polyp formation, which did not resolve during the recovery period.  Also, there was hyalinization of the cytoplasm of olfactory mucosal cells, the significance of which is unknown, which did not resolve during the recovery period.  A minimum-effect or no-effect level with respect to the incidence of granulomas could not be determined from this study, since the histologic findings were similar from all three concentrations studied.
Dose descriptor:
LOAEC
Effect level:
50 mg/m³ air
Sex:
male/female
Basis for effect level:
other: Granulomatous laryngitis with polyp formation was noted at all dose levels, including the lowest dose level.
Critical effects observed:
not specified
Conclusions:
In a 14-week inhalation study (reliability score 2), conducted in a similar manner to OECD 413 and in compliance with GLP, a LOAEC was determined to 50 mg/m3 air in males and females. This was based on granulomatous laryngitis with polyp formation at all dose levels. A minimum-effect or no-effect level cannot be determined from this study, since the lowest exposure concentration produced a similar incidence of granulomatous laryngitis to that seen in the intermediate and highest concentration groups.The NOAEC for general systemic effects was 100 mg/m3.
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
01.11.1982 to 12.11.1982
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
The study did not meet current guideline requirements for repeated dose toxicity, primarily because it is only nine days in duration. It does, however, add weight of evidence for determining whether laryngeal granuloma formation is related to exposure duration.
Qualifier:
no guideline followed
Principles of method if other than guideline:
This study was designed to determine the toxic effects in rats resulting from nine days of repeated inhalation of 3-trimethoxysilylpropyl methacrylate hydrolysate aerosol.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hilltop Lab animals, Inc., Scottdale, PA.
- Age at study initiation: 54 days
- Weight at study initiation: Males: 175-176 g; Females: 130-132 g.
- Fasting period before study: None
- Housing: Individually housed in stainless steel wire-mesh cages in glove-box housing units.
- Diet: Ad libitum (except during exposure)
- Water: Ad libitum (except during exposure)
- Acclimation period: Three days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22.7 °C
- Humidity (%): 40-50 %
- Air changes (per hr): During exposure (12-17), no further information.
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 01.11.1982 To: 12.11.1982
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: Distilled water and acetic acid (pH 4); used 15% solution
Remarks on MMAD:
MMAD / GSD: The mass median aerodynamic diametres for the aerosols ranged between 1.04 and 1.4 micrometers with geometric standard deviations of 2.16 to 2.4.
Details on inhalation exposure:
Inhalation chambers were constructed from stainless steel with glass windows for animal observations. The volume was approximately 900 litres and the airflow between 180 and 250 litres/minute. Temperature and humidity readings were taken at least four times during each six-hour exposure. The aerosol was generated from a 15% (w/w) solution of 3-(trimethoxysilyl)propyl methacrylate in distilled water titrated to pH 4 with acetic acid, using a nebuliser. The aerosol from each nebuliser was conducted through glass tubes into a stainless steel mixer at the inlet to each chamber. The aerosol was mixed with additional air prior to entering the chambers.

Particle size analysis was determined with a cascade impactor. Glass fibre filters were used as the collection substrates. The filters were assayed for 3-(trimethoxysilyl)propyl methacrylate in the same way as the filters for the concentration analyses. The mass median aerodynamic diameters and geometric standard deviations for the different exposure groups were determined from log-probability plots of the cumulative percentage mass collected versus the effective cut-off diameters for the impactor stages.

Chamber temperature: 21.1-25 °C
Chamber humidity: 35-66 %
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chambers were monitored approximately once each hour for 3-(trimethoxysilyl)propyl methacrylate aerosol and methanol vapour concentrations. Aerosol samples were collected from the centre of the chamber on pre-weighed glass fibre filters and dried to a constant weight before reweighing. Methanol concentrations were determined by gas chromatography.
Duration of treatment / exposure:
9 days (five days exposure, two days rest, four days exposure)
Frequency of treatment:
6 hours/day
Dose / conc.:
50 mg/m³ air
Dose / conc.:
100 mg/m³ air
Dose / conc.:
300 mg/m³ air
No. of animals per sex per dose:
Ten
Control animals:
yes, concurrent no treatment
Details on study design:
No satellite groups.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed prior to, during and following each exposure for signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on the morning preceding the first exposure. The animals were also weighed on the morning preceding the second, fifth, sixth and seventh exposures, and prior to sacrifice.

FOOD CONSUMPTION: Yes
- Food consumption was measured for approximately 16 hours following eight exposures for the female rats and following nine exposures for the male rats.

WATER CONSUMPTION: Yes
- Water consumption was measured for approximately 16 hours following eight exposures for the female rats and following nine exposures for the male rats.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the first exposure and at sacrifice.
- Dose groups that were examined: All

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At sacrifice
- Animals fasted: No
- How many animals: All animals
- Parameters checked in Table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: All animals approximately 16 hours following eight exposures for the female rats and following nine exposures for the male rats.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in Table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Gross examinations were performed and retained.
Histopathological examinations of tissues, and general observations of the animals were assessed to determine the toxicity of the test material (Table 2).
Selected organs were weighed at sacrifice: liver, heart, brain, lungs, kidneys and testes from all animals (absolute and percentage of body weight).

Addendum: After fourteen weeks of inhalation of gamma-methacryloxypropyl-trimethoxysilane (3-(trimethoxysilyl)propyl methacrylate) hydrolysate aerosols (Report #47-78), numerous rats in all exposure groups (i.e. 250, 100, and 50 mg/m3) were found to have developed granulomatous laryngitis. This lesion was present in a specific portion of the larynx. Because of the marked specificity in the anatomical location of this lesion, it was considered advisable to examine step sections of the available laryngeal tissues from all rats of the nine-day aerosol study in order to substantiate the presence or absence of this lesion.
Statistics:
Results for quantitative continuous variables (such as body weight changes) were intercompared among the three concentration groups and one control group by the following tests: analysis of variance, Bartlett's homogeneity of variance and Duncan's multiple range.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
No animals died during the exposures.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a significant depression in body weight gain for the males and females in the high exposure concentration group, and a transient depression for the females at the intermediate concentration.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food and water consumption were slightly elevated for the male rats at the low and intermediate exposure concentrations.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were statistically significant elevations in serum albumin for the male rats at all exposure concentrations. This was accompanied by a significant decrease in total protein, resulting in a significant and dose-related decrease in the calculated total serum globulin. A similar effect was not observed in the female rats.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No statistically significant alterations were found in any of the urinalyses.
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Description (incidence and severity):
These results could indicate a possible immunosuppressive action in male rats; however, there were no other indications of disturbed immunological functions in these animals.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no histopathological lesions observed which were attributable to the 3-(trimethoxysilyl)propyl methacrylate exposures. Addendum result: The re-evaluatlon indicates that the development of granulomatous laryngitis is present after nine days of inhalation of 3-(trimethoxysilyl)propyl methacrylate aerosol even at the lowest concentration tested, 50 mg/m3.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
The mean (± standard deviation) exposure concentrations were 302 (+/- 16), 98.8 (+/- 10.7), and 49.6 (+/- 4.3) mg/m3. The mass median aerodynamic diameters ranged between 1.04 and 1.40 micrometres with geometric standard deviations between 2.16 and 2.40. The mean (+/- standard deviation) methanol concentrations were 210 (+/- 38), 185 (+/- 32), and 75 (+/- 23) for the three exposure groups.
Dose descriptor:
LOAEC
Effect level:
50 mg/m³ air
Sex:
male/female
Basis for effect level:
other: Development of granulomatous laryngitis is present after nine days of Inhalation of 3-trimethoxysilylpropyl methacrylate aerosol even at the lowest concentration tested, 50 mg/m3.
Critical effects observed:
not specified
Conclusions:
In a well documented nine-day aerosol inhalation study, not conducted according to any guideline but in compliance with GLP (reliability score 4), a LOAEC was concluded to be 50 mg/m3 air. This was based on minimal biological effects (decreased body weight gain in high and mid-dose group on days 5-12 although close to controls by day 12, dose-related decrease in total serum globulins at all concentrations) were seen as a result of exposure of Fisher 344 rats to 3-(trimethoxysilyl)propyl methacrylate aerosol at concentrations of 50, 100 and 300 mg/m3. Re-evaluation of the laryngeal tissues revealed that laryngeal granulomas were apparent after nine days exposure with all concentrations tested.
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12.04.1982 to 23.04.1982
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
The study did not meet current guideline requirements for repeated dose toxicity, primarily because it is only nine days in duration. It does, however, add weight of evidence for determining whether laryngeal granuloma formation is related to exposure duration.
Qualifier:
no guideline followed
Principles of method if other than guideline:
This study was designed to determine and evaluate the toxic effects in rats resulting from nine days of repeated inhalation of 3-trimethoxysilylpropyl methacrylate vapour.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI
- Age at study initiation: 76 days
- Weight at study initiation: Males: 246-268 g; Females: 154-158 g.
- Fasting period before study: None
- Housing: Two per cage in stainless steel wire-mesh cages
- Diet: Ad libitum (except during exposure)
- Water: Ad libitum (except during exposure)
- Acclimation period: Two days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 8ppm: 26-27; 20ppm: 31.5-34.5; 40ppm: 41-46.5 (during exposure)
- Humidity (%): Approximately 35 %
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 12.04.1982 To: 23.04.1982
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Inhalation chambers were constructed from stainless steel with glass windows for animal observation. The volume was approximately 4400 litres and the airflow was 1000 L/min (14 air changes per hour). Temperature and relative humidity gauges were placed inside chambers. The temperature and relative humidity within the chambers were recorded at least four times during each six-hour exposure.

The vapour was generated with S.I.D. vapour generators. Dehumidified air was blown through a heater and into th evaporator section of the generator. The liquid test substance pumped into the air outlet end of the rotating evaporator tube. The heated air passed countercurrent to the fluid flow and carried the vapour into the exposure chamber. The vapour concentration was controlled by varying the fluid feed rate, the evaporator temperature, or the airflow through the evaporator.

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations were analysed approximately once each hour by gas chromatography.
- Samples taken from breathing zone: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations were analysed approximately once each hour by gas chromatography.
Duration of treatment / exposure:
Nine days (five days exposure initially, two days rest, then further four days of exposure).
Frequency of treatment:
6 hours/day
Dose / conc.:
8 ppm (nominal)
Dose / conc.:
20 ppm (nominal)
Dose / conc.:
40 ppm (nominal)
Dose / conc.:
7.5 ppm (analytical)
Dose / conc.:
23 ppm (analytical)
Dose / conc.:
42 ppm (analytical)
No. of animals per sex per dose:
Ten
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: No data
- Rationale for selecting satellite groups: No satellite groups
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Prior to, during and following exposure.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: morning before first exposure, prior to second, fifth, sixth and seventh exposures and prior to sacrifice.

FOOD AND WATER CONSUMPTION:
- The rats were individually housed in metabolism cages. Food and water consumption was measured for approximately 16 hours following eight exposures for the female rats and following nine exposures for the male rats.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to first exposure and at sacrifice.
- Dose groups that were examined: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The day of sacrifice
- Animals fasted: No
- How many animals: All surviving rats
- Parameters evaluated not given.

