Registration Dossier

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Administrative data

Description of key information

The key study for the inhalation route is a 90-day inhalation test (Charles River, 2017) conducted to OECD TG 413 and in compliance with GLP in which Sprague Dawley rats were exposed to an aerosol of N-[3-(trimethoxysilyl)propyl] ethylenediamine at concentration of 5, 15 or 45 mg/m3, 5 days per week. The NOAEC for local effects was 15 mg/m3, based on effects on the respiratory tract. There were no adverse systemic effects.

The key study for the oral route is a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test conducted to OECD TG 422 and in compliance with GLP (Dow Corning Corporation, 2002a). The NOAEL from this study was at least 500 mg/kg bw/day as no adverse effects were observed at any dose tested.

A nine-day dermal toxicity study (Bushy Run Research Center, 1993) supported the finding of low repeated dose systemic toxicity. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24.09.2001 to 06.09.2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Modified OECD 422 and USEPA Health Effects test guideline OPPTS 870.3650 (2000)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc.
- Age at study initiation: At least 8 weeks
- Weight at study initiation: Males: 253-301 g; Females: 181-229 g
- Fasting period before study: None
- Housing: Individually housed in suspended wire-mesh cages. Pregnant females were moved into shoebox cages no later than three days prior to their expected delivery date
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01.10.2001 To: 11.04.2002
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing formulations were prepared daily by adding an appropriate amount of test substance to a measured amount of vehicle (dried and de-acidified) under nitrogen atmosphere.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was dried and de-acidified to removal residual water before use as test substance hydrolyses in contact with moisture.
- Lot/batch no. (if required): 070K0127
- Purity: 100 %
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability of the test substance in the vehicle were verified by gas chromatopgraphy with a flame ionisation detection (FID). Concentration verification was conducted on a weekly basis.
Duration of treatment / exposure:
Males: 28 days (beginning 2 weeks prior to mating)
Toxicity phase female: 29 days (beginning 2 week prior to mating)
Reproductive phase females: 39-44 days (2 weeks prior to mating, throughout mating and pregnancy until day 3 postpartum
Frequency of treatment:
Daily (seven days/week)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10/sex/dose (with an females used for toxicity and reproductive phase groups only)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on a range-finding study (Dow Corning Corporation, 2002a; study number 9618)
- Rationale for selecting satellite groups: Toxicity phase females used to assess effects in females without the influence of pregnancy, so help investigate whether pregnant animals might be more susceptibe to toxicity.
- Post-exposure recovery period in satellite groups: No post-exposure recovery group
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for morbidity, moribundity and mortality. General clinical observations were made at least once per day, approximately one hour after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the first dosing and weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly beginning approximately one week prior to test substance administration, on the first day of dosing and just prior to scheduled necropsy.

FOOD CONSUMPTION: Individual food consumption was recorded weekly for female animals in the toxicity group for four weeks. Individual food consumption for the male animals and the reproductive group females were recorded during the first two weeks of treatment. Food consumption was not measured during the cohabitation period. Food consumption was measured for the reproductive group females throughout gestation until terminal sacrifice.

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: Yes
- Time schedule for collection of blood: On the day of scheduled sacrifice
- Anaesthetic used for blood collection: Yes, ketamine HCl/Xylazine
- Animals fasted: Yes, overnight
- How many animals: All male and toxicity group females
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled sacrifice
- Animals fasted: Yes
- How many animals: All male and toxicity group females
- Parameters checked in table 1 were examined.

URINALYSIS: No

FUNCTIONAL OBSERVATIONAL BATTERY (FOB): Yes
- Time schedule for examinations: Prior to the start of dosing and during the fourth week of the treatment. The assessments were conducted following the daily dose administration.
- Dose groups that were examined: All male and toxicity group females.
- Battery of functions tested: cageside observations, hand-held observations, open field observations, categorical observations, measurements/counts, motor activity.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 2). Males and toxicity group females were subjected to a complete gross necropsy that included examination of the cranial, thoracic and abdominal cavities and their contents.
HISTOPATHOLOGY: Yes (see table 2). A thorough histopathological examination was performed on the tissues collected for the control and high dose male and female toxicity group animals.
Other examinations:
See Section 7.8 for examinations relating to reproductive and developmental toxicity.
Statistics:
All data analysis was carried out using SAS. In all comparisons, the family wise error rate was held at 5% (alpha=0.05). Prior to this analysis, an exploratory analysis was carried out on all variables tested. Bartlett's test was used to check for heterogeneity of variances and a Kolmogorov-smirnov test was used to test normality of the data. Parametric data was then tested using one-way Analysis of Variance (ANOVA) followed by Dunnett's Test (if significant). Non-parametric data were tested by Kruskal Wallis Test followed by Wilcoxon (if significant). For variables that had multiple measurements across time Repeated Measurement ANOVA was used to analyse the data. Categorical data were tested for equal proportions using the Fisher's Exact Test.

Statistically significant probabilities are reported at either the p≤0.05 or p≤0.01 levels.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: One male in the 125 mg/kg bw/day dose group was found dead on study day 17 due to renal disease unrelated to treatment. There were two females in the reproductive groups that died due to dosing errors. All other animals survived to scheduled sacrifice. Clear perioral soiling was slightly more common in the high dose group. In the high dose groups there were increased nasal sounds, laboured breathing and/or soft squeaky vocalisation for one male and four toxicity andfive reproductive group females. At 125 mg/kg bw/day, one toxicity group and one reproductive group female had increased nasal sounds. At 25 mg/kg bw/day, one toxicity group female exhibited soft squeaky vocalisation for three days of the study. The incidence of nasal sounds/squeaky vocalisation was noted in some animals and several times in others to a maximum of 18 days during the study. These findings were not observed in control rats.

BODY WEIGHT AND WEIGHT GAIN: Mean body weights in males and females in the 25, 125 and 500 mg/kg bw/day groups were comparable to the control group values throughout the study; no statistically significant differences were noted. Reductions (not statistically significant) in weekly body weight gain were observed in the 25 and 125 mg/kg bw/day group males during week 4. These reductions were not considered to be test substance-related since the difference did not occur in a dose-dependent manner and no reductions were noted in female body weight gains during this time period.

FOOD CONSUMPTION: There were no statistically significant differences in food consumption in any group.

HAEMATOLOGY: There were no treatment-related effects on haematology parameters. However, in the toxicity group females there was a statistically-significant increase in platelet counts compared to controls. The counts for the treated groups were within published historical control ranges, whereas controls in the present study were somewhat below those ranges. No biological/toxicological significance is attributed to treatment.

CLINICAL CHEMISTRY: There were no treatment-related effects on clinical chemistry. However, in females, there was a statistically significant decrease in the chloride value (1.9%) in the high dose group, and a slight decrease (1.4%) in sodium in the middle and high dose groups. There was no dose-response. Sodium values for all female groups, including controls, were within or slightly below published historical control ranges. Chloride values for all groups were slightly above published control ranges. There were no associated clinical or morphological findings, so the findings were not thought to be biologically or toxicologically significant.

FOB: Cage side observations: There were no treatment-related changes noted in unusual body movements, abnormal behaviour, posture or resistance to removal.
Handling observations: Palpebral closure, lacrimation, pupil size and reactivity, salivation, muscle tone, extensor thrust response and reactivity to handling were not affected by the treatment.
Open Field Observations: No differences were apparent between the control and treated groups in the open field observations.
Categorical Observations: No differences between control and treated groups when skin or hair coat, behaviour, respiration, muscle movements, eyes, urine or faeces, soiling, posture or general abnormalities were evaluated.
Measurements/counts: There was no effect on rectal temperature, hindlimb grip strength or landing foot splay assessments.
Motor activity: There were no effects on motor activity.

ORGAN WEIGHTS: There were no treatment-related effects on organ weights. There was no dose-response associated with a statistically-significant small increase in relative prostate weight in the 25 mg/kg bw/day group.

GROSS PATHOLOGY: There were no findings attributable to the test substance.

HISTOPATHOLOGY: There were no treatment-related findings.
Dose descriptor:
NOAEL
Effect level:
>= 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse effects observed.
Critical effects observed:
not specified
Conclusions:
In an oral gavage study conducted to OECD 422 and to GLP (reliability score 1) the NOAEL for repeated dose toxicity was at least 500 mg/kg bw/day for N-(3-(trimethoxysilyl)propyl)ethylenediamine in rats.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 February 2017 to December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
ECHA decision number: TPE-D-2114300905-56-01/F
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
not specified
Remarks:
There are no deviations from the test guideline that affect the integrity of the study
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a room with controls set to maintain 18°C to 24°C and under dry nitrogen gas, keep away from moisture with dessicant.
- Stability under test conditions: The test article is considered to be stable.
- Solubility and stability of the test substance in the solvent/vehicle: Filtered air was used as vehicle.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not applicable
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 227 to 307 g (males)/149 to 192 g (females)
- Fasting period before study: Food and water were not available during the exposure period.
- Housing: All animals were housed in groups of 2 to 4 per cage following receipt in clean, solid bottom cages with bedding material.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 meal was available ad libitum
- Water: Reverse osmosis-treated water was available ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY: Filters servicing the automatic watering system were changed regularly according to SOPs. The municipal water supplying the laboratory was analyzed for contaminants according to SOPs. SOPs provide specifications for acceptable levels of heavy metals and pesticides that are reasonably expected to be present in the diet without interfering with the purpose or conduct of the study. No contaminants were reasonably expected to be present in water or diet that would interfere with the objectives of the study, therefore, no testing was conducted as part of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 20°C and 26°C
- Humidity (%): 15-30%
- Air changes (per hr): 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
> 1.3 - <= 2.1 µm
Geometric standard deviation (GSD):
2.47
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel, conventional nose-only exposure systems
- Method of holding animals in test chamber: nose-only exposure restraint tubes
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols: Liquid droplet aerosol atmospheres of the test substance were generated using single-jet Collision nebulizers. Test substance exposure atmospheres were generated by mixing the aerosolized test substance with nitrogen and/or breathing-quality dry, compressed air in the appropriate ratio to achieve the target concentrations. Specific exposure atmosphere generation equipment, operating parameters and methods were defined during method development.
- Temperature, humidity, pressure in air chamber: Average daily temperature and relative humidity of the exposure atmospheres were 23 ± 3ºC and 15%-30%, respectively. Temperature and relative humidity were monitored continuously and recorded at approximately 60-minute intervals during each exposure.
- Air flow rate: Airflow rate through the system was set based on output from the generation equipment and the dilution airflow to provide sufficient volumes for the number of animals to be exposed and for test atmosphere sampling.
- Air change rate: 10 per hour
- Method of particle size determination: Aerosol particle size measurements were conducted during method development as needed and at least twice weekly during the 13-week exposure phase for all test substance exposure groups. Aerosol particle size measurements were conducted using a 7-stage stainless steel cascade impactor. The aerosol size was expressed in terms of the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD). The target range for MMAD is 1.0 to 3.0 microns and GSD is 1.5 to 3.5. The mean aerosol particle size exposure system 2, 3 and 4 were 2.1, 1.9 and 1.3 microns, respectively. Mean GSD for exposure system 2, 3 and 4 were 2.35, 2.47 and 2.15, respectively.
- Treatment of exhaust air: exhaust passed through a Solberg canister filter prior to entering the facility exhaust system, which consists of redundant exhaust blowers preceded by activated charcoal- and HEPA-filtration units.

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: filtered air
- Composition of vehicle: not applicable
- Type and concentration of dispersant aid (if powder): not applicable
- Concentration of test material in vehicle: not applicable
- Lot/batch no. of vehicle (if required): not applicable
- Purity of vehicle: not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were collected using pre-weighed filters held in a filter holder and placed in the animal breathing zone of an exposure system. A volume of the test atmosphere is pulled through the filter. Following sample collection, the sample is reweighed. The exposure concentration (mg/m3) were calculated by dividing the gravimetrically determined mass of test substance aerosol by the sample volume. The mass was determined by subtracting the initial filter weight from the final weight of the post-sample filter. Sample volume was calculated by multiplying the sample flow rate by the length of the sampling period. Concentrations were not be adjusted for test substance purity. For the test substance exposure systems, samples were collected daily at appropriate intervals based on the target exposure concentrations. For the control group (Group 1), gravimetric samples were collected at least twice weekly during the 13-week exposure period.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days a week for 6 hours a day
Dose / conc.:
5 mg/m³ air (nominal)
Dose / conc.:
15 mg/m³ air (nominal)
Dose / conc.:
45 mg/m³ air (nominal)
Dose / conc.:
5.1 mg/m³ air (analytical)
Dose / conc.:
14.9 mg/m³ air (analytical)
Dose / conc.:
44 mg/m³ air (analytical)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Exposure concentrations were selected to cover a range extending from an anticipated no observed-adverse-effect-concentration (NOAEC) to a concentration in which toxic effects are seen in the highest exposure concentration. Specific concentrations were based on the results of a 28-day range-finding study in which microscopic findings were noted in the lungs, trachea larynx and nasal levels at a concentration of 100 mg/m3.
- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: The animals scheduled for the recovery evaluations will be subjected to necropsy following a 4-week non-exposure period (minimum of 27 days).
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily, once in the morning and once in the afternoon. Moribund animals were euthanized and necropsied as soon as possible. Animals found dead were necropsied as soon as possible to ensure that tissues will not be lost due to autolysis.
- Cage side observations checked in table were included: mortality, abnormalities, and signs of pain and distress

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to treatment, weekly during the study period and on the day of necropsy

BODY WEIGHT: Yes
- Time schedule: Prior to treatment, twice weekly during the study period and on the day of necropsy

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: Once during acclimation prior to randomization and all surviving animals near the end of the exposure period or near the end of the recovery period.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.1] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: not conducted, as not triggered by clinical observations

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No 2)
HISTOPATHOLOGY: Yes (see table No 2)
Statistics:
All analyses were two-tailed for significance levels of 5% and 1%. All means were presented with standard deviations. All statistical tests were performed by a computer with appropriate programming as referenced below.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical observations attributable to treatment in the low and mid-dose groups, or in the recovery groups. There were no clinical signs noted 0-1 hour following exposure in any group.

The only group with clinical signs of toxicity due to treatment was the high dose (45 mg/m3) group during the treatment period. Males and females of this group had multiple instances of red, brown, and/or yellow material on various body surfaces and clear discharge around the eyes. The staining was judged by the study author to be indicative of test substance-related stress or general ill health. This finding was observed prior to dosing and on non-exposure days during the treatment period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male in the 15 mg/m3 group was euthanized in extremis on Study Day 61 due to a clinical observation of red penile discharge. At necropsy, this male was noted with gross observations of dilated pelvis, distended bladder, distended ureters, and red fluid in the bladder and ureters. This death was not considered to be test substance-related. In addition, a single male in the control group was found dead on Study Day 65; there were no remarkable clinical observations or gross observations noted for this animal. All other animals survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related effects on body weight or body weight gain in the low and mid-dose groups.

Test substance-related effects on body weights were noted in the 45 mg/m3 group males and females. Lower mean body weights and cumulative body weight gains were noted beginning as early as Study Week 1 in males and females, which continued until the primary necropsy (Study Week 13); the differences were generally significant compared to the control group. On Study Week 13 (the day of the primary necropsy), mean body weights in the 45 mg/m3 males and females were 9.7% and 6.4% lower, respectively, than the mean control group body weights. In addition, body weight gains in the 45 mg/m3 group males and females were comparable or higher than the control group values during the recovery period; while mean body weights in the males remained lower (not statistically significant) than the control group. Therefore, the animals recovered once exposure stopped, but males were slightly slower to recover than females.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The only treatment-related effect on organ weights occurred in the high dose group and was limited to the lungs. There were no adverse findings in the low and mid-dose groups.

At the end of the treatment period: Lung weights (absolute and relative to body and brain weights) tended to be higher with exposure concentration across all groups with statistical significance achieved only at 45 mg/m3. For the males, all group mean and individual animal absolute lung weights, as examples, were within historical control reference ranges. In contrast for the 45 mg/m3 group females, mean absolute and both mean relative weights exceeded their historical control reference ranges and, as an example, 6 individual animal absolute lung weights also exceeded the historical control reference range. The most likely microscopic correlations for the higher lung weights were the findings affecting the bronchioles (epithelial hyperplasia, mixed cell infiltrates and luminal debris). Since none of these findings were seen in either sex at exposure concentrations < 45 mg/m3, the 8% to 10% higher lung weights seen in the 15 mg/m3 group males (and lesser magnitudes of change seen at lower exposure concentrations in both sexes), which were reversible, were considered incidental and unrelated to treatment.