URINALYSIS: Yes
- Time schedule for collection of urine: Following eight exposures while animals were in the metabolism cages.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters evaluated not given.
Sacrifice and pathology:
All surviving animals were sacrificed on the morning following the final exposure.
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Larynges of all animals. No other organs/tissues were examined.
ORGAN WEIGHTS: The liver, heart, brain, lungs, kidneys and testes from all animals were weighed at sacrifice (absolute and as a percentage of body weight).
Statistics:
Results for quantitative continuous variables (such as body weight changes) were intercompared among the three concentration groups and one control group by the following tests: analysis of variance, Bartlett's homogeneity of variance and Duncan's multiple range.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was an apparent increase in the mean body weight of the male animals in the high concentration exposure group when compared to the control animals. However, this is explained by a drop in the body weight of one control male animal at the ninth day, causing the mean body weight of the control group to decrease.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was increased 36 %, respectively, for males of the high exposure group, when compared to the values in the control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was increased 48%, respectively, for males of the high exposure group, when compared to the values in the control group.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant findings in the eye examinations.
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no adverse findings in the serum chemistry investigations.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The mean total urine volume for the male rats in the 40 ppm group was 44% greater than the mean control values.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were statistically significant decreases in the relative mean kidney weights for male rats in the highest and lowest exposure groups but not the intermediate exposure concentration group. The relative mean heart weight decreased in the males of the high exposure concentration group and the absolute heart weight decreased for the female rats in the high and intermediate concentration groups.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no histologic lesions encountered which could be attributed to the exposures.
Histopathological findings: neoplastic:
not examined
Details on results:
The mean (+/- standard deviation) exposure concentrations were 42 (2), 23 (2), 7.5 (1.4), and 0 (0) ppm.
Dose descriptor:
LOAEC
Effect level:
ca. 8 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
food consumption and compound intake
water consumption and compound intake
organ weights and organ / body weight ratios
Critical effects observed:
not specified
Conclusions:
In a well documented nine-day vapour inhalation study, not conducted according to any guideline but in compliance with GLP (reliability score 4), minimal biological effects (increased food and water consumption, decreased heart and kidney weights, increased urine output) were observed as a result of exposure of Fisher 344 rats to 3-(trimethoxysilyl)propyl methacrylate vapor at concentrations up to 42 ppm (approximately equivalent to 400 mg/m3). There were no laryngeal granulomas.
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
21.03.1988 to 15.04.1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted specifically to identify whether an aerosol of the test substance induced laryngeal granulomas. While this study does not meet standards of the current guidelines in terms of the diversity of the examinations conducted, it is a well conducted study that achieves its objective.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study was to confirm the development of laryngeal granulomas in rats following short-term inhalation exposures to aerosol of hydrolyzed and condensed 3-trimethoxysilylpropyl methacrylate. It was also the purpose of this study to further characterize the initial development of this lesion in the laboratory rodent.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Fischer 344/CDF
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY.
- Age at study initiation: 56 days
- Weight at study initiation: Approximately 188 g.
- Fasting period before study: None
- Housing: Two per cage 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: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-23.3 °C
- Humidity (%): 46-62 %
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 21.03.1988 To: 15.04.1988
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: 1% or 15% solution in distilled water and acetic acid (pH=5)
Remarks on MMAD:
MMAD / GSD: 2.09-2.54 microns
Details on inhalation exposure:
The inhalation chambers were constructed from stainless steel and glass windows for animal observations. The volume was approximately 1330 litres, and the airflow was approximately 300 litres/minutes (13.5 air changes/hour). Temperature and relative humidity measurements were recorded at least twelve times per exposure.

A 1 or 15% liquid solution of 3-(trimethoxysilyl)propyl methacrylate hydrolysate was metered from a piston pump into an atomiser. The atomiser was inserted into the top of the inhalation chamber turret where the liquid aerosol was dispersed throughout the chambers by filtered chamber supply air.

The particle size distribution was measured using a cascade impactor. These determinations were made three times per week throughout the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of 3-(trimethoxysilyl)propyl methacrylate hydrolysate were determined by gravimetric methods. Four samples were obtained each day. Two samples were obtained from each 3-(trimethoxysilyl)propyl methacrylate aerosol exposure chamber each day.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
14 mg/m³ air (analytical)
Remarks:
1% solution; equivalent concentration=gravimetric concentration/0.7217
Dose / conc.:
48.9 mg/m³ air (analytical)
Remarks:
1% solution; equivalent concentration=gravimetric concentration/0.7217
Dose / conc.:
70.2 mg/m³ air (analytical)
Remarks:
1% solution; equivalent concentration=gravimetric concentration/0.7217
Dose / conc.:
97 mg/m³ air (analytical)
Remarks:
1% solution; equivalent concentration=gravimetric concentration/0.7217
Dose / conc.:
100.9 mg/m³ air
Remarks:
15% solution; equivalent concentration=gravimetric concentration/0.7217
Dose / conc.:
15 mg/m³ air (nominal)
Remarks:
1% solution
Dose / conc.:
50 mg/m³ air (nominal)
Remarks:
1% solution
Dose / conc.:
70 mg/m³ air (nominal)
Remarks:
1% solution
Dose / conc.:
100 mg/m³ air (nominal)
Remarks:
1% solution
Dose / conc.:
100 mg/m³ air (nominal)
Remarks:
15% solution
No. of animals per sex per dose:
24 to 28 male rats/dose group
Control animals:
yes, concurrent no treatment
Details on study design:
No further information.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Preceding and following each exposure, observations were recorded for animals exhibiting overt clinical signs. The animals were examined for mortality at the weekends.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the time of body weight measurements and just preceding sacrifice, detailed observations were performed on all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on the morning prior to the inhalation of their first exposure. Then weekly during the exposure period and just prior to sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Macroscopic observations, microscopic evaluation of the respiratory tract, and transmission electron microscopy of larynges were conducted.
Statistics:
The data for continuous, parametric variables were intercompared for the exposure and control groups by use of Levene's test for homogeneity of variances and by t-tests.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects on absolute body weight; slight depressions in body weight gain occurred but not in a concentration-related manner.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Goblet cell hyperplasia occurred in the nasal mucosa of all groups of rats exposed to aerosols of hydrolyzed and condensed 3-(trimethoxysilyl)propyl methacrylate; cytoplasmic hyalinization of the nasal mucosa was generally limited to rats exposed to aerosol generated from the 15% solution. Laryngeal lesions included squamous metaplasia in rats exposed to concentrations of 70 mg/m3 or greater, and granulomatous laryngitis in rats exposed to 100 mg/m3, regardless of whether the aerosol was generated from a 1% or a 15% solution. However, the granulomatous laryngitis lesions were larger in rats exposed to aerosol generated from a 15% solution and also occurred at a slightly higher frequency (100%) than those rats exposed to an aerosol generated from a 1% solution (85%). At the site of the larynx where the granulomas formed in the 100 mg/m3 groups, another lesion, squamous metaplasia, occurred at a high incidence (>=70%) in rats exposed to the aerosol at 70 mg/m3 or greater. There was also a low incidence (15-20%) of squamous metaplasia at the site in rats exposed to 15 or 50 mg/m3 3-(trimethoxysilyl)propyl methacrylate. The incidence and severity of this lesion increased with increasing exposure concentration.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
LOAEC
Effect level:
15 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Goblet cell hyperplasia occurred in the nasal mucosa of all groups of rats exposed to aerosols of hydrolyzed and condensed 3-trimethoxysilylpropyl methacrylate
Dose descriptor:
NOAEC
Effect level:
15 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on observed laryngeal granulomas at concentrations of 70 mg/m3.
Critical effects observed:
not specified
Conclusions:
In a four-week (5 days/week) inhalation study, not conducted according to any guideline but in compliance with GLP (reliability score 2), rats were exposed to aerosol of hydrolyzed and condensed 3-(trimethoxysilyl)propyl methacrylate at concentrations of 100 mg/m3. Regardless of whether 1% or 15% solutions were used, granulomas were produced in rats. Aerosol generated from a 1% solution at atmosphere concentrations of 70 mg/m3 and below did not produce granulomas in rats.
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
22.07.1991 to 23.10.1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
restrictions in the range of examinations performed.
Qualifier:
equivalent or similar to
Guideline:
EPA OPP 82-4 (90-Day Inhalation Toxicity)
Deviations:
yes
Remarks:
Limited investigations as study had a specific purpose. Post-exposure period of 12 months.
Principles of method if other than guideline:
Purpose: To determine the fate of laryngeal granulomas during a 1-year recovery period (persist unchanged, regress and/or resolve, progress and become more severe, or result in carcinomas). Multiple toxicity parameters were assessed.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Indianapolis, IN.
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: Males: 142.2-199.8 grams; Female: 106.6-144.5 grams.
- Fasting period before study: No
- Housing: Individually in stainless steel, wire mesh cages.
- Diet: Ad libitum (except during exposure)
- Water: Ad libitum (except during exposure)
- Acclimation period: Three weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.7-26.1 °C
- Humidity (%): 40-70 %
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 22.07.1991 To: 23.10.1992
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: distilled water adjusted to pH 5
Remarks on MMAD:
MMAD / GSD: Mean concentrations of 0 and 102.0 (± 5.76) mg/m3 were measured; mean methanol concentrations of 0 and 36 (± 3.7) ppm were also measured. Mean mass median aerodynamic diameter for the exposed group was 1.7.
Details on inhalation exposure:
The inhalation chambers were stainless steel with glass windows for animal observation. The volume of each chamber was approximately 1330 litres and the airflow rate was approximately 300l/min (13 air changes/hour).

A 2% solution of H-MPTMS (pH5) was metered from a piston pump into an atomiser. The atomiser was inserted into the top of the inhalation chamber turret, where the liquid aerosol was dispersed throughout the chamber by filtered chamber supply air.

The particle size distribution was measured using a TSI Aerodynamic Particle Sizer in conjunction with a 20:1 dilutor. These determinations were made each exposure day for the chambers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
3-(Trimethoxysilyl)propyl methacrylate condensate was measured gravimetrically. Four samples from the exposure chamber were obtained during each 6-hour exposure.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
6 hours/day, 5 days/week for 13 weeks and for 2 days during fourteenth week
Dose / conc.:
105 mg/m³ air (analytical)
Dose / conc.:
100 mg/m³ air (nominal)
No. of animals per sex per dose:
Total of 40 animals (20 male and 20 female): 8/sex terminated day after last exposure
8/sex terminated after 1, 4, 8 or 12 month recovery periods
Control animals:
yes, concurrent no treatment
Details on study design:
One group, containing 40 male and female Fischer 344 rats, was exposed to aerosolized H-MPTMS (gamma-methacryloxypropyltrimethoxysilane [3-(trimethoxysilyl)propyl methacrylate] hydrolysate) for 6 hours/day, 5 days/week for 13 weeks and for 2 days during the fourteenth week. The concentration was 100 mg/m3. A control group of the same size was exposed to filtered air alone. Eight animals/sex/group were sacrificed at the end of the exposure period and after 1, 4, 8, and 12 month recovery periods.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the time of body weight measurements and just preceding sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to first exposure, and then weekly for the duration of the study.