At the end of the recovery period: treatment-related and statistically significant higher lung weights (absolute and/or relative to body and brain weights) persisted in both sexes at exposure concentrations of 45 mg/m3. Although mean absolute lung weight was not statistically significant in the 45 mg/m3 group males, the approximately 10% increase was considered sufficient to be considered an effect of test substance exposure. Peribronchiolar chronic inflammation was considered the microscopic correlate for the persistent high lung weights in this group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic findings were observed in the high dose group only at the end of the treatment period and were limited to the lungs. There were no adverse findings in any of the groups at the end of the recovery period.

Treatment-related findings of not fully collapsed (3/10 males and 8/10 females) and pale lungs (1/10 males and 2/10 females) were observed exclusively in the 45 mg/m3 group at the primary necropsy; luminal debris in the bronchioles and peribronchiolar inflammatory cell infiltrates appeared to be the microscopic correlates for lungs not fully collapsed while a correlation was not observed for pale lungs. At the Study Week 17 recovery necropsy, not fully collapsed lungs
was seen in 2 males and 1 female from the 45 mg/m3 group but also 1 male each from the control and 5 mg/m3 groups. Therefore at the recovery necropsy, lungs not fully collapsed was not conclusively associated with exposure to the test substance aerosol.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Tables 3 to 6.
Treatment-related microscopic findings were limited to the respiratory tract. These findings were observed in all groups at the end of the treatment period, but they were only observed in the high dose group at the end of the recovery period.

At the end of the treatment period: In the nasal cavity treatment-related epithelial (transitional, respiratory, or olfactory) degeneration/regeneration was characterized by loss of normal tissue architecture, most commonly by cell loss and replacement by a single layer of attenuated epithelium; these changes were typically bilateral. Specific to the respiratory epithelium was loss of cilia and reduction/loss of goblet cells. The sites of respiratory epithelial degeneration/regeneration were most commonly the dorsal aspect of the nasal septum and lining the dorsal meatus, including nasal turbinate(s). Olfactory epithelial degeneration/regeneration was limited to 1 female in the 45 mg/m3 group and was diffuse within the dorsal meatus of nasal level III while focal and unilateral in the posterior nasal levels. Transitional epithelial degeneration/regeneration was most commonly limited to the tissues lining the lateral meatus (nasal turbinates and lateral wall). Squamous cell metaplasia and mixed cell inflammation were usually associated with areas of epithelial degeneration/regeneration. Squamous cell metaplasia represented a change from the normal epithelium lining a region to an attenuated stratified and keratinized squamous epithelium. Mixed cell inflammation was characterized by a mononuclear cell (lymphocytes, plasma cells and macrophages) population admixed with variable numbers of neutrophils. In contrast to epithelial degeneration/regeneration seen most commonly in the females, transitional and respiratory epithelial hyperplasia was the most common epithelial change seen in the males exposed to test substance aerosol; affected regions were comparable to those seen with degeneration/regeneration in the females. Several of these findings (transitional epithelial hyperplasia, mixed cell inflammation, and squamous cell metaplasia) at nasal level II were seen in one or both sexes at exposure concentrations of 5, 15, and 45 mg/m3.

In the larynx, trachea, and bronchi (recorded under lungs) treatment-related respiratory epithelial degeneration/regeneration and mixed cell inflammation were comparable to that seen in the nasal cavity. It was not uncommon in control group animals to see respiratory epithelial loss and/or attenuation at these sites. Loss was clearly an artifact of tissue collection/processing while the cause of the thinned epithelium was less certain. However, respiratory epithelial degeneration/regeneration was diagnosed only when there was single cell necrosis, mitotic figure(s) and/or rare neutrophils (sub- or intra-epithelial) associated with the attenuated epithelium. Disruption of the normal epithelial architecture with loss of cilia was also considered degeneration/regeneration. In the larynx, squamous metaplasia was similar to that
seen in the nasal cavity except that it was more commonly seen as a change from the normal non-keratinized stratified epithelium to an attenuated and keratinized stratified squamous epithelium. In addition to mixed cell inflammation in the larynx, chronic active inflammation was also an effect of exposure to the test substance, particularly in the 45 mg/m3 group females, and the epiglottis was a common site of this inflammation. Chronic active inflammation was similar to mixed cell inflammation except in the former, fibroblasts/fibroplasia were present generally indicating inflammation of longer duration. Several of these findings (respiratory epithelial
degeneration of bronchi and mixed cell inflammation of the larynx and bronchi) were seen in one or both sexes at exposure concentrations of 5, 15, and 45 mg/m3.

At the end of the recovery period: In the nasal cavity (levels II and III) most previous effects of exposure persisted in one or both sexes from the 45 mg/m3 group with
incidences and severities that were generally lower than those seen at the primary necropsy. A new effect, and evidence of chronic irritation, was mucous cell hyperplasia of the nasal septum at nasal level II in the males. Due to the low incidence and minimal to mild severity and resolution during the 4-week recovery period, findings in the nasal cavity were considered to be nonadverse at 5 and 15 mg/m3.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
In the lungs at the primary necropsy, treatment-related effects were centered on the bronchi and/or bronchioles. Changes in the bronchi were comparable to and considered an extension of the changes seen in the trachea. The most prominent change was noted at the periphery of the lungs at the level of the terminal bronchioles. In control group animals, terminal bronchioles rarely had any associated inflammatory or mononuclear cell infiltrates and the epithelial lining
was an admixture of ciliated respiratory epithelial cells and cells with distinct apical blebbing (club/Clara cells). Exposure-related effects included variably dense perivascular and/or peribronchiolar mixed cell infiltrates characterized by mononuclear cells with generally fewer eosinophils and/or neutrophils and in some cases the suggestion of increased fibrosis/fibroplasia (Masson’s stain suggested this was true in a 45 mg/m3 group female evaluated). There was loss of the normal epithelium lining the terminal bronchioles with replacement by a prominent respiratory epithelium with a robustly ciliated epithelium; club cells were generally absent (mild respiratory epithelial hyperplasia). With minimal respiratory epithelial hyperplasia, club cells were occasionally present and/or there was epithelial alteration comparable to the respiratory epithelial degeneration/regeneration noted in the bronchi. Some animals had bronchiolar luminal debris (exudate) consisting of a mucinous material admixed with foamy alveolar macrophages and less commonly neutrophils. It was this exudate in combination with the peribronchiolar cellular infiltrates and respiratory epithelial hyperplasia that were the most likely microscopic correlations for the lungs noted at necropsy as not fully collapsed. Other than the changes affecting the bronchi, discussed above, all treatment-related effects to the lower airways were limited to the 45 mg/m3 group.

In the lungs at the recovery necropsy, some of the previous effects of exposure persisted in one or both sexes from the 45 mg/m3 group with incidences and severities that were generally lower than those seen at the primary necropsy. A new effect, and evidence of chronic irritation, was the shift from mixed cell peribronchiolar infiltrates to chronic inflammation of the bronchioles. Chronic inflammation was characterized by peribronchiolar fibrosis admixed with mononuclear cell infiltrates. Although the absence of club cells was a component of respiratory epithelial hyperplasia of the bronchioles at Study Week 13, they remained reduced in number to absent at the recovery necropsy. Aggregates of pigmented alveolar macrophages, most likely containing hemosiderin, was also a new apparent effect of test substance administration in the 45 mg/m3 group males. Two males from the 45 mg/m3 group had proliferative changes characterized as ‘atypical’ including bronchiolo-alveolar hyperplasia and bronchial mucous cell metaplasia.
Key result
Dose descriptor:
NOAEC
Effect level:
ca. 15 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
45 mg/m³ air (nominal)
System:
other: whole respiratory tract
Organ:
larynx
lungs
nasal cavity
trachea
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 3 Incidence of Selected Histopathologic Findings, Study Week 13 Primary Necropsy - males

Males

Exposure Concentration (mg/m3)