FOOD AND WATER CONSUMPTION: First four weeks, then only if body weight gain was statistically different from the control groups.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Following the last exposure and at the end of the recovery months 1, 4, 8 and 12.
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes
- How many animals: 8 rats/sex/group following last exposure and at the end of recovery months 1 and 4. From 8 males/group, 8 female control rats and 7 female exposed rats at the end of recovery months 8 and 12.
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Following the last exposure and at the end of the recovery months 1, 4, 8 and 12.
- Animals fasted: Yes
- How many animals: 8 rats/sex/group following last exposure and at the end of recovery months 1 and 4. From 8 males/group, 8 female control rats and 7 female exposed rats at the end of recovery months 8 and 12.
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Eight animals/sex/group were sacrificed at the end of the exposure period and after 1, 4, 8, and 12 month recovery periods.
Gross and microscopic pathology were assessed.
Statistics:
The data for quantitative continuous variables were intercompared for the H-MPTMS exposure and the control group by use of the t-test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only exposure-related clinical sign was periocular encrustation, which was probably due to mild irritation.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females in the 100 mg/m3 group died or were sacrificed moribund during the study (Study Days 304 and 319). These deaths were not related to the exposure, since they occurred during the recovery period and the histopathologic data did not suggest an exposure-related death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreases in body weight and body weight gain for males in the 100 mg/m3 group occurred only during the exposure period.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative lung weight increases at the end of the exposure period were related to the exposure.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mild to marked laryngeal granulomas, mild to moderate hyaline epithelial inclusions within the epithelia lining the sections of the nasal cavity, and minimum to mild alveolar histiocytosis were observed in the animals at the end of exposure and also at the different time points during the recovery period. Of the animals in the 100 mg/m3 group, 94% had laryngeal granulomas, 100% had hyaline epithelial inclusions, and 96% had alveolar histiocytosis. Mild to marked squamous metaplasia of the mucosal epithelium overlying the granulomas was also present at the Study Week 14 termination; however, except for 1 male and 2 females, it was not observed during the recovery period. Minimum to moderate granulomatous lymphadenitis within the mediastinal lymph nodes appeared in almost 50% of the animals sacrificed during the recovery period. The hyaline epithelial inclusions within the nasal epithelia, granulomatous laryngitis, and alveolar histiocytosis did not appear to change in frequency of occurrence or severity during the recovery period. No evidence of neoplastic lesions was observed for H-MPTMS exposed animals.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Dose descriptor:
LOAEC
Effect level:
ca. 100 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Local effects on the respiratory tract
Remarks on result:
other: Local effects
Dose descriptor:
LOAEC
Effect level:
ca. 100 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Systemic effects
Critical effects observed:
not specified
Conclusions:
In a 90-day inhalation study with up to one year recovery period, conducted according to EPA OPP 82-4 and in compliance with GLP (reliability score 2), the NOAEC for these local effects was <100 mg/m3, and the NOAEC for general systemic toxicity was <100 mg/m3 based on reduced body weight/gain. Additionally, exposure of rats to 100 mg/m3 (2%, pH 5) H-MPTMS for 13 weeks, produced mild to marked laryngeal granulomas, mild to moderate hyaline epithelial inclusions and minimum to mild alveolar histiocytosis. The hyaline epithelial inclusions within the nasal epithelia, granulomatous laryngitis and alveolar histiocytosis did not appear to change in frequency of occurrence or severity during the recovery period. No evidence of laryngeal carcinomas was noted.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
50 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
14.08.1989 to 08.09.1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted specifically to identify whether an aerosol of the test substance induced laryngeal granulomas. While this study does not meet standards of the current guidelines in terms of the diversity of the examinations conducted, it is a well conducted study that achieves its objective.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of this study was to confirm the development of laryngeal granulomas in rats following short-term repeated inhalation exposures to 3-trimethoxysilylpropyl methacrylate-hydrolysate aerosol.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY.
- Age at study initiation: 41 days
- Weight at study initiation: Males: 130-131 grams
- Fasting period before study: none
- Housing: One or two per cage 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 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.5-21 °C
- Humidity (%): 45-77 %
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 14.08.1989 To: 08.09.1989
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: distilled water
Remarks on MMAD:
MMAD / GSD: 2.65 microns with standard deviation of 1.74.
Details on inhalation exposure:
The inhalation chambers were constructed from stainless steel and glass windows for animal observations. The volume was approximately 1330 litres, and the airflow was approximately 300 litres/minutes (13.5 air changes/hour). Temperature and relative humidity measurements were recorded at least twelve times per exposure.

A 2% solution of 3-(trimethoxysilyl)propyl methacrylate hydrolysate was metered from a piston pump into an atomiser. The atomiser was inserted into the top of the inhalation chamber turret where the liquid aerosol was dispersed throughout the chambers by filtered chamber supply air.

The particle size distribution was measured using a TSI Aerodynamic Particle Sizer and a 20:1 diluter. These determinations were made once per day for the duration of the study (except on day 3 due to equipment failure).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of 3-(trimethoxysilyl)propyl methacrylate hydrolysate were determined by gravimetric methods. Four samples were obtained each day.
Duration of treatment / exposure:
4 weeks (19 days of exposure over a 4-week period, 18 days of exposure for satellite group)
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
143 mg/m³ air
Dose / conc.:
150 mg/m³ air
No. of animals per sex per dose:
15 males rats/treated group; 15 male rats/control group
5 animals from each group were designated to a satellite group for further ultrastructural evaluation of the larynges (this was cancelled)
Control animals:
yes, concurrent no treatment
Details on study design:
A group of 15 male Fischer 344 rats was exposed 6 hours per day for 19 days of exposure (18 days of exposure for the satellite group) over a 4-week period to an aerosol of 3-(trimethoxysilyl)propyl methacrylate hydrolysate (3-(trimethoxysilyl)propyl methacrylate hydrolysate; gamma-methacryloxypropyltrimethoxysilane hydrolysate; CAS: 2530-85-0) at a target concentration of 150 mg/m3. A control group of 15 male rats was exposed with the same exposure regimen to filtered air only. Five animals of the control and 3-(trimethoxysilyl)propyl methacrylate hydrolysate exposure groups were assigned to a satellite group for possible ultrastructural evaluation of the larynges, but not subsequently evaluated. 
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Preceding and following each exposure. On non-exposure days, animals were observed once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the time of body weight collection and just preceding sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on the morning prior to initiation of the first exposure. Then weekly during the four week exposure period and immediately preceding sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes a full necropsy was performed.
HISTOPATHOLOGY: Yes, of gross lesions, larynx, lungs, trachea, nasal turbinates and kidneys.
Statistics:
The data for continuous, parametric variables were intercompared for the exposure and control groups by use of Levene's test for homogeneity of variances and by t-tests.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only exposure-related clinical sign, observed in most of the animals, was perinasal encrustation.
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreases in body weight gain were observed for the 3-(trimethoxysilyl)propyl methacrylate-hydrolysate exposure group during the first two weeks of the study.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Nasal crust around the nares of the 3-(trimethoxysilyl)propyl methacrylate-hydrolysate exposed rats was the only gross lesion noted at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Upon histological examination, changes were noted within the respiratory tracts of the3-(trimethoxysilyl)propyl methacrylate-hydrolysate exposed rats. Cytoplasmic hyalinization was observed within the olfactory mucosa of the nasal cavity. In addition, squamous metaplasia and foci of minimal to mild granulomatous laryngitis (laryngeal granulomas) were present within the larynges of 3-(trimethoxysilyl)propyl methacrylate-hydrolysate exposed rats. These findings concur with microscopic changes observed in previous 3-(trimethoxysilyl)propyl methacrylate-hydrolysate aerosol studies.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
LOAEL
Effect level:
143 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Laryngeal granulomas developed.
Critical effects observed:
not specified
Conclusions:
In a four week study aerosol inhalation study, not conducted according to any guideline but in compliance with GLP, a LOAEL was concluded to be 143 mg/m3 in males, based on the development of laryngeal granulomas.
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 15.09.1986 to 05.06.1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: unknown
Principles of method if other than guideline:
The purpose of this study was to determine a no-observable-effect level for the development of laryngeal granulomas in rats following inhalation exposures to aerosol of hydrolyzed and condensed 3-trimethoxysilylpropyl methacrylate. It was also an additional objective of this study to further characterize the initial development of this lesion and the potential progression of this lesion during the lifetime of the laboratory rodent. However, because no laryngeal granulomas were developed in this study, all rats held for recovery were sacrificed approximately 6 months after the termination of exposures.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Two-hundred male and fifty-three female Fischer 344/CDF rats, 40 days of age, were received on August 25, 1986, from Charles River Breeding Laboratories, Inc. (Kingston, NY). Three male and three female rats were sacrificed on August 26, 1986, and the trachea, lungs, liver, kidneys, heart, spleen, salivary glands, submandibular lymph nodes, and nasal cavities were fixed and examined microscopically. Fecal samples were collected from these animals and examined for intestinal parasites by zinc sulfate flotation, and a Scotch~ tape test was performed on the perineum for examination of pinworms. Prior to sacrifice, blood samples were obtained for possible serologic evaluations. Blood samples were collected from five male and five female rats two weeks later for serologic evaluations. In addition, body weight determinations, clinical observations, and ophthalmic evaluations of all animals were performed prior to the initiation of the 14-week exposure regimen. Physical examination, parasitology, and histopathology indicated these animals were free of infectious disease and were suitable for use on this study.
An additional 42 male Fischer 344/CDF rats, 39 days of age, were received on September IS, 1986, fro~ Charles River Breeding Laboratories, Inc. (Kingston, NY). Three rats were sacrificed for quality control on September 16, 1986, and the same tissues as described above were fixed and examined micro¬scopically. Fecal samples were collected and examined using the zinc sulfate flotation method and these animals were examined for pinworms. Prior to the sacrifice, blood samples were collected for possible serologic evaluation. Blood samples were collected from five animals on September 30, 1986, for serologic evaluations. In addition, body weight determinations and clinical observations were performed on these male rats prior to the initiation of their 7-week exposure regimen (this group of rats started exposure on October 6, 1986, and was exposed for 7 weeks).

Animal Husbandry:
The rats were housed two per cage in stainless steel wire-mesh cages, 23.5 cm x 20 cm x 18 cm high in Room 165 for approximately 16 weeks. The rats were housed individually during the recovery period in the same type of cages as during the 14-week exposure regimen. They were located in Room 165 for approximately one week, then moved to Room 164 Bioclean Unit F for the duration of the recovery period. All animals were separated by test group and sex. Deotized Animal Cage Board. (Shepherd Specialty Papers, Inc., Kalamazoo, HI) was placed under each row of cages to collect excrement. The room temperature and relative humidity were monitored continuously by a Hygrothermograph. Seven-Day Continuous Recorder, Model #8368-00 (Cole-Parmer Instrument Company, Chicago, IL).The animals were kept on a 12-hour photoperiod throughout the study.
The additional male rats received on September 15, 1986, were housed two per cage in stainless steel, wire-mesh cages, 23.5 cm x 20 cm x 18 cm high in Room 164 Bioclean Unit B for 15 days, then transferred to Room 165 for approximately 12 weeks, then moved to Room 164 Bioclean Unit F for the duration of their recovery period. The animal husbandry procedures for these additional male rats were similar to those used for the animals received on July 17, 1986.
During 3-(trimethoxysilyl)propyl methacrylate exposures, the animals were transferred to.9.5 cm x 18 cm x 17.5 cm stainless steel, wire-mesh cages, separated by test group, and housed one per cage. Some of the high concentration group male rats were individually housed in 21 cm x 12.5 cm x 18 cm stainless steel, wire-mesh cages.
During nonexposure periods, water (Municipal Authority of Westmoreland County, Greensburg, PA), supplied by an automatic watering system, and powdered food (Purina Certified Rodent Chow '5002, Ralston Purina Company, St. Louis, MO) were available to the animals ad libitum. Analyses of the food and water showed no contaminants at concentrations high enough to interfere with the outcome of the study. The animal husbandry procedures for the recovery animals were similar to those used during nonexposure periods. At the time of group assignment, only animals with body weights within two standard deviations of the group mean for each sex were used in the study. Any animal in poor health was rejected from group -assignment.

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: distilled water, pH=5
Remarks on MMAD:
MMAD / GSD: Aerosol Generation: Aqueous solution of test substance was metered from a piston pump (Fluid Metering, Inc., Oyster Bay, NY), equipped with a 3/8-inch (50 mg/m ) or lIS-inch (5 and 15 mg/m3) piston. The solution was metered into an atomizer (Spraying Systems Company, -Wheaton, IL) fitted with a No. 1650 liquid nozzle and a No. 64 air nozzle. The atomizer was inserted into the top of the inhalation turret where the liquid aerosol was dispersed throughout the chambers by filtered chamber supply air. The operating pressure of the atomizers used to generate the solution of test substance for each target concentration was 20 psig.
Details on inhalation exposure:
Inhalation Chamber Design and Operation: The chambers were constructed from stainless steel with glass windows for animal observation. The volume of the chambers was approximately 1330 liters and a typical airflow was approximately 300 L/min (13 air changes per hour). Chamber temperature and relative humidity were recorded using a minimum-maximum thermometer (Brooklyn Thermometer Company, Inc., Farmingdale, NY) and an Airguide hunidity indicator (Airguide Instrument Company, Chicago, IL). Temperature and relative humidity measurements were recorded 12 times per exposure.