0

5

15

45

Nasal level IIa

11b

10

11b

10

   Degeneration/regeneration, respiratory epithelium

0

0

0

2

       Minimal

-

-

-

2

       Mild

-

-

-

0

       Moderate

-

-

-

0

   Degeneration/regeneration, transitional epithelium

0

0

0

0

       Minimal

-

-

-

-

       Mild

-

-

-

-

       Moderate

-

-

-

-

       Marked

-

-

-

-

   Hyperplasia, respiratory epithelium

0

0

0

6

       Minimal

-

-

-

4

       Mild

-

-

-

2

   Hyperplasia, transitional epithelium

1

4

6b

3

       Minimal

1

4

6b

2

       Mild

0

0

0

1

   Inflammation, mixed cell

0

2

5

10

       Minimal

-

2

4

10

       Mild

-

0

1

0

   Metaplasia, squamous cell

0

1

4

6

       Minimal

-

1

4

4

       Mild

-

0

0

2

Nasal level IIIa

11b

NA

NA

10

   Degeneration/regeneration, olfactory epithelium (marked)          

0

-

-

0

   Degeneration/regeneration, respiratory epithelium

0

-

-

1

       Minimal

-

-

-

1

       Mild

-

-

-

0

Larynxa

11b

10

10b

10

   Degeneration/regeneration, respiratory epithelium(minimal)

0

0

1

4

   Inflammation, chronic active

0

0

2

1

       Minimal

-

-

0

0

       Mild

-

-

2

1

   Inflammation, mixed cell

2

2

5b

6

       Minimal

2

2

3b

4

       Mild

0

0

2

2

   Metaplasia, squamous cell

0

0

0

2

       Minimal

-

-

-

1

       Mild

-

-

-

1

Tracheaa

11b

9

11b

10

   Degeneration/regeneration, respiratory epithelium

0

0

3b

7

       Minimal

-

-

2b

5

       Mild

-

-

1

2

       Moderate

-

-

0

0

       Inflammation, mixed cell (minimal)

1b

0

1

3

Lungsa

11b

10

11b

10

   Degeneration/regeneration, respiratory epithelium, bronchus

0

4

9b

7

       Minimal

-

4

9b

5

       Mild

-

0

0

2

   Hyperplasia, respiratory epithelium, bronchioles

0

0

0

7

       Minimal

-

-

-

6

       Mild

-

-

-

1

   Infiltrate, mixed cell, peribronchiolar

0

0

0

7

       Minimal

-

-

-

3

       Mild

-

-

-

4

 

 

 

 

 

   Inflammation, mixed cell, bronchus

0

3

3

4

       Minimal

-

3

3

3

       Mild

-

0

0

1

   Luminal debris, bronchioles

0

0

0

2

       Minimal

-

-

-

0

       Mild

-

-

-

2

a Number of tissues examined from each group.

b Includes 1 FD/EE rat.

BOLDED incidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine, CAS 1760-24-3

Table 4 Incidence of Selected Histopathologic Findings, Study Week 13 Primary Necropsy - females

 

Females

Exposure Concentration (mg/m3)

0

5

15

45

Nasal level IIa

10

10

10

10

   Degeneration/regeneration, respiratory epithelium

0

0

0

8

       Minimal

-

-

-

2

       Mild

-

-

-

2

       Moderate

-

-

-

4

   Degeneration/regeneration, transitional epithelium

0

0

1

8

       Minimal

-

-

1

1

       Mild

-

-

0

1

       Moderate

-

-

0

2

       Marked

-

-

0

4

   Hyperplasia, respiratory epithelium

0

0

0

0

       Minimal

-

-

-

-

       Mild

-

-

-

-

   Hyperplasia, transitional epithelium

0

0

0

0

       Minimal

-

-

-

-

       Mild

-

-

-

-

   Inflammation, mixed cell

2

5

4

8

       Minimal

2

3

4

3

       Mild

0

2

0

5

   Metaplasia, squamous cell

0

0

1

4

       Minimal

-

-

0

4

       Mild

-

-

1

0

Nasal level IIIa

10

10

10

10

   Degeneration/regeneration, olfactory epithelium (marked)          

0

0

0

1

   Degeneration/regeneration, respiratory epithelium

0

0

0

3

       Minimal

-

-

-

1

       Mild

-

-

-

2

Larynxa

10

10

10

10

   Degeneration/regeneration, respiratory epithelium(minimal)

0

0

1

1

   Inflammation, chronic active

0

0

0

5

       Minimal

-

-

-

2

       Mild

-

-

-

3

   Inflammation, mixed cell

1

4

7

7

       Minimal

1

4

5

6

       Mild

0

0

2

1

   Metaplasia, squamous cell

0

0

0

5

       Minimal

-

-

-

5

       Mild

-

-

-

0

Tracheaa

10

10

10

10

   Degeneration/regeneration, respiratory epithelium

0

0

2

8

       Minimal

-

-

1

3

       Mild

-

-

1

4

       Moderate

-

-

0

1

       Inflammation, mixed cell (minimal)

0

0

1

5

Lungsa

10

10

10

10

   Degeneration/regeneration, respiratory epithelium, bronchus

0

1

5

9

       Minimal

-

1

5

7

       Mild

-

0

0

2

   Hyperplasia, respiratory epithelium, bronchioles

0

0

0

10

       Minimal

-

-

-

10

       Mild

-

-

-

0

   Infiltrate, mixed cell, peribronchiolar

0

0

0

10

       Minimal

-

-

-

5

       Mild

-

-

-

5

 

 

 

 

 

   Inflammation, mixed cell, bronchus

0

1

0

6

       Minimal

-

1

-

6

       Mild

-

0

-

0

   Luminal debris, bronchioles

0

0

0

7

       Minimal

-

-

-

3

       Mild

-

-

-

4

a Number of tissues examined from each group.

b Includes 1 FD/EE rat.

BOLDEDincidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine, CAS 1760-24-3

Table 5 Incidence of Selected Histopathologic Findings, Study Week 17 Recovery Necropsy - males

 

Males

Exposure Concentration (mg/m3)

0

5

15

45

Nasal level IIa

9

10

9

10

   Degeneration/regeneration, respiratory epithelium(minimal)

0

0

0

1

   Degeneration/regeneration, transitional epithelium

0

0

0

1

       Minimal

-

-

-

1

       Mild

-

-

-

0

   Hyperplasia, mucous cell (minimal)

0

1

1

5

   Hyperplasia, respiratory epithelium (mild)

0

0

0

2

   Inflammation, mixed cell

2

1

1

3

       Minimal

2

1

1

1

       Mild

0

0

0

2

Nasal level IIIa

NA

NA

NA

NA

   Degeneration/regeneration, olfactory epithelium (moderate)       

-

-

-

-

   Inflammation, mixed cell (mild)

-

-

-

-

Larynxa

9

10

9

10

   Degeneration/regeneration, respiratory epithelium(minimal)

0

0

0

0

   Inflammation, chronic

0

0

0

4

       Minimal

-

-

-

1

       Mild

-

-

-

3

Tracheaa

9

10

9

10

   Degeneration/regeneration, respiratory epithelium (Minimal)

0

0

0

0

       Inflammation, mixed cell (minimal)

0

0

0

0

Lungsa

9

10

9

10

   Decreased club cells, terminal bronchioles (Present)

0

0

0

8

   Degeneration/regeneration, respiratory epithelium, bronchus (Minimal)

0

0

0

0

   Inflammation, chronic, bronchioles (Minimal)

0

0

0

7

   Luminal debris, bronchioles (Minimal)

0

0

0

2

   Macrophages, pigmented (Minimal)

0

1

0

3

a Number of tissues examined from each group.

BOLDEDincidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine,

CAS 1760-24-3

There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Table 6 Incidence of Selected Histopathologic Findings, Study Week 17 Recovery Necropsy - females

 

Females

Exposure Concentration (mg/m3)

0

5

15

45

Nasal level IIa

10

10

10

10

   Degeneration/regeneration, respiratory epithelium(minimal)

0

0

0

1

   Degeneration/regeneration, transitional epithelium

0

0

0

2

       Minimal

-

-

-

0

       Mild

-

-

-

2

   Hyperplasia, mucous cell (minimal)

0

0

0

0

   Hyperplasia, respiratory epithelium (mild)

0

0

0

0

   Inflammation, mixed cell

2

0

1

5

       Minimal

2

-

1

4

       Mild

0

-

0

1

Nasal level IIIa

10

10

10

10

   Degeneration/regeneration, olfactory epithelium (moderate)       

0

0

0

1

   Inflammation, mixed cell (mild)

0

0

0

1

Larynxa

10

10

10

10

   Degeneration/regeneration, respiratory epithelium(minimal)

0

0

0

3

   Inflammation, chronic

0

0

0

6

       Minimal

-

-

-

3

       Mild

-

-

-

3

Tracheaa

10

10

10

10

   Degeneration/regeneration, respiratory epithelium (Minimal)

0

0

0

1

       Inflammation, mixed cell (minimal)

0

0

0

1

Lungsa

10

10

10

10

   Decreased club cells, terminal bronchioles (Present)

0

0

0

10

   Degeneration/regeneration, respiratory epithelium, bronchus (Minimal)

0

0

0

1

   Inflammation, chronic, bronchioles (Minimal)

0

0

0

9

   Luminal debris, bronchioles (Minimal)

0

0

0

5

   Macrophages, pigmented (Minimal)

0

0

0

0

a Number of tissues examined from each group.

BOLDEDincidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine,

CAS 1760-24-3

There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Table 7 Correlation of Selected Observations

 

Necropsy

Organ Weight

Clinical Pathology

Histopathology

Lung- not fully collapsed (PN; 45 mg/m3)

↑ lung weight

-

Lung - Luminal debris, bronchioles; respiratory epithelial hyperplasia of the bronchioles and mixed cell infiltrate, peribronchiolar

-

↑ lung weight (RN;

 45 mg/m3)

-

Lung- Luminal debris, bronchioles and chronic inflammation, bronchioles

 

 Relationships were suspected between gross necropsy, organ weight, clinical pathology, and histopathology observations, as presented in Table 7. These proposed relationships were based on subjective interpretation rather than a statistical analysis of correlation.

Conclusions:
In a 90-day repeated dose toxicity study conducted to OECD TG 413 and in compliance with GLP, exposure of Sprague-Dawley rats to N-[3-(trimethoxysilyl)propyl] ethylenediamine via nose-only inhalation for 6 hours per day on a 5-day basis for 13 weeks at target exposure concentrations of 5, 15, and 45 mg/m3 resulted in non-adverse test substance-related effects at 5 and 15 mg/m3. Adverse test substance-related clinical observations, lower body weights and body weight gains, macroscopic findings in the lungs, increased lung weights, and microscopic findings in the nasal cavity, larynx, trachea, and lungs were noted in the 45 mg/m3 group males and females at the end of the treatment period. At the recovery necropsy, many of the effects of test substance exposure, including higher lung weights, persisted in the nasal cavity, larynx, trachea, and lungs in the 45 mg/m3 group and were noted at generally a lower incidence and severity than that seen at the primary necropsy. Therefore, the NOAEC was 15 mg/m3.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
45 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 February 2017 to December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
ECHA decision number: TPE-D-2114300905-56-01/F
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
not specified
Remarks:
There are no deviations from the test guideline that affect the integrity of the study
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a room with controls set to maintain 18°C to 24°C and under dry nitrogen gas, keep away from moisture with dessicant.
- Stability under test conditions: The test article is considered to be stable.
- Solubility and stability of the test substance in the solvent/vehicle: Filtered air was used as vehicle.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: not applicable
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 227 to 307 g (males)/149 to 192 g (females)
- Fasting period before study: Food and water were not available during the exposure period.
- Housing: All animals were housed in groups of 2 to 4 per cage following receipt in clean, solid bottom cages with bedding material.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 meal was available ad libitum
- Water: Reverse osmosis-treated water was available ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY: Filters servicing the automatic watering system were changed regularly according to SOPs. The municipal water supplying the laboratory was analyzed for contaminants according to SOPs. SOPs provide specifications for acceptable levels of heavy metals and pesticides that are reasonably expected to be present in the diet without interfering with the purpose or conduct of the study. No contaminants were reasonably expected to be present in water or diet that would interfere with the objectives of the study, therefore, no testing was conducted as part of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 20°C and 26°C
- Humidity (%): 15-30%
- Air changes (per hr): 10 per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
> 1.3 - <= 2.1 µm
Geometric standard deviation (GSD):
2.47
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel, conventional nose-only exposure systems
- Method of holding animals in test chamber: nose-only exposure restraint tubes
- Source and rate of air:
- Method of conditioning air:
- System of generating particulates/aerosols: Liquid droplet aerosol atmospheres of the test substance were generated using single-jet Collision nebulizers. Test substance exposure atmospheres were generated by mixing the aerosolized test substance with nitrogen and/or breathing-quality dry, compressed air in the appropriate ratio to achieve the target concentrations. Specific exposure atmosphere generation equipment, operating parameters and methods were defined during method development.
- Temperature, humidity, pressure in air chamber: Average daily temperature and relative humidity of the exposure atmospheres were 23 ± 3ºC and 15%-30%, respectively. Temperature and relative humidity were monitored continuously and recorded at approximately 60-minute intervals during each exposure.
- Air flow rate: Airflow rate through the system was set based on output from the generation equipment and the dilution airflow to provide sufficient volumes for the number of animals to be exposed and for test atmosphere sampling.
- Air change rate: 10 per hour
- Method of particle size determination: Aerosol particle size measurements were conducted during method development as needed and at least twice weekly during the 13-week exposure phase for all test substance exposure groups. Aerosol particle size measurements were conducted using a 7-stage stainless steel cascade impactor. The aerosol size was expressed in terms of the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD). The target range for MMAD is 1.0 to 3.0 microns and GSD is 1.5 to 3.5. The mean aerosol particle size exposure system 2, 3 and 4 were 2.1, 1.9 and 1.3 microns, respectively. Mean GSD for exposure system 2, 3 and 4 were 2.35, 2.47 and 2.15, respectively.
- Treatment of exhaust air: exhaust passed through a Solberg canister filter prior to entering the facility exhaust system, which consists of redundant exhaust blowers preceded by activated charcoal- and HEPA-filtration units.

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: filtered air
- Composition of vehicle: not applicable
- Type and concentration of dispersant aid (if powder): not applicable
- Concentration of test material in vehicle: not applicable
- Lot/batch no. of vehicle (if required): not applicable
- Purity of vehicle: not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were collected using pre-weighed filters held in a filter holder and placed in the animal breathing zone of an exposure system. A volume of the test atmosphere is pulled through the filter. Following sample collection, the sample is reweighed. The exposure concentration (mg/m3) were calculated by dividing the gravimetrically determined mass of test substance aerosol by the sample volume. The mass was determined by subtracting the initial filter weight from the final weight of the post-sample filter. Sample volume was calculated by multiplying the sample flow rate by the length of the sampling period. Concentrations were not be adjusted for test substance purity. For the test substance exposure systems, samples were collected daily at appropriate intervals based on the target exposure concentrations. For the control group (Group 1), gravimetric samples were collected at least twice weekly during the 13-week exposure period.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days a week for 6 hours a day
Dose / conc.:
5 mg/m³ air (nominal)
Dose / conc.:
15 mg/m³ air (nominal)
Dose / conc.:
45 mg/m³ air (nominal)
Dose / conc.:
5.1 mg/m³ air (analytical)
Dose / conc.:
14.9 mg/m³ air (analytical)
Dose / conc.:
44 mg/m³ air (analytical)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Exposure concentrations were selected to cover a range extending from an anticipated no observed-adverse-effect-concentration (NOAEC) to a concentration in which toxic effects are seen in the highest exposure concentration. Specific concentrations were based on the results of a 28-day range-finding study in which microscopic findings were noted in the lungs, trachea larynx and nasal levels at a concentration of 100 mg/m3.
- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: The animals scheduled for the recovery evaluations will be subjected to necropsy following a 4-week non-exposure period (minimum of 27 days).
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily, once in the morning and once in the afternoon. Moribund animals were euthanized and necropsied as soon as possible. Animals found dead were necropsied as soon as possible to ensure that tissues will not be lost due to autolysis.
- Cage side observations checked in table were included: mortality, abnormalities, and signs of pain and distress

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to treatment, weekly during the study period and on the day of necropsy

BODY WEIGHT: Yes
- Time schedule: Prior to treatment, twice weekly during the study period and on the day of necropsy

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: Once during acclimation prior to randomization and all surviving animals near the end of the exposure period or near the end of the recovery period.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.1] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations: not conducted, as not triggered by clinical observations

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table No 2)
HISTOPATHOLOGY: Yes (see table No 2)
Statistics:
All analyses were two-tailed for significance levels of 5% and 1%. All means were presented with standard deviations. All statistical tests were performed by a computer with appropriate programming as referenced below.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were no clinical observations attributable to treatment in the low and mid-dose groups, or in the recovery groups. There were no clinical signs noted 0-1 hour following exposure in any group.