Target Concentrations and Exposure Regimen: The animals were acclimated to the exposure chambers located in Room 139 (air-only exposure) for two days prior to the initiation of the exposure regimen. Target concentrations of 0 (control), 5, 15, and 50 mg/m3 were selected for this study. Two chambers were required for the 50 mg/m3 target concentration group because of the large number of animals. Equal numbers of animals were placed in each of the 50 mg/m3 group chambers. The rats (60 days of age) were exposed for six hours per day beginning on September 15, 1986. Fourteen male rats (61 days of age) were exposed for one six-hour exposure on September 16, 1986. Eighteen male rats (62 days of age) were exposed for three six-hour exposures beginning on September 17, 1986. Beginning on September 22, 1986, 14 male rats (67 days of age) were exposed for five days. Twenty-two male rats (60 days of age) were exposed for six hours per day for 34 days beginning on October 6, 1986. The balance of the animals were exposed five days per week for 13 weeks, and the males were exposed for two days and the females for three days during the final (14th) week. The six-hour chamber exposure interval was defined as the time when the aerosol generation system was turned on and subsequently turned off.
The cage placement pattern was changed weekly in a predetermined manner within each chamber to compensate for any possible, but undetected, variations in chamber exposure conditions.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber Concentrations. Particle Size Analyses. and Chamber Aerosol Distribution:
Chamber concentrations of the test substance (1% solution) were determined by gravimetric methods. Two samples were obtained from each test substance aerosol exposure chamber each day. The sample flowrates for the 5, 15, 50 (chamber 137-4), and 50 mg/m3 (chamber 137-5) concentrations were 23.20, 11.11, 4.30, and 3.95 L/min, respectively. The sample collection time was 150 minutes for all groups. The glass fiber filters (47 mm, Type A-E, Gelman Instrument Company, Ann Arbor, MI) used to collect the test substance aerosols were connected to a dry gas meter (Rockwell International, Pittsburgh, PA), a critical orifice, and a vacuum pump (Terracon Corp., Waltham, MA). The filters with the collected A-174 were dried for 20 minutes in a drying oven (approximate temperature l20°C) and then cooled for 10 minutes in a desiccator with anhydrous calcium sulfate. The amount of A-174 in the chamber was determined from the change in weight according to the formula:
Equivalent test substance Concentration = Measured Concentration / 0.7217
where the resulting silicone formed on the dried filter paper represents approximately 72.17% of the test substance. The desiccation procedure and adjustment of the gravimetric concentration reflect procedures that have been followed in previous studies (BRRC Report Nos. 46-44, 1983, and 47-78, 1984).
The particle size distribution was measured using a TSI Aerodynamic Particle Sizer (Model APS 33, TSI Incorporated, St. Paul, MN). These determinations were made two times per week for the first 13 weeks and once during the final week of the exposure. The daily dats collected from each test substance exposure chamber were analyzed by probit analysis to obtain the count median aerodynamic diameter (CMAD), mass median aerodynamic diameter (HMAD), and their respective geometric standard deviations (ag).
Prior to the initiation of exposures, the distribution of the aerosol was evaluated for one of the 50 mg/m3 group chambers. The results of the chamber aerosol distribution assessment indicated that there was acceptable uniformity of distribution for the aerosol.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
5 mg/m³ air
Dose / conc.:
15 mg/m³ air
Dose / conc.:
50 mg/m³ air
No. of animals per sex per dose:
See study design.
Control animals:
yes, concurrent no treatment
Details on study design:
Four groups of F-344 rats were exposed for 6 hours/day, 5 days/week, for up to 14 weeks to filtered air (control group) or to aerosols of hydrolyzed and condensed test substance (in a 1% aqueous solution) at target concentrations of 5, 15, or 50 mg/m3. Selected animals were then maintained for approximately six months after the termination of the exposure regimen.  

Tables 1 and 2 provide information on the original study design which is briefly described below.
All male rats of the 5 and 15 mg/m3 groups and all 40 female rats on study started exposures on September 15, 1986. All but 4 male rats of the control group also started the exposure regimen on September 15. Because of the large number of rats assigned to the 50 mg/m3 group, the first day of exposure was staggered for some subgroups: 62 rats started exposure on September 15 and received 13-1/2 weeks (66-67 days) of exposure; 14 rats started exposure on September 16 and received 1 exposure; 14 rats started exposure on September 17 and received 3 exposures (additionally, the only 4 control male rats that did not start the exposure regimen on September 15 started on September 17 and had 3 days of the exposure regimen); 14 rats started exposure on September 22 and received 5 exposures; and 22 rats started exposure on October 6 and had 7 weeks (34 days) of exposure.

Forty-two of the male rats were sacrificed on December 17, 1986, two of whiCh were selected for perfusion fixation of the laryngeal tissue (Tables 1 and 2). All the female rats were sacrificed on December 18, 1986. Since no laryngeal granulomas were induced, the male rats held for recovery (35 rats of the control group and 50 rats of the 50 mg/m3 group) were sacrificed and discarded on June 5, 1987. The male rats exposed for 1, 3, 5, or 34 days' starting on September 16, 17, or 22, 1986, or on October 6, 1986, respectively, and which were involved in perfusion fixation for possible evaluation of the larynx by transmission electron microscopy (TEM), scanning electron microscopy/energy dispersive X-ray diffraction (SEM/EDX), scanning electron microscopy (SEM), or light microscopy techniques were sacrificed on various dates as described in Table 2.
Observations and examinations performed and frequency:
All animals were individually observed for signs of toxic effects except during exposure. During the exposures, observations were recorded on a group basis. Preceding and following each exposure, observations were recorded for animals exhibiting overt clinical signs. The animals were examined for mortality on weekends and holidays. At the time of body weight collection and just preceding sacrifice, detailed observations were performed on all animals. All male animals held for recovery were observed every Monday, Friday, and alternate Wednesdays, except holidays, for overt clinical signs. A mortality check was conducted daily on all animals during the recovery period.

Ophthalmic Examinations: Prior to the first exposure, the anterior chambers and corneas of the eyes of each animal received on August 25, 1986, were examined by a veterinarian using an external light source and a magnifying lens. The animals scheduled for a complete necropsy during the 14th week of the study had an ophthalmic examination performed prior to sacrifice.

Body Weights: All animals were weighed on the morning prior to the initiation of their first exposure. These values were used as the pre exposure reference weights and were subtracted from each subsequent weight to determine the change in body weight. The animals were also weighed weekly during the exposure regimen of the study and prior to sacrifice. The male animals held for recovery were weighed every other week throughout that postexposure period.

Food and Water Consumption: Food and water consumption were measured while the rats were in the metabolism cages (see Urinalysis). The rats were fed powdered food (Certified Rodent Chow #5002, Ralston Purina Company).

Blood Analysis: Serum chemistry and hematology evaluations were performed on blood samples collected from 40 male and 40 female rats (10/group) sacrificed at the end of the 14-week exposure regimen. Blood was obtained from the orbital sinuses of methoxyflurane-anesthetized animals immediately before sacrifice. Food was removed from all cages at the start of the blood collection period, but water was supplied ad libitum.

Urinalysis: Urine was collected from 10 animals/sex/group. The anima~s were housed individually in polycarbonate metabolism cages with stainless steel, wire-mesh bottoms approximately 20 cm in diameter x 11 cm high (Nalge Company, Rochester, NY). Food and water were available ad libitum. Approximately two to three thymol crystals were added to the urine collection tube as a preservative. Urine was collected for approximately 15 hours following 66 exposures for male rats and for approximately 16 hours following 67 exposures for female rats.

Organ Weights: The brain, liver, kidneys, lungs, spleen, heart, adrenal glands, and testes (males) from the 10 rats/s~x/group sacrificed-during the 14th week were weighed at necropsy. Organ weights were statistically compared to those of the control animals both as absolute weights and as a percentage of body weight.
Sacrifice and pathology:
Necropsy and Histopathology: Ten rats per exposure group were subjected to a complete necropsy on December 17, 1986 (males), and December 18, 1986 (females). Except for the rats designated for perfusion fixation, rats were killed by exsanguination via the brachial blood vessels following anesthesia with methoxyflurane. Gross examinations were performed on selected animals (rats sacrificed at the end of the 14-week exposure regimen) and the tissues listed below were saved in 10% neutral buffered formalin (except eyes - preserved in Bouin's solution) for histologic evaluations. Histologic evaluations were performed on selected tissues embedded in paraffin and stained with hematoxylin and eosin from selected animals in the control and high concentration croups. The larnynx was also examined from animals of the low and middle concentration croups. The larynges of selected male rats were perfused for possible examination by SEM/EDX.

In addition, 10 high concentration group male rats (start of exposures on October 6, 1986) were subjected to a minimal necropsy on November 11, 1986, following 34 exposures and 10 high concentration group male rats were subjected to a minimal necropsy on January 8, 1987, following approximately seven weeks of nonexposure. The larynges were evaluated by light microscopy.

The purpose of the recovery group was to determine the regression of laryngeal cranulomas over the lifetime of these laboratory rodents; however, since no laryngeal granulomas were detected macroscopically or microscopically, the Sponsor determined there was no reason to maintain these rats. Therefore, the study was terminated on June 3, 1987, and all surviving male rats (except for one rat sacrificed as moribund on May 29, 1987) were removed from the study on June 5, 1987, killed by carbon dioxide asphyxiation, and discarded.
Statistics:
For statistical purposes, to negate the constant fluctuations in size of the groups, and hence mean body weight values, a core group of animals was chosen for the control and 50 mg/m3 group male rats for conducting statistics on body weight data. The core group for the control males consisted of 45 rats and excluded the 4 rats that received just 3 exposures. The core group for the 50 mg/m3 group males consisted of the 62 rats which received the entire 14 weeks of exposure. The body weights for all males of the 5 and 15 mg/m3 groups and all female rats on study were used for the statistical analyses.

Statistical Procedures: Results of quantitative continuous variables (e.g., body weight change) were intercompared for the three exposure groups and one control group by use of Bartlett's test for homogeneity of variances (Sokal and Rohlf, 1969), analysis of variances (ANOVA) (Sokal and Rohlf, 1969), and Duncan's multiple range test (Snedecor and Cochran, 1967). The latter was used, if F for the !NOVA was significant, to delineate which groups differed from the control. If Bartlett's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances (Welch and Brown and Forsythe, 1974) followed, if necessary, by t-tests. Bonferroni corrections were applied to all t-test comparisons. The fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
of statistical significance
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Actual mean chamber aerosol concentrations were 4.8, 14.7, and 50.1 mg/m3. The mass median aerodynamic diameter for the particle size distribution for the groups ranged from 2.3 to 2.9 microns. The only exposure-related effects were in the serum protein electrophoresis and histologic evaluations. With regard to serum protein electrophoresis, males of the 50 mg/m3 group had a 27% decrease in globulin concentration as determined by protein electrophoresis. There was also a 20% decrease in the globulin concentration for female rats of both the 15 and 50 mg/m3 groups, but the change was not statistically significant. The biological significance of this finding is unknown. However, decreased globulin can be an indication of liver of kidney dysfunction. There were no adverse microscopic findings, therefore this finding is probably not toxicologically significant (additional comment of study reviewer). The principal histologic change in the olfactory mucosa, cytoplasmic hyalinization without any indication of cellular necrosis, was found in all but one of the aerosol-exposed animals.  Additionally, macrophage infiltration was found in the lungs of the 50 mg/m3 group, a normal local biological response to foreign particle deposition in the lungs.  However, the major finding in this study was that laryngeal granulomas were not produced in rats.  

Therefore the NOAEC for laryngeal granulomas and general systemic toxicity was >=50 mg/m3.
Dose descriptor:
NOAEL
Effect level:
50 mg/m³ air
Sex:
male/female
Basis for effect level:
other: Laryngeal granulomas were not produced in rats inhaling aerosols generated from a 1% pH=5 solution of 3-trimethoxysilylpropyl methacrylate at concentrations up to 50 mg/m3.
Critical effects observed:
not specified

Chamber Concentrations. Particle Size Analyses. Temperature, and Relative Humidity:

The following table presents data for the nominal and gravimetric exposure concentrations obtained for the study:

Target Concentration (mg/m3)

Nominal Concentration of test substance solution (mg/m3)

Corrected* Nominal Concentration (mg/m3)

Gravimetric Concentration (mg/m3)

Equivalent(a) test substance Concentration (mg/m3)

5

692

6.92

3.5+/-0.29

4.9

15

1671

16.71

10.6+/-0.66

14.7

50(A)**

5881

58.81

36.1+/-1.27

50.0

50(B)**

5821

58.21

36.2+/-1.65

50.2

*Corrected Nominal Concentration = Nominal Concentration x 0.01 for 1% solutions

**Two chambers had to be used for this group because of the large number of animals.

(a)Equivalent test substance concentration = Gravimetric Concentration + 0.7217

 

The following table summarizes the particle size data collected during the study:

Target Conc. (mg/m3)

CMAD (µ)

sg

MMAS (µ)

sg

5

1.2

1.5

2.3

1.7

15

1.2

1.5

2.6

1.7

50A

1.2

1.5

2.9

1.8

50(B)

1.2

1.8

2.9

1.8

Where

CMAD = Count Median Aerodynamic Diameter

MMAD = Mass Median Aerodynamic Diameter

sg = Geometric Standard Deviation

The means of the daily mean chamber temperature and relative humidity ranged from 21.2 to 23.8°C and from 40-56%,

respectively.

Conclusions:
In a repeated inhalation dose toxicity study, ot conducted according to any guideline but in compliance with GLP, a NOAEC of 50 mg/m3 was concluded. This was based on that laryngeal granulomas were not produced in rats inhaling aerosols generated from a 1% pH=5 solution of test substance at concentrations up to 50 mg/m3.
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
20.05.1985 to 14.06.1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted specifically to identify whether an aerosol of the test substance induced laryngeal granulomas. While this study does not meet standards of the current guidelines in terms of the diversity of the examinations conducted, it is a well conducted study that achieves its objective.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study was to characterize factors relating to the development of laryngeal granulomas in male rats following repeated inhalation exposures (four weeks) of gamma-methacryloxypropyltrimethoxysilane 3-trimethoxysilylpropyl methacrylate aerosol. Because the rate of hydrolysis of the methoxy groups of 3-trimethoxysilylpropyl methacrylate, with resultant polymerization and change from a liquid to a solid state, is increased as the pH of the solution becomes more acidic, there was concern that the methods for preparation of the generation solution (i.e. acidic pH of 3-4 and recirculation of the test material) for studies conducted prior to the present study may have resulted in aerosols of a solid, polymerized material that was not representative of the formulation, usage, and exposure hazard in the industrial setting. Therefore, this study was designed to answer several major questions as follows:
1) Are the laryngeal granulomas reproducible using concentrations and generation parameters employed in previous studies?;
2) Will there be a significant difference in biological response in rats exposed to aerosols derived from a formulation more typical of industrial usage (1%, pH 5, non-recirculated solution) versus that used for previous studies (1%, pH 3-4, recirculated solution)?;
3) Does the recirculation of a 15%, pH 3 solution hasten the polymerization of 3-trimethoxysilylpropyl methacrylate and thereby become an important factor in the production of the laryngeal granulomas?;
4) If differences in the biological response to inhalation of aerosols from a 1%, pH 5, non-recirculated solution versus a 15%, pH 3, non-recirculated solution versus a 15%, pH 3, recirculated solution were found, could those differences be related to different A-174 moieties in the exposure atmosphere and/or the laryngeal tissue?;
5) Does a dose-response relationship exist for inhalation of 3-trimethoxysilylpropyl methacrylate aerosol?;
6) Can the presence of 3-trimethoxysilylpropyl methacrylate be analytically verified in the sites of these granulomas and what form does it assume in these sites?
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY
- Age at study initiation: 42 days
- Weight at study initiation: Approximately 100 grams
- Fasting period before study: None
- Housing: Two per cage in stainless steel wire-mesh cages.
- Diet: Ad libitum (except during exposure)
- Water: Ad libitum (except during exposure)
- Acclimation period: Two days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5-23 °C
- Humidity (%): 28-71 %
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 20.05.1985 To: 14.06.1985
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: 1% solution in distilled water and acetic acid (pH 5) or 15% solution in distilled water and acetic acid (pH 3)
Remarks on MMAD:
MMAD / GSD: Mean 2.92 to 5.2 microns
Details on inhalation exposure:
The inhalation chambers were constructed from stainless steel and glass windows for animal observations. The volume was approximately 1330 litres, and the airflow was approximately 360 litres/minutes (16 air changes/hour). Temperature and relative humidity measurements were recorded at least five times per exposure.

A solution of 3-(trimethoxysilyl)propyl methacrylate was metered from a piston pump directly into an atomiser for the non-recirculating method of aerosol generation. The atomiser was inserted into the top of the inhalation chamber turret where the liquid aerosol was diluted to the desired concentration and dispersed throughout the chambers by filtered chamber supply air. The recirculation system for generation (Group IV only) used one piston pump and atomiser to continuously spray the 3-(trimethoxysilyl)propyl methacrylate solution back into the reservoir, A second piston pump and atomiser were then used in a similar manner to the non-circulated method, with the pump drawing the continuously recirculated fluid from the reservoir.

The particle size distribution was measured daily in each chamber with a TSI Aerodynamic Particle Sizer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of 3-(trimethoxysilyl)propyl methacrylate hydrolysate were determined by gravimetric methods. Samples were obtained twice per day from the 3-(trimethoxysilyl)propyl methacrylate test chamber and once per day from the air-control chamber.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
See methods
No. of animals per sex per dose:
5 males/dose
Control animals:
yes, concurrent no treatment
Details on study design:
Five groups of 20 to 40 male F-344 rats were exposed for 6 hours/day, 5 days/week for up to 4 weeks to filtered air (control group; Group I) or respirable aerosols of 3-(trimethoxysilyl)propyl methacrylate (Groups II - V). The 3-(trimethoxysilyl)propyl methacrylate exposure groups were exposed to target concentrations generated from solutions varying in percent concentration, pH, and method of aerosol generation: Group II - 10 mg/m3, 1% (v/v), pH 5, non-recirculated solution; Group III - 50 mg/m3, 1%, pH 5, non-recirculated solution; Group IV - 50 mg/m3, 15%, pH 3, recirculated solution; and Group V - 50 mg/m3, 15%, pH 3, non-recirculated solution. 
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals observed prior to, during and following each exposure.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on the morning prior to initiation of the first exposure, then weekly and preceding sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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: No

CLINICAL CHEMISTRY: YNo

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Macroscopic observations and microscopic evaluation of the respiratory tract were conducted. Additionally, tissue samples (laryngeal granulomas) were evaluated by SEM/EDX, as well as by transmission electron microscopy.
Other examinations:
Samples from the chamber atmosphere were evaluated by SEM/EDX (scanning electron microscopy/ energy dispersive X-ray analysis) and infrared (IR) spectroscopy.
Statistics:
The data for continuous, parametric variables were intercompared for the exposure and control groups by use of Levene's test for homogeneity of variances and by t-tests.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
Actual mean equivalent 3-(trimethoxysilyl)propyl methacrylate concentrations obtained for Groups II through V were 13.5, 68.2, 67.8, and 69.0 mg/m3, respectively.  

Laryngeal granulomas were found in all groups (III, IV, and V) of rats exposed to approximately 70 mg/m3 of 3-(trimethoxysilyl)propyl methacrylate, regardless of the formulation of the generation solution. No granulomas were found in control rats (Group I) or rats exposed to approximately 14 mg/m3 of the aerosol generated from a 1%, pH 5, non-recirculated solution (Group II). At the site of laryngeal granulomas, spherical siliceous particles were observed ultrastructurally, these being present both extracellularly and within the cytoplasm of histiocytes and fibroblasts. Additionally, there was generally an increased incidence of hyaline bodies observed in the nasal mucosa of rats exposed to 3-(trimethoxysilyl)propyl methacrylate when compared to control rats. Transient decreases in body weight gain occurred in rats exposed to approximately 70 mg/m3 of 3-(trimethoxysilyl)propyl methacrylate (Groups III, IV, and V), but no exposure-related clinical signs or macroscopic abnormalities were observed.
Dose descriptor:
NOAEC
Effect level:
13.5 mg/m³ air
Sex:
male
Basis for effect level:
other: Aerosol exposure at 13.5 mg/m3 did not produce laryngeal granulomas to rats under the conditions of this study.
Critical effects observed:
not specified

Discussion of study author regarding formation of laryngeal granuloma

The formulation of generated solution had no biological significance; rats from all three groups exposed to approximately 70 mg/m3 of 3-(trimethoxysilyl)propyl methacrylate developed laryngeal granulomas after 2 and/or 4 weeks of exposure. The granulomas from rats exposed to aerosols of the 1%, pH5 solution (Group III) were smaller than those observed for both groups exposed to aerosols of the 15%, pH3 solution. However, no qualitative difference in the IR spectra was observed in samples obtained from the chamber atmosphere of Groups III and IV to explain these slight differences in granuloma incidence (at 2 weeks) and size (at both 2 and 4 weeks). Similarly, no difference was observed in the appearance of the material in the granuloma, or the morphological and cytological characteristics of the granuloma itself, to account for these changes. The incidence and severity of the lesion was very similar in both groups exposed to the 15%, pH3 solution (Groups IV and V). Therefore, the recirculation of the 15% solution (Group IV) produced no apparent biological differences from the non-recirculated solution (Group V).

The mechanism whereby the laryngeal granuloma is produced remains unknown. The reasons for the unusual location of the lesion (larynx) and the presence of particles (of approximately 2 microns) in the submucosa under the intact epithelium are not apparent. Some factors that could relate to the site of the lesion may be an unusual sensitivity of these cells to 3-(trimethoxysilyl)propyl methacrylate toxicity, or an increased deposition and/or decreased clearance of particles in this area. Possible explanations for the appearance of spherical siliceous particles in the submucosa include the transport of particles across the epithelium or an initial necrosis and ulceration of the epithelium, followed by particulate deposition, with reepithelialization of the ulcerated site(s). However, there was no evidence of focal necrosis, reepithelialization, or a prominent change in cell type was observed in this study. An additional explanation, which gains support from the fact that the material was determined not to be a solid particulate in the chamber atmosphere, is that the 3-(trimethoxysilyl)propyl methacrylate is inhaled as a liquid aerosol. It then somehow crosses the intact epithelium to the submucosa, where it eventually polymerizes into a solid particulate.

Conclusions:
In a four-week inhalation study, not conducted to any guideline but in compliance with GLP (reliability score 2) an aerosol concentration of approximately 70 mg/m3 of A-174 caused laryngeal granulomas. Altering the percent concentration, pH, or method of aerosol generation did not significantly alter the production of laryngeal granulomas at 70 mg/m3. However, no granulomas were observed at a concentration of 15 mg/m3 (pH 5; 1% solution). At the site of laryngeal granulomas, spherical siliceous particles were observed ultrastructurally, these being present both extracellularly and within the cytoplasm of histiocytes and fibroblasts. The only sytemic effect was on body weight, which has resolved in all but only in the 50 mg/m3 (pH3, 5% solution). The NOAEC for laryngeal granulomas and general toxicity (reduced body weights) was 13.5 mg/m3.
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
11.04.1983 to 14.10.1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
some restrictions in the range of examinations performed.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
unknown
Principles of method if other than guideline:
This study was designed to determine the toxic effects, if any, in rats resulting from 14 weeks of repeated inhalation of 3-trimethoxysilylpropyl methacrylate aerosol.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hilltop Lab Animals, Inc., Scottdale, PA.
- Age at study initiation: No data
- Weight at study initiation: No data
- Fasting period before study: No
- Housing: Individually housed 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 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 11.04.1983 To: 14.10.1983
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: distilled water and acetic acid (pH 4)
Remarks on MMAD:
MMAD / GSD: 1.6 microns
Details on inhalation exposure:
Inhalation chambers were constructed from stainless steel with glass windows for animal observation. The volume was approximately 900 litres and the airflow was approximately 200-260 l/min (13-17 air changes per hour).