The only group with clinical signs of toxicity due to treatment was the high dose (45 mg/m3) group during the treatment period. Males and females of this group had multiple instances of red, brown, and/or yellow material on various body surfaces and clear discharge around the eyes. The staining was judged by the study author to be indicative of test substance-related stress or general ill health. This finding was observed prior to dosing and on non-exposure days during the treatment period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male in the 15 mg/m3 group was euthanized in extremis on Study Day 61 due to a clinical observation of red penile discharge. At necropsy, this male was noted with gross observations of dilated pelvis, distended bladder, distended ureters, and red fluid in the bladder and ureters. This death was not considered to be test substance-related. In addition, a single male in the control group was found dead on Study Day 65; there were no remarkable clinical observations or gross observations noted for this animal. All other animals survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related effects on body weight or body weight gain in the low and mid-dose groups.

Test substance-related effects on body weights were noted in the 45 mg/m3 group males and females. Lower mean body weights and cumulative body weight gains were noted beginning as early as Study Week 1 in males and females, which continued until the primary necropsy (Study Week 13); the differences were generally significant compared to the control group. On Study Week 13 (the day of the primary necropsy), mean body weights in the 45 mg/m3 males and females were 9.7% and 6.4% lower, respectively, than the mean control group body weights. In addition, body weight gains in the 45 mg/m3 group males and females were comparable or higher than the control group values during the recovery period; while mean body weights in the males remained lower (not statistically significant) than the control group. Therefore, the animals recovered once exposure stopped, but males were slightly slower to recover than females.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The only treatment-related effect on organ weights occurred in the high dose group and was limited to the lungs. There were no adverse findings in the low and mid-dose groups.

At the end of the treatment period: Lung weights (absolute and relative to body and brain weights) tended to be higher with exposure concentration across all groups with statistical significance achieved only at 45 mg/m3. For the males, all group mean and individual animal absolute lung weights, as examples, were within historical control reference ranges. In contrast for the 45 mg/m3 group females, mean absolute and both mean relative weights exceeded their historical control reference ranges and, as an example, 6 individual animal absolute lung weights also exceeded the historical control reference range. The most likely microscopic correlations for the higher lung weights were the findings affecting the bronchioles (epithelial hyperplasia, mixed cell infiltrates and luminal debris). Since none of these findings were seen in either sex at exposure concentrations < 45 mg/m3, the 8% to 10% higher lung weights seen in the 15 mg/m3 group males (and lesser magnitudes of change seen at lower exposure concentrations in both sexes), which were reversible, were considered incidental and unrelated to treatment.

At the end of the recovery period: treatment-related and statistically significant higher lung weights (absolute and/or relative to body and brain weights) persisted in both sexes at exposure concentrations of 45 mg/m3. Although mean absolute lung weight was not statistically significant in the 45 mg/m3 group males, the approximately 10% increase was considered sufficient to be considered an effect of test substance exposure. Peribronchiolar chronic inflammation was considered the microscopic correlate for the persistent high lung weights in this group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic findings were observed in the high dose group only at the end of the treatment period and were limited to the lungs. There were no adverse findings in any of the groups at the end of the recovery period.

Treatment-related findings of not fully collapsed (3/10 males and 8/10 females) and pale lungs (1/10 males and 2/10 females) were observed exclusively in the 45 mg/m3 group at the primary necropsy; luminal debris in the bronchioles and peribronchiolar inflammatory cell infiltrates appeared to be the microscopic correlates for lungs not fully collapsed while a correlation was not observed for pale lungs. At the Study Week 17 recovery necropsy, not fully collapsed lungs
was seen in 2 males and 1 female from the 45 mg/m3 group but also 1 male each from the control and 5 mg/m3 groups. Therefore at the recovery necropsy, lungs not fully collapsed was not conclusively associated with exposure to the test substance aerosol.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Tables 3 to 6.
Treatment-related microscopic findings were limited to the respiratory tract. These findings were observed in all groups at the end of the treatment period, but they were only observed in the high dose group at the end of the recovery period.

At the end of the treatment period: In the nasal cavity treatment-related epithelial (transitional, respiratory, or olfactory) degeneration/regeneration was characterized by loss of normal tissue architecture, most commonly by cell loss and replacement by a single layer of attenuated epithelium; these changes were typically bilateral. Specific to the respiratory epithelium was loss of cilia and reduction/loss of goblet cells. The sites of respiratory epithelial degeneration/regeneration were most commonly the dorsal aspect of the nasal septum and lining the dorsal meatus, including nasal turbinate(s). Olfactory epithelial degeneration/regeneration was limited to 1 female in the 45 mg/m3 group and was diffuse within the dorsal meatus of nasal level III while focal and unilateral in the posterior nasal levels. Transitional epithelial degeneration/regeneration was most commonly limited to the tissues lining the lateral meatus (nasal turbinates and lateral wall). Squamous cell metaplasia and mixed cell inflammation were usually associated with areas of epithelial degeneration/regeneration. Squamous cell metaplasia represented a change from the normal epithelium lining a region to an attenuated stratified and keratinized squamous epithelium. Mixed cell inflammation was characterized by a mononuclear cell (lymphocytes, plasma cells and macrophages) population admixed with variable numbers of neutrophils. In contrast to epithelial degeneration/regeneration seen most commonly in the females, transitional and respiratory epithelial hyperplasia was the most common epithelial change seen in the males exposed to test substance aerosol; affected regions were comparable to those seen with degeneration/regeneration in the females. Several of these findings (transitional epithelial hyperplasia, mixed cell inflammation, and squamous cell metaplasia) at nasal level II were seen in one or both sexes at exposure concentrations of 5, 15, and 45 mg/m3.

In the larynx, trachea, and bronchi (recorded under lungs) treatment-related respiratory epithelial degeneration/regeneration and mixed cell inflammation were comparable to that seen in the nasal cavity. It was not uncommon in control group animals to see respiratory epithelial loss and/or attenuation at these sites. Loss was clearly an artifact of tissue collection/processing while the cause of the thinned epithelium was less certain. However, respiratory epithelial degeneration/regeneration was diagnosed only when there was single cell necrosis, mitotic figure(s) and/or rare neutrophils (sub- or intra-epithelial) associated with the attenuated epithelium. Disruption of the normal epithelial architecture with loss of cilia was also considered degeneration/regeneration. In the larynx, squamous metaplasia was similar to that
seen in the nasal cavity except that it was more commonly seen as a change from the normal non-keratinized stratified epithelium to an attenuated and keratinized stratified squamous epithelium. In addition to mixed cell inflammation in the larynx, chronic active inflammation was also an effect of exposure to the test substance, particularly in the 45 mg/m3 group females, and the epiglottis was a common site of this inflammation. Chronic active inflammation was similar to mixed cell inflammation except in the former, fibroblasts/fibroplasia were present generally indicating inflammation of longer duration. Several of these findings (respiratory epithelial
degeneration of bronchi and mixed cell inflammation of the larynx and bronchi) were seen in one or both sexes at exposure concentrations of 5, 15, and 45 mg/m3.