The test substance aerosol was generated using a Laskin nebuliser. A double aspirator tube was used to generate the 250 mg/m3 test substance target aerosol concentration. single aspirator tubes were used for generation of the 100 and 50 mg/m3 test substance aerosol target concentrations. A predetermined amount of test solution was added once or twice to each flask for the 6-hour exposure. Compressed air supplied to each nebuliser created a negative pressure such that the liquid solution was aspirated into the tube where it was then dispersed as a fine liquid aerosol. The liquid aerosol was then introduced into the top of the exposure chamber where it was diluted to the desired concentration and dispersed through the chamber by filtered chamber supply air.

Particle size distribution was measured daily in each chamber with a TSI Aerodynamic Particle Sizer.

The mean daily chamber temperature was 23-24oC for each chamber. The mean chamber relative humidity increased as the concentration increased. The mean for the control chamber was 45% with a daily mean range of 32-58%.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of test substance were analysed by gravimetric determination approximately once per hour from the test substance chambers and twice per day from the air control chamber.
Duration of treatment / exposure:
14 weeks exposure followed by 91 day recovery/observation period for 8 animals/sex/group.
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
244 mg/m³ air (analytical)
Dose / conc.:
100 mg/m³ air (analytical)
Dose / conc.:
50 mg/m³ air (analytical)
Dose / conc.:
250 mg/m³ air (nominal)
Dose / conc.:
100 mg/m³ air (nominal)
Dose / conc.:
50 mg/m³ air (nominal)
No. of animals per sex per dose:
18
Control animals:
yes, concurrent no treatment
Details on study design:
Groups of 18 male and 18 female F-344 rats were exposed to gamma-methacryloxypropyltrimethoxysllane (3-trimethoxysilylpropyl methacrylate) aerosol, six hours per day, five days per week, for 14 weeks. A control group was exposed to chamber air only.  
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed prior to, during and following each exposure. During the recovery period, all animals were observed at least once per day.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on the morning prior to initiation of the first exposure. Then weekly during the exposure and preceding sacrifice. Animals in the recovery phase were weighed weekly for the first month and biweekly thereafter, and just prior to sacrifice.

FOOD AND WATER CONSUMPTION: Ten rats (per sex/group) were housed in metabolism cages. Food and water consumption were measured for approximately 16 hours following 66 exposures for the female rats and following 67 exposures for the male rats. During the recovery period all animals were housed in metabolism cages and food/water consumption measured for approximately 18 hours following 88 postexposure days for the females and 89 postexposure days for the males.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to first exposure and at sacrifice.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Just prior to sacrifice
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: No
- How many animals: 10/sex/group after exposure period and 8/sex/group after recovery period.
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Just prior to sacrifice
- Animals fasted: No
- How many animals: 10/sex/group after exposure period and 8/sex/group after recovery period.
- Parameters checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: While in metabolism cages for food/water consumption.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Full necropsy and histopathologic examination of tissues were assessed to determined the toxicity of the test material.
Statistics:
The data for continuous, parametric variables were intercompared for the exposure and control groups by use of analysis of variance, Bartlett's homogeneity of variance and Duncan's multiple range.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
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:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
The mean (+/- standard deviation) of the measured exposure concentrations were 244 (+/- 9), 100 (+/- 6), and 50 (+/- 2) mg/m3. The mass median aerodynamic diameter was 1.6 micrometers with a geometric standard deviation in the range of 0.43 to 0.65 for the three concentrations. The mean (± standard deviation) methanol concentrations were 353 (+/- 70), 232 (+/- 40), and 130 (+/- 17) ppm for the three exposure groups.

Body weight gain was reduced by the fourth exposure at the highest concentration in males; following termination of exposures, the body weights returned to 
those seen for the controls.  No other in-life signs of adverse effects were noted in any exposure group.  There were no biologically significant alterations in  clinical pathology evaluations, including serum chemistry, protein electrophoresis, haematology or urinanalysis noted at either sacrifice interval.  
Statistically significant differences for liver weights of the male rats in the highest exposure group were noted.  There were no significant histologic findings for  the liver, but there were treatment-related upper respiratory tract lesions observed in all three exposure groups.  The most biologically significant lesion was granulomatous laryngitis with polyp formation, which did not resolve during the recovery period.  Also, there was hyalinization of the cytoplasm of olfactory mucosal cells, the significance of which is unknown, which did not resolve during the recovery period.  A minimum-effect or no-effect level with respect to the incidence of granulomas could not be determined from this study, since the histologic findings were similar from all three concentrations studied.
Dose descriptor:
LOAEC
Effect level:
50 mg/m³ air
Sex:
male/female
Basis for effect level:
other: Granulomatous laryngitis with polyp formation was noted at all dose levels, including the lowest dose level.
Critical effects observed:
not specified
Conclusions:
In a 14-week inhalation study (reliability score 2), conducted in a similar manner to OECD 413 and in compliance with GLP, a LOAEC was determined to 50 mg/m3 air in males and females. This was based on granulomatous laryngitis with polyp formation at all dose levels. A minimum-effect or no-effect level cannot be determined from this study, since the lowest exposure concentration produced a similar incidence of granulomatous laryngitis to that seen in the intermediate and highest concentration groups.The NOAEC for general systemic effects was 100 mg/m3.
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
01.11.1982 to 12.11.1982
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
The study did not meet current guideline requirements for repeated dose toxicity, primarily because it is only nine days in duration. It does, however, add weight of evidence for determining whether laryngeal granuloma formation is related to exposure duration.
Qualifier:
no guideline followed
Principles of method if other than guideline:
This study was designed to determine the toxic effects in rats resulting from nine days of repeated inhalation of 3-trimethoxysilylpropyl methacrylate hydrolysate aerosol.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hilltop Lab animals, Inc., Scottdale, PA.
- Age at study initiation: 54 days
- Weight at study initiation: Males: 175-176 g; Females: 130-132 g.
- Fasting period before study: None
- Housing: Individually housed in stainless steel wire-mesh cages in glove-box housing units.
- Diet: Ad libitum (except during exposure)
- Water: Ad libitum (except during exposure)
- Acclimation period: Three days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22.7 °C
- Humidity (%): 40-50 %
- Air changes (per hr): During exposure (12-17), no further information.
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 01.11.1982 To: 12.11.1982
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: Distilled water and acetic acid (pH 4); used 15% solution
Remarks on MMAD:
MMAD / GSD: The mass median aerodynamic diametres for the aerosols ranged between 1.04 and 1.4 micrometers with geometric standard deviations of 2.16 to 2.4.
Details on inhalation exposure:
Inhalation chambers were constructed from stainless steel with glass windows for animal observations. The volume was approximately 900 litres and the airflow between 180 and 250 litres/minute. Temperature and humidity readings were taken at least four times during each six-hour exposure. The aerosol was generated from a 15% (w/w) solution of 3-(trimethoxysilyl)propyl methacrylate in distilled water titrated to pH 4 with acetic acid, using a nebuliser. The aerosol from each nebuliser was conducted through glass tubes into a stainless steel mixer at the inlet to each chamber. The aerosol was mixed with additional air prior to entering the chambers.

Particle size analysis was determined with a cascade impactor. Glass fibre filters were used as the collection substrates. The filters were assayed for 3-(trimethoxysilyl)propyl methacrylate in the same way as the filters for the concentration analyses. The mass median aerodynamic diameters and geometric standard deviations for the different exposure groups were determined from log-probability plots of the cumulative percentage mass collected versus the effective cut-off diameters for the impactor stages.

Chamber temperature: 21.1-25 °C
Chamber humidity: 35-66 %
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The chambers were monitored approximately once each hour for 3-(trimethoxysilyl)propyl methacrylate aerosol and methanol vapour concentrations. Aerosol samples were collected from the centre of the chamber on pre-weighed glass fibre filters and dried to a constant weight before reweighing. Methanol concentrations were determined by gas chromatography.
Duration of treatment / exposure:
9 days (five days exposure, two days rest, four days exposure)
Frequency of treatment:
6 hours/day
Dose / conc.:
50 mg/m³ air
Dose / conc.:
100 mg/m³ air
Dose / conc.:
300 mg/m³ air
No. of animals per sex per dose:
Ten
Control animals:
yes, concurrent no treatment
Details on study design:
No satellite groups.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed prior to, during and following each exposure for signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on the morning preceding the first exposure. The animals were also weighed on the morning preceding the second, fifth, sixth and seventh exposures, and prior to sacrifice.

FOOD CONSUMPTION: Yes
- Food consumption was measured for approximately 16 hours following eight exposures for the female rats and following nine exposures for the male rats.

WATER CONSUMPTION: Yes
- Water consumption was measured for approximately 16 hours following eight exposures for the female rats and following nine exposures for the male rats.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the first exposure and at sacrifice.
- Dose groups that were examined: All

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At sacrifice
- Animals fasted: No
- How many animals: All animals
- Parameters checked in Table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: All animals approximately 16 hours following eight exposures for the female rats and following nine exposures for the male rats.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in Table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Gross examinations were performed and retained.
Histopathological examinations of tissues, and general observations of the animals were assessed to determine the toxicity of the test material (Table 2).
Selected organs were weighed at sacrifice: liver, heart, brain, lungs, kidneys and testes from all animals (absolute and percentage of body weight).

Addendum: After fourteen weeks of inhalation of gamma-methacryloxypropyl-trimethoxysilane (3-(trimethoxysilyl)propyl methacrylate) hydrolysate aerosols (Report #47-78), numerous rats in all exposure groups (i.e. 250, 100, and 50 mg/m3) were found to have developed granulomatous laryngitis. This lesion was present in a specific portion of the larynx. Because of the marked specificity in the anatomical location of this lesion, it was considered advisable to examine step sections of the available laryngeal tissues from all rats of the nine-day aerosol study in order to substantiate the presence or absence of this lesion.
Statistics:
Results for quantitative continuous variables (such as body weight changes) were intercompared among the three concentration groups and one control group by the following tests: analysis of variance, Bartlett's homogeneity of variance and Duncan's multiple range.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
No animals died during the exposures.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was a significant depression in body weight gain for the males and females in the high exposure concentration group, and a transient depression for the females at the intermediate concentration.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food and water consumption were slightly elevated for the male rats at the low and intermediate exposure concentrations.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were statistically significant elevations in serum albumin for the male rats at all exposure concentrations. This was accompanied by a significant decrease in total protein, resulting in a significant and dose-related decrease in the calculated total serum globulin. A similar effect was not observed in the female rats.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No statistically significant alterations were found in any of the urinalyses.
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Description (incidence and severity):
These results could indicate a possible immunosuppressive action in male rats; however, there were no other indications of disturbed immunological functions in these animals.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no histopathological lesions observed which were attributable to the 3-(trimethoxysilyl)propyl methacrylate exposures. Addendum result: The re-evaluatlon indicates that the development of granulomatous laryngitis is present after nine days of inhalation of 3-(trimethoxysilyl)propyl methacrylate aerosol even at the lowest concentration tested, 50 mg/m3.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
The mean (± standard deviation) exposure concentrations were 302 (+/- 16), 98.8 (+/- 10.7), and 49.6 (+/- 4.3) mg/m3. The mass median aerodynamic diameters ranged between 1.04 and 1.40 micrometres with geometric standard deviations between 2.16 and 2.40. The mean (+/- standard deviation) methanol concentrations were 210 (+/- 38), 185 (+/- 32), and 75 (+/- 23) for the three exposure groups.
Dose descriptor:
LOAEC
Effect level:
50 mg/m³ air
Sex:
male/female
Basis for effect level:
other: Development of granulomatous laryngitis is present after nine days of Inhalation of 3-trimethoxysilylpropyl methacrylate aerosol even at the lowest concentration tested, 50 mg/m3.
Critical effects observed:
not specified
Conclusions:
In a well documented nine-day aerosol inhalation study, not conducted according to any guideline but in compliance with GLP (reliability score 4), a LOAEC was concluded to be 50 mg/m3 air. This was based on minimal biological effects (decreased body weight gain in high and mid-dose group on days 5-12 although close to controls by day 12, dose-related decrease in total serum globulins at all concentrations) were seen as a result of exposure of Fisher 344 rats to 3-(trimethoxysilyl)propyl methacrylate aerosol at concentrations of 50, 100 and 300 mg/m3. Re-evaluation of the laryngeal tissues revealed that laryngeal granulomas were apparent after nine days exposure with all concentrations tested.
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12.04.1982 to 23.04.1982
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
The study did not meet current guideline requirements for repeated dose toxicity, primarily because it is only nine days in duration. It does, however, add weight of evidence for determining whether laryngeal granuloma formation is related to exposure duration.
Qualifier:
no guideline followed
Principles of method if other than guideline:
This study was designed to determine and evaluate the toxic effects in rats resulting from nine days of repeated inhalation of 3-trimethoxysilylpropyl methacrylate vapour.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI
- Age at study initiation: 76 days
- Weight at study initiation: Males: 246-268 g; Females: 154-158 g.
- Fasting period before study: None
- Housing: Two per cage in stainless steel wire-mesh cages
- Diet: Ad libitum (except during exposure)
- Water: Ad libitum (except during exposure)
- Acclimation period: Two days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 8ppm: 26-27; 20ppm: 31.5-34.5; 40ppm: 41-46.5 (during exposure)
- Humidity (%): Approximately 35 %
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 12.04.1982 To: 23.04.1982
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Inhalation chambers were constructed from stainless steel with glass windows for animal observation. The volume was approximately 4400 litres and the airflow was 1000 L/min (14 air changes per hour). Temperature and relative humidity gauges were placed inside chambers. The temperature and relative humidity within the chambers were recorded at least four times during each six-hour exposure.

The vapour was generated with S.I.D. vapour generators. Dehumidified air was blown through a heater and into th evaporator section of the generator. The liquid test substance pumped into the air outlet end of the rotating evaporator tube. The heated air passed countercurrent to the fluid flow and carried the vapour into the exposure chamber. The vapour concentration was controlled by varying the fluid feed rate, the evaporator temperature, or the airflow through the evaporator.

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations were analysed approximately once each hour by gas chromatography.
- Samples taken from breathing zone: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations were analysed approximately once each hour by gas chromatography.
Duration of treatment / exposure:
Nine days (five days exposure initially, two days rest, then further four days of exposure).
Frequency of treatment:
6 hours/day
Dose / conc.:
8 ppm (nominal)
Dose / conc.:
20 ppm (nominal)
Dose / conc.:
40 ppm (nominal)
Dose / conc.:
7.5 ppm (analytical)
Dose / conc.:
23 ppm (analytical)
Dose / conc.:
42 ppm (analytical)
No. of animals per sex per dose:
Ten
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: No data
- Rationale for selecting satellite groups: No satellite groups
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Prior to, during and following exposure.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: morning before first exposure, prior to second, fifth, sixth and seventh exposures and prior to sacrifice.

FOOD AND WATER CONSUMPTION:
- The rats were individually housed in metabolism cages. Food and water consumption was measured for approximately 16 hours following eight exposures for the female rats and following nine exposures for the male rats.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to first exposure and at sacrifice.
- Dose groups that were examined: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The day of sacrifice
- Animals fasted: No
- How many animals: All surviving rats
- Parameters evaluated not given.

URINALYSIS: Yes
- Time schedule for collection of urine: Following eight exposures while animals were in the metabolism cages.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters evaluated not given.
Sacrifice and pathology:
All surviving animals were sacrificed on the morning following the final exposure.
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Larynges of all animals. No other organs/tissues were examined.
ORGAN WEIGHTS: The liver, heart, brain, lungs, kidneys and testes from all animals were weighed at sacrifice (absolute and as a percentage of body weight).
Statistics:
Results for quantitative continuous variables (such as body weight changes) were intercompared among the three concentration groups and one control group by the following tests: analysis of variance, Bartlett's homogeneity of variance and Duncan's multiple range.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was an apparent increase in the mean body weight of the male animals in the high concentration exposure group when compared to the control animals. However, this is explained by a drop in the body weight of one control male animal at the ninth day, causing the mean body weight of the control group to decrease.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was increased 36 %, respectively, for males of the high exposure group, when compared to the values in the control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was increased 48%, respectively, for males of the high exposure group, when compared to the values in the control group.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant findings in the eye examinations.
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no adverse findings in the serum chemistry investigations.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The mean total urine volume for the male rats in the 40 ppm group was 44% greater than the mean control values.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were statistically significant decreases in the relative mean kidney weights for male rats in the highest and lowest exposure groups but not the intermediate exposure concentration group. The relative mean heart weight decreased in the males of the high exposure concentration group and the absolute heart weight decreased for the female rats in the high and intermediate concentration groups.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no histologic lesions encountered which could be attributed to the exposures.
Histopathological findings: neoplastic:
not examined
Details on results:
The mean (+/- standard deviation) exposure concentrations were 42 (2), 23 (2), 7.5 (1.4), and 0 (0) ppm.
Dose descriptor:
LOAEC
Effect level:
ca. 8 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
food consumption and compound intake
water consumption and compound intake
organ weights and organ / body weight ratios
Critical effects observed:
not specified
Conclusions:
In a well documented nine-day vapour inhalation study, not conducted according to any guideline but in compliance with GLP (reliability score 4), minimal biological effects (increased food and water consumption, decreased heart and kidney weights, increased urine output) were observed as a result of exposure of Fisher 344 rats to 3-(trimethoxysilyl)propyl methacrylate vapor at concentrations up to 42 ppm (approximately equivalent to 400 mg/m3). There were no laryngeal granulomas.
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
21.03.1988 to 15.04.1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted specifically to identify whether an aerosol of the test substance induced laryngeal granulomas. While this study does not meet standards of the current guidelines in terms of the diversity of the examinations conducted, it is a well conducted study that achieves its objective.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of this study was to confirm the development of laryngeal granulomas in rats following short-term inhalation exposures to aerosol of hydrolyzed and condensed 3-trimethoxysilylpropyl methacrylate. It was also the purpose of this study to further characterize the initial development of this lesion in the laboratory rodent.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Fischer 344/CDF
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY.
- Age at study initiation: 56 days
- Weight at study initiation: Approximately 188 g.
- Fasting period before study: None
- Housing: Two per cage 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: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-23.3 °C
- Humidity (%): 46-62 %
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 21.03.1988 To: 15.04.1988
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: 1% or 15% solution in distilled water and acetic acid (pH=5)
Remarks on MMAD:
MMAD / GSD: 2.09-2.54 microns
Details on inhalation exposure:
The inhalation chambers were constructed from stainless steel and glass windows for animal observations. The volume was approximately 1330 litres, and the airflow was approximately 300 litres/minutes (13.5 air changes/hour). Temperature and relative humidity measurements were recorded at least twelve times per exposure.

A 1 or 15% liquid solution of 3-(trimethoxysilyl)propyl methacrylate hydrolysate was metered from a piston pump into an atomiser. The atomiser was inserted into the top of the inhalation chamber turret where the liquid aerosol was dispersed throughout the chambers by filtered chamber supply air.

The particle size distribution was measured using a cascade impactor. These determinations were made three times per week throughout the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of 3-(trimethoxysilyl)propyl methacrylate hydrolysate were determined by gravimetric methods. Four samples were obtained each day. Two samples were obtained from each 3-(trimethoxysilyl)propyl methacrylate aerosol exposure chamber each day.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
14 mg/m³ air (analytical)
Remarks:
1% solution; equivalent concentration=gravimetric concentration/0.7217
Dose / conc.:
48.9 mg/m³ air (analytical)
Remarks:
1% solution; equivalent concentration=gravimetric concentration/0.7217
Dose / conc.:
70.2 mg/m³ air (analytical)
Remarks:
1% solution; equivalent concentration=gravimetric concentration/0.7217
Dose / conc.:
97 mg/m³ air (analytical)
Remarks:
1% solution; equivalent concentration=gravimetric concentration/0.7217
Dose / conc.:
100.9 mg/m³ air
Remarks:
15% solution; equivalent concentration=gravimetric concentration/0.7217
Dose / conc.:
15 mg/m³ air (nominal)
Remarks:
1% solution
Dose / conc.:
50 mg/m³ air (nominal)
Remarks:
1% solution
Dose / conc.:
70 mg/m³ air (nominal)
Remarks:
1% solution
Dose / conc.:
100 mg/m³ air (nominal)
Remarks:
1% solution
Dose / conc.:
100 mg/m³ air (nominal)
Remarks:
15% solution
No. of animals per sex per dose:
24 to 28 male rats/dose group
Control animals:
yes, concurrent no treatment
Details on study design:
No further information.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Preceding and following each exposure, observations were recorded for animals exhibiting overt clinical signs. The animals were examined for mortality at the weekends.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the time of body weight measurements and just preceding sacrifice, detailed observations were performed on all animals.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on the morning prior to the inhalation of their first exposure. Then weekly during the exposure period and just prior to sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Macroscopic observations, microscopic evaluation of the respiratory tract, and transmission electron microscopy of larynges were conducted.
Statistics:
The data for continuous, parametric variables were intercompared for the exposure and control groups by use of Levene's test for homogeneity of variances and by t-tests.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no effects on absolute body weight; slight depressions in body weight gain occurred but not in a concentration-related manner.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Goblet cell hyperplasia occurred in the nasal mucosa of all groups of rats exposed to aerosols of hydrolyzed and condensed 3-(trimethoxysilyl)propyl methacrylate; cytoplasmic hyalinization of the nasal mucosa was generally limited to rats exposed to aerosol generated from the 15% solution. Laryngeal lesions included squamous metaplasia in rats exposed to concentrations of 70 mg/m3 or greater, and granulomatous laryngitis in rats exposed to 100 mg/m3, regardless of whether the aerosol was generated from a 1% or a 15% solution. However, the granulomatous laryngitis lesions were larger in rats exposed to aerosol generated from a 15% solution and also occurred at a slightly higher frequency (100%) than those rats exposed to an aerosol generated from a 1% solution (85%). At the site of the larynx where the granulomas formed in the 100 mg/m3 groups, another lesion, squamous metaplasia, occurred at a high incidence (>=70%) in rats exposed to the aerosol at 70 mg/m3 or greater. There was also a low incidence (15-20%) of squamous metaplasia at the site in rats exposed to 15 or 50 mg/m3 3-(trimethoxysilyl)propyl methacrylate. The incidence and severity of this lesion increased with increasing exposure concentration.
Histopathological findings: neoplastic:
not examined
Dose descriptor:
LOAEC
Effect level:
15 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Goblet cell hyperplasia occurred in the nasal mucosa of all groups of rats exposed to aerosols of hydrolyzed and condensed 3-trimethoxysilylpropyl methacrylate
Dose descriptor:
NOAEC
Effect level:
15 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on observed laryngeal granulomas at concentrations of 70 mg/m3.
Critical effects observed:
not specified
Conclusions:
In a four-week (5 days/week) inhalation study, not conducted according to any guideline but in compliance with GLP (reliability score 2), rats were exposed to aerosol of hydrolyzed and condensed 3-(trimethoxysilyl)propyl methacrylate at concentrations of 100 mg/m3. Regardless of whether 1% or 15% solutions were used, granulomas were produced in rats. Aerosol generated from a 1% solution at atmosphere concentrations of 70 mg/m3 and below did not produce granulomas in rats.
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
22.07.1991 to 23.10.1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
restrictions in the range of examinations performed.
Qualifier:
equivalent or similar to
Guideline:
EPA OPP 82-4 (90-Day Inhalation Toxicity)
Deviations:
yes
Remarks:
Limited investigations as study had a specific purpose. Post-exposure period of 12 months.
Principles of method if other than guideline:
Purpose: To determine the fate of laryngeal granulomas during a 1-year recovery period (persist unchanged, regress and/or resolve, progress and become more severe, or result in carcinomas). Multiple toxicity parameters were assessed.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Indianapolis, IN.
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: Males: 142.2-199.8 grams; Female: 106.6-144.5 grams.
- Fasting period before study: No
- Housing: Individually in stainless steel, wire mesh cages.
- Diet: Ad libitum (except during exposure)
- Water: Ad libitum (except during exposure)
- Acclimation period: Three weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.7-26.1 °C
- Humidity (%): 40-70 %
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From: 22.07.1991 To: 23.10.1992
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
other: distilled water adjusted to pH 5
Remarks on MMAD:
MMAD / GSD: Mean concentrations of 0 and 102.0 (± 5.76) mg/m3 were measured; mean methanol concentrations of 0 and 36 (± 3.7) ppm were also measured. Mean mass median aerodynamic diameter for the exposed group was 1.7.
Details on inhalation exposure:
The inhalation chambers were stainless steel with glass windows for animal observation. The volume of each chamber was approximately 1330 litres and the airflow rate was approximately 300l/min (13 air changes/hour).