At the end of the recovery period: In the nasal cavity (levels II and III) most previous effects of exposure persisted in one or both sexes from the 45 mg/m3 group with
incidences and severities that were generally lower than those seen at the primary necropsy. A new effect, and evidence of chronic irritation, was mucous cell hyperplasia of the nasal septum at nasal level II in the males. Due to the low incidence and minimal to mild severity and resolution during the 4-week recovery period, findings in the nasal cavity were considered to be nonadverse at 5 and 15 mg/m3.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
In the lungs at the primary necropsy, treatment-related effects were centered on the bronchi and/or bronchioles. Changes in the bronchi were comparable to and considered an extension of the changes seen in the trachea. The most prominent change was noted at the periphery of the lungs at the level of the terminal bronchioles. In control group animals, terminal bronchioles rarely had any associated inflammatory or mononuclear cell infiltrates and the epithelial lining
was an admixture of ciliated respiratory epithelial cells and cells with distinct apical blebbing (club/Clara cells). Exposure-related effects included variably dense perivascular and/or peribronchiolar mixed cell infiltrates characterized by mononuclear cells with generally fewer eosinophils and/or neutrophils and in some cases the suggestion of increased fibrosis/fibroplasia (Masson’s stain suggested this was true in a 45 mg/m3 group female evaluated). There was loss of the normal epithelium lining the terminal bronchioles with replacement by a prominent respiratory epithelium with a robustly ciliated epithelium; club cells were generally absent (mild respiratory epithelial hyperplasia). With minimal respiratory epithelial hyperplasia, club cells were occasionally present and/or there was epithelial alteration comparable to the respiratory epithelial degeneration/regeneration noted in the bronchi. Some animals had bronchiolar luminal debris (exudate) consisting of a mucinous material admixed with foamy alveolar macrophages and less commonly neutrophils. It was this exudate in combination with the peribronchiolar cellular infiltrates and respiratory epithelial hyperplasia that were the most likely microscopic correlations for the lungs noted at necropsy as not fully collapsed. Other than the changes affecting the bronchi, discussed above, all treatment-related effects to the lower airways were limited to the 45 mg/m3 group.

In the lungs at the recovery necropsy, some of the previous effects of exposure persisted in one or both sexes from the 45 mg/m3 group with incidences and severities that were generally lower than those seen at the primary necropsy. A new effect, and evidence of chronic irritation, was the shift from mixed cell peribronchiolar infiltrates to chronic inflammation of the bronchioles. Chronic inflammation was characterized by peribronchiolar fibrosis admixed with mononuclear cell infiltrates. Although the absence of club cells was a component of respiratory epithelial hyperplasia of the bronchioles at Study Week 13, they remained reduced in number to absent at the recovery necropsy. Aggregates of pigmented alveolar macrophages, most likely containing hemosiderin, was also a new apparent effect of test substance administration in the 45 mg/m3 group males. Two males from the 45 mg/m3 group had proliferative changes characterized as ‘atypical’ including bronchiolo-alveolar hyperplasia and bronchial mucous cell metaplasia.
Key result
Dose descriptor:
NOAEC
Effect level:
ca. 15 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
45 mg/m³ air (nominal)
System:
other: whole respiratory tract
Organ:
larynx
lungs
nasal cavity
trachea
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Table 3 Incidence of Selected Histopathologic Findings, Study Week 13 Primary Necropsy - males

Males

Exposure Concentration (mg/m3)

0

5

15

45

Nasal level IIa

11b

10

11b

10

   Degeneration/regeneration, respiratory epithelium

0

0

0

2

       Minimal

-

-

-

2

       Mild

-

-

-

0

       Moderate

-

-

-

0

   Degeneration/regeneration, transitional epithelium

0

0

0

0

       Minimal

-

-

-

-

       Mild

-

-

-

-

       Moderate

-

-

-

-

       Marked

-

-

-

-

   Hyperplasia, respiratory epithelium

0

0

0

6

       Minimal

-

-

-

4

       Mild

-

-

-

2

   Hyperplasia, transitional epithelium

1

4

6b

3

       Minimal

1

4

6b

2

       Mild

0

0

0

1

   Inflammation, mixed cell

0

2

5

10

       Minimal

-

2

4

10

       Mild

-

0

1

0

   Metaplasia, squamous cell

0

1

4

6

       Minimal

-

1

4

4

       Mild

-

0

0

2

Nasal level IIIa

11b

NA

NA

10

   Degeneration/regeneration, olfactory epithelium (marked)          

0

-

-

0

   Degeneration/regeneration, respiratory epithelium

0

-

-

1

       Minimal

-

-

-

1

       Mild

-

-

-

0

Larynxa

11b

10

10b

10

   Degeneration/regeneration, respiratory epithelium(minimal)

0

0

1

4

   Inflammation, chronic active

0

0

2

1

       Minimal

-

-

0

0

       Mild

-

-

2

1

   Inflammation, mixed cell

2

2

5b

6

       Minimal

2

2

3b

4

       Mild

0

0

2

2

   Metaplasia, squamous cell

0

0

0

2

       Minimal

-

-

-

1

       Mild

-

-

-

1

Tracheaa

11b

9

11b

10

   Degeneration/regeneration, respiratory epithelium

0

0

3b

7

       Minimal

-

-

2b

5

       Mild

-

-

1

2

       Moderate

-

-

0

0

       Inflammation, mixed cell (minimal)

1b

0

1

3

Lungsa

11b

10

11b

10

   Degeneration/regeneration, respiratory epithelium, bronchus

0

4

9b

7

       Minimal

-

4

9b

5

       Mild

-

0

0

2

   Hyperplasia, respiratory epithelium, bronchioles

0

0

0

7

       Minimal

-

-

-

6

       Mild

-

-

-

1

   Infiltrate, mixed cell, peribronchiolar

0

0

0

7

       Minimal

-

-

-

3

       Mild

-

-

-

4

 

 

 

 

 

   Inflammation, mixed cell, bronchus

0

3

3

4

       Minimal

-

3

3

3

       Mild

-

0

0

1

   Luminal debris, bronchioles

0

0

0

2

       Minimal

-

-

-

0

       Mild

-

-

-

2

a Number of tissues examined from each group.

b Includes 1 FD/EE rat.

BOLDED incidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine, CAS 1760-24-3

Table 4 Incidence of Selected Histopathologic Findings, Study Week 13 Primary Necropsy - females

 

Females

Exposure Concentration (mg/m3)

0

5

15

45

Nasal level IIa

10

10

10

10

   Degeneration/regeneration, respiratory epithelium

0

0

0

8

       Minimal

-

-

-

2

       Mild

-

-

-

2

       Moderate

-

-

-

4

   Degeneration/regeneration, transitional epithelium

0

0

1

8

       Minimal

-

-

1

1

       Mild

-

-

0

1

       Moderate

-

-

0

2

       Marked

-

-

0

4

   Hyperplasia, respiratory epithelium

0

0

0

0

       Minimal

-

-

-

-

       Mild

-

-

-

-

   Hyperplasia, transitional epithelium

0

0

0

0

       Minimal

-

-

-

-

       Mild

-

-

-

-

   Inflammation, mixed cell

2

5

4

8

       Minimal

2

3

4

3

       Mild

0

2

0

5

   Metaplasia, squamous cell

0

0

1

4

       Minimal

-

-

0

4

       Mild

-

-

1

0

Nasal level IIIa

10

10

10

10

   Degeneration/regeneration, olfactory epithelium (marked)          

0

0

0

1

   Degeneration/regeneration, respiratory epithelium

0

0

0

3

       Minimal

-

-

-

1

       Mild

-

-

-

2

Larynxa

10

10

10

10

   Degeneration/regeneration, respiratory epithelium(minimal)

0

0

1

1

   Inflammation, chronic active

0

0

0

5

       Minimal

-

-

-

2

       Mild

-

-

-

3

   Inflammation, mixed cell

1

4

7

7

       Minimal

1

4

5

6

       Mild

0

0

2

1

   Metaplasia, squamous cell

0

0

0

5

       Minimal

-

-

-

5

       Mild

-

-

-

0

Tracheaa

10

10

10

10

   Degeneration/regeneration, respiratory epithelium

0

0

2

8

       Minimal

-

-

1

3

       Mild

-

-

1

4

       Moderate

-

-

0

1

       Inflammation, mixed cell (minimal)

0

0

1

5

Lungsa

10

10

10

10

   Degeneration/regeneration, respiratory epithelium, bronchus

0

1

5

9

       Minimal

-

1

5

7

       Mild

-

0

0

2

   Hyperplasia, respiratory epithelium, bronchioles

0

0

0

10

       Minimal

-

-

-

10

       Mild

-

-

-

0

   Infiltrate, mixed cell, peribronchiolar

0

0

0

10

       Minimal

-

-

-

5

       Mild

-

-

-

5

 

 

 

 

 

   Inflammation, mixed cell, bronchus

0

1

0

6

       Minimal

-

1

-

6

       Mild

-

0

-

0

   Luminal debris, bronchioles

0

0

0

7

       Minimal

-

-

-

3

       Mild

-

-

-

4

a Number of tissues examined from each group.

b Includes 1 FD/EE rat.

BOLDEDincidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine, CAS 1760-24-3

Table 5 Incidence of Selected Histopathologic Findings, Study Week 17 Recovery Necropsy - males

 

Males

Exposure Concentration (mg/m3)

0

5

15

45

Nasal level IIa

9

10

9

10

   Degeneration/regeneration, respiratory epithelium(minimal)

0

0

0

1

   Degeneration/regeneration, transitional epithelium

0

0

0

1

       Minimal

-

-

-

1

       Mild

-

-

-

0

   Hyperplasia, mucous cell (minimal)

0

1

1

5

   Hyperplasia, respiratory epithelium (mild)

0

0

0

2

   Inflammation, mixed cell

2

1

1

3

       Minimal

2

1

1

1

       Mild

0

0

0

2

Nasal level IIIa

NA

NA

NA

NA

   Degeneration/regeneration, olfactory epithelium (moderate)       

-

-

-

-

   Inflammation, mixed cell (mild)

-

-

-

-

Larynxa

9

10

9

10

   Degeneration/regeneration, respiratory epithelium(minimal)

0

0

0

0

   Inflammation, chronic

0

0

0

4

       Minimal

-

-

-

1

       Mild

-

-

-

3

Tracheaa

9

10

9

10

   Degeneration/regeneration, respiratory epithelium (Minimal)

0

0

0

0

       Inflammation, mixed cell (minimal)

0

0

0

0

Lungsa

9

10

9

10

   Decreased club cells, terminal bronchioles (Present)

0

0

0

8

   Degeneration/regeneration, respiratory epithelium, bronchus (Minimal)

0

0

0

0

   Inflammation, chronic, bronchioles (Minimal)

0

0

0

7

   Luminal debris, bronchioles (Minimal)

0

0

0

2

   Macrophages, pigmented (Minimal)

0

1

0

3

a Number of tissues examined from each group.

BOLDEDincidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine,

CAS 1760-24-3

There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Table 6 Incidence of Selected Histopathologic Findings, Study Week 17 Recovery Necropsy - females

 

Females

Exposure Concentration (mg/m3)

0

5

15

45

Nasal level IIa

10

10

10

10

   Degeneration/regeneration, respiratory epithelium(minimal)

0

0

0

1

   Degeneration/regeneration, transitional epithelium

0

0

0

2

       Minimal

-

-

-

0

       Mild

-

-

-

2

   Hyperplasia, mucous cell (minimal)

0

0

0

0

   Hyperplasia, respiratory epithelium (mild)

0

0

0

0

   Inflammation, mixed cell

2

0

1

5

       Minimal

2

-

1

4

       Mild

0

-

0

1

Nasal level IIIa

10

10

10

10

   Degeneration/regeneration, olfactory epithelium (moderate)       

0

0

0

1

   Inflammation, mixed cell (mild)

0

0

0

1

Larynxa

10

10

10

10

   Degeneration/regeneration, respiratory epithelium(minimal)

0

0

0

3

   Inflammation, chronic

0

0

0

6

       Minimal

-

-

-

3

       Mild

-

-

-

3

Tracheaa

10

10

10

10

   Degeneration/regeneration, respiratory epithelium (Minimal)

0

0

0

1

       Inflammation, mixed cell (minimal)

0

0

0

1

Lungsa

10

10

10

10

   Decreased club cells, terminal bronchioles (Present)

0

0

0

10

   Degeneration/regeneration, respiratory epithelium, bronchus (Minimal)

0

0

0

1

   Inflammation, chronic, bronchioles (Minimal)

0

0

0

9

   Luminal debris, bronchioles (Minimal)

0

0

0

5

   Macrophages, pigmented (Minimal)

0

0

0

0

a Number of tissues examined from each group.

BOLDEDincidences represent effects of exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine,

CAS 1760-24-3

There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to N-[3-(trimethoxysilyl)propyl]ethylenediamine. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Table 7 Correlation of Selected Observations

 

Necropsy

Organ Weight

Clinical Pathology

Histopathology

Lung- not fully collapsed (PN; 45 mg/m3)

↑ lung weight

-

Lung - Luminal debris, bronchioles; respiratory epithelial hyperplasia of the bronchioles and mixed cell infiltrate, peribronchiolar

-

↑ lung weight (RN;

 45 mg/m3)

-

Lung- Luminal debris, bronchioles and chronic inflammation, bronchioles

 

 Relationships were suspected between gross necropsy, organ weight, clinical pathology, and histopathology observations, as presented in Table 7. These proposed relationships were based on subjective interpretation rather than a statistical analysis of correlation.

Conclusions:
In a 90-day repeated dose toxicity study conducted to OECD TG 413 and in compliance with GLP, exposure of Sprague-Dawley rats to N-[3-(trimethoxysilyl)propyl] ethylenediamine via nose-only inhalation for 6 hours per day on a 5-day basis for 13 weeks at target exposure concentrations of 5, 15, and 45 mg/m3 resulted in non-adverse test substance-related effects at 5 and 15 mg/m3. Adverse test substance-related clinical observations, lower body weights and body weight gains, macroscopic findings in the lungs, increased lung weights, and microscopic findings in the nasal cavity, larynx, trachea, and lungs were noted in the 45 mg/m3 group males and females at the end of the treatment period. At the recovery necropsy, many of the effects of test substance exposure, including higher lung weights, persisted in the nasal cavity, larynx, trachea, and lungs in the 45 mg/m3 group and were noted at generally a lower incidence and severity than that seen at the primary necropsy. Therefore, the NOAEC was 15 mg/m3.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
15 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
This study was designed to determine the effects in rats resulting from repeated percutaneous exposure to A-1120 for 9 doses over an 11 day period.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
11 days
Frequency of treatment:
9 applications
Dose / conc.:
257.5 mg/kg bw/day (nominal)
Dose / conc.:
772.5 mg/kg bw/day (nominal)
Dose / conc.:
1 545 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20/sex/control and high dose
10/sex/ mid and low dose
Control animals:
other: filtered water
Dose descriptor:
NOAEL
Effect level:
>= 1 545 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No signs of systemic toxicity.
Critical effects observed:
no

No clinical signs of toxicity except local effects on skin. Barely perceptible erythema was observed occasionally in males of the 772.5 and 1545 mg/kg bw/day groups and in females of the 1545 mg/kg bw/day group during the first week of treatment. Exfoliation and/or excoriation, were observed during the treatment period in males and females of the 772.5 and 1545 mg/kg bw/day groups. One female of the 257.5 mg/kg bw/day group also showed excoriation during the treatment period. During the 19 -day recovery period, exfoliation and/or excoriation were observed in males and females of the 1545 mg/kg bw/day group. No skin reactions were observed after day 17.

Decreases in food consumption were observed in males of the 772.5 and 1545 mg/kg bw/day during the treatment period. Some body weight decreases were observed but there was no clear dose-response. Slight increases in water consumption were observed in females of the 772.5 and 1545 mg/kg bw/day groups during the study. However, the increases were not considered to be toxicologically significant due to the lack of any associated changes in clinical pathology parameters and the small magnitude of the changes. There were no treatment-related effects on haematology, clinical chemistry, isoenzymes or urinalysis at the end of the treatment period. There was no effect on haematological parameters at the end of the recovery period.

There were slight increases in adrenal weights in the mid and high dose groups, but in light of the lack of pathological findings these were not considered to be toxicologically significant.

Various signs of skin irritation were observed. Exfoliation and excoriation were noted at the end of the treatment period of males in the mid and high dose groups, and females of all groups. Microscopic findings in these groups were hyperkeratosis, acanthosis, epidermitis and dermatitis. Ulceration and dermal fibrosis were observed occasionally in the same treated groups. Residual effects, as indicated by minimal hyperkeratosis and dermatitis were observed in males and females of the high dose group at the end of the 19 -day recovery period.

Conclusions:
Treatment of rats to N-(3-(trimethoxysilyl)propyl)ethylenediamine for nine cutaneous applications during an 11-day period produced transient clinical, necropsy and microscopic observations indicative of mild to moderate skin irritation in males treated with 772.5 and 1545 mg/kg bw/day and females treated with 257.5 mg/kg bw/day and above. There was no clear indication of systemic toxicity. Therefore the NOAEL for systemic effects is at least 1545 mg/kg bw/day in rats.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
This study was designed to determine the effects in rats resulting from repeated percutaneous exposure