A 2% solution of H-MPTMS (pH5) was metered from a piston pump into an atomiser. The atomiser was inserted into the top of the inhalation chamber turret, where the liquid aerosol was dispersed throughout the chamber by filtered chamber supply air.

The particle size distribution was measured using a TSI Aerodynamic Particle Sizer in conjunction with a 20:1 dilutor. These determinations were made each exposure day for the chambers.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
3-(Trimethoxysilyl)propyl methacrylate condensate was measured gravimetrically. Four samples from the exposure chamber were obtained during each 6-hour exposure.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
6 hours/day, 5 days/week for 13 weeks and for 2 days during fourteenth week
Dose / conc.:
105 mg/m³ air (analytical)
Dose / conc.:
100 mg/m³ air (nominal)
No. of animals per sex per dose:
Total of 40 animals (20 male and 20 female): 8/sex terminated day after last exposure
8/sex terminated after 1, 4, 8 or 12 month recovery periods
Control animals:
yes, concurrent no treatment
Details on study design:
One group, containing 40 male and female Fischer 344 rats, was exposed to aerosolized H-MPTMS (gamma-methacryloxypropyltrimethoxysilane [3-(trimethoxysilyl)propyl methacrylate] hydrolysate) for 6 hours/day, 5 days/week for 13 weeks and for 2 days during the fourteenth week. The concentration was 100 mg/m3. A control group of the same size was exposed to filtered air alone. Eight animals/sex/group were sacrificed at the end of the exposure period and after 1, 4, 8, and 12 month recovery periods.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At the time of body weight measurements and just preceding sacrifice.

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to first exposure, and then weekly for the duration of the study.

FOOD AND WATER CONSUMPTION: First four weeks, then only if body weight gain was statistically different from the control groups.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Following the last exposure and at the end of the recovery months 1, 4, 8 and 12.
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes
- How many animals: 8 rats/sex/group following last exposure and at the end of recovery months 1 and 4. From 8 males/group, 8 female control rats and 7 female exposed rats at the end of recovery months 8 and 12.
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Following the last exposure and at the end of the recovery months 1, 4, 8 and 12.
- Animals fasted: Yes
- How many animals: 8 rats/sex/group following last exposure and at the end of recovery months 1 and 4. From 8 males/group, 8 female control rats and 7 female exposed rats at the end of recovery months 8 and 12.
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Eight animals/sex/group were sacrificed at the end of the exposure period and after 1, 4, 8, and 12 month recovery periods.
Gross and microscopic pathology were assessed.
Statistics:
The data for quantitative continuous variables were intercompared for the H-MPTMS exposure and the control group by use of the t-test.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only exposure-related clinical sign was periocular encrustation, which was probably due to mild irritation.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females in the 100 mg/m3 group died or were sacrificed moribund during the study (Study Days 304 and 319). These deaths were not related to the exposure, since they occurred during the recovery period and the histopathologic data did not suggest an exposure-related death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreases in body weight and body weight gain for males in the 100 mg/m3 group occurred only during the exposure period.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and relative lung weight increases at the end of the exposure period were related to the exposure.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mild to marked laryngeal granulomas, mild to moderate hyaline epithelial inclusions within the epithelia lining the sections of the nasal cavity, and minimum to mild alveolar histiocytosis were observed in the animals at the end of exposure and also at the different time points during the recovery period. Of the animals in the 100 mg/m3 group, 94% had laryngeal granulomas, 100% had hyaline epithelial inclusions, and 96% had alveolar histiocytosis. Mild to marked squamous metaplasia of the mucosal epithelium overlying the granulomas was also present at the Study Week 14 termination; however, except for 1 male and 2 females, it was not observed during the recovery period. Minimum to moderate granulomatous lymphadenitis within the mediastinal lymph nodes appeared in almost 50% of the animals sacrificed during the recovery period. The hyaline epithelial inclusions within the nasal epithelia, granulomatous laryngitis, and alveolar histiocytosis did not appear to change in frequency of occurrence or severity during the recovery period. No evidence of neoplastic lesions was observed for H-MPTMS exposed animals.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Dose descriptor:
LOAEC
Effect level:
ca. 100 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Local effects on the respiratory tract
Remarks on result:
other: Local effects
Dose descriptor:
LOAEC
Effect level:
ca. 100 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Systemic effects
Critical effects observed:
not specified
Conclusions:
In a 90-day inhalation study with up to one year recovery period, conducted according to EPA OPP 82-4 and in compliance with GLP (reliability score 2), the NOAEC for these local effects was <100 mg/m3, and the NOAEC for general systemic toxicity was <100 mg/m3 based on reduced body weight/gain. Additionally, exposure of rats to 100 mg/m3 (2%, pH 5) H-MPTMS for 13 weeks, produced mild to marked laryngeal granulomas, mild to moderate hyaline epithelial inclusions and minimum to mild alveolar histiocytosis. The hyaline epithelial inclusions within the nasal epithelia, granulomatous laryngitis and alveolar histiocytosis did not appear to change in frequency of occurrence or severity during the recovery period. No evidence of laryngeal carcinomas was noted.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
15 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

All of the exposure atmosphere concentrations described in this discussion (as stated in the study reports) relate to corrected concentrations of parent test material, calculated according to:

 

Equivalent MPTMS Concentration = Measured Concentration / 0.7217

 

where the resulting silicone formed on the dried filter paper used for gravimetric analysis represents approximately 72.17% of the test atmosphere.

For all of the available aerosol inhalation studies, the test atmosphere was generated by first preparing an aqueous solution of 3-trimethoxysilylpropyl methacrylate, adjusting to pH 4 or 5 (or in some cases pH3) and stirring for a period of 30 minutes or until solutions were clear, prior to aerosolisation. The starting concentration of parent material was generally 1, 2, 5 or 15% but since the parent test substance is hydrolytically unstable (measured half-life 1.87 hours at pH 7, expected to be faster at pH 3, 4 or 5), a significant amount of hydrolysis would have occurred prior to exposure, generating 3-trimethoxysilylpropyl methacrylate and methanol. Furthermore, at the relatively high aqueous concentrations of the solutions used to generate the aerosol atmospheres (approximately 10, 20 or 150 g/l), significant condensation or polymerisation of the silanol hydrolysis product would have taken place. Hydrolysis and condensation are discussed further in Sections 1.3 and 4.1. Gravimetric analysis of the chamber atmosphere confirmed that the test animals were exposed to approximately 72% silicone products of condensation; the remaining 28% would therefore have consisted of parent substance.  

 

Analysis of the methanol concentration in the test chambers (e.g. in BRRC, 1994) does not allow the relative proportions of parent and hydrolysis product to be determined. The measured methanol concentration in the 100 mg/m3 test chamber exceeded the maximum theoretical concentration that could be generated from 100 mg/m3 parent substance, since additional methanol would also have been generated during the stirring period. Methanol concentrations levelled out during the exposure period, indicating that further hydrolysis was not occurring.

The most significant effect observed in the aerosol inhalation studies was the formation of laryngeal granulomas, which is considered to be a local effect on the respiratory system. It is not possible to determine definitively if the effects seen in these studies were caused by exposure to condensate, hydrolysis product or parent material, or a combination of these, although the study author of BRRC, 1989b postulates that granuloma formation is initiated by deposition and polymerisation of liquid test material. The formation of laryngeal granulomas is therefore apparently linked specifically to aerosol exposure, which is supported by the absence of these effects in a 9-day vapour inhalation study. Several observations can be made from the available aerosol studies:

1) Solution concentrations of 15% caused larger granulomas than 1% with the same aerosol concentration;

2) Granulomas have been observed after 2 days exposure (BRRC, 1989b), so it can be assumed that the effect is not exposure duration dependent; and

3) Granulomas produced with a 90-day inhalation of 100 mg/m3 (2% solution) did not change/reverse/progress after a 1-year post-exposure period, i.e. no change in frequency of occurrence or severity during the recovery period, and there was no evidence of laryngeal carcinomas (BRRC, 1994).

Due to the choice of dose levels and examinations performed, a NOAEC for effects on the larynx was not determined in all of the available studies. In addition, laryngeal granuloma formation was not observed at any concentration up to 50 mg/m3 in one of the 14-week studies (BRRC, 1989a). Clear evidence of effects at this level were seen in another 14 -week study (BRRC, 1984). An overall NOAEC of 15 mg/m3 was established for effects on the larynx in a 4-week study (BRRC, 1986), also supported by the absence of granulomas in the 90-day study (BRRC, 1989a), and since there was no apparent duration dependency seen between 9-day, 4-week and 14-week study duration, this result can be considered as a subchronic NOAEC value.

End use of 3-trimethoxysilylpropyl methacrylate relates typically to aqueous-based products containing 1-5% parent substance in the formulation. Partial curing of the parent material prior to use is to be expected, and thus the mixture of parent, hydrolysis product and condensate tested in the available aerosol inhalation studies is considered to be representative of the exposure atmosphere experienced by workers, professionals or consumers in situations where aerosol formation is possible. Use of the NOAEC established for A-174 hydrolysate mixture is therefore suitable for risk characterisation purposes in these scenarios.

For applications where there is no potential for aerosol formation, the laryngeal granuloma effect is not relevant. Laryngeal granuloma formation was not observed in a vapour inhalation study with parent 3-trimethoxysilylpropyl methacrylate, and the only effects seen at the maximum attainable concentration of 42 ppm (approximately 426 mg/m3) were minor organ weight and relative organ weight changes (Bushy Run Research Center, 1982). It is therefore necessary to determine a separate NOAEC for systemic toxicity. There were very few clinical signs of toxicity in any of the available repeated dose toxicity studies. The only consistently significant finding was a reduction in body weight gains. The overall NOAEC for this effect appears to be between 50 and 100 mg/m3 for a 1 or 2% solution. However, in the absence of granuloma formation the effects on body weight were not observed (Bushy Run Research Centre, 1989a), thus there might be a link between these two adverse effects in the other studies. There was no apparent duration-dependent increase in severity of clinical effects. As a worst-case interpretation, it is assumed that the body weight effects are not linked to granuloma formation and the lowest available systemic NOAEC of 50 mg/m3 is used as a starting point for risk characterisation purposes.

Justification for classification or non-classification

Based on the available data 3-trimethoxysilylpropyl methacrylate does not require classification for repeated dose toxicity according to Regulation (EC) No 1272/2008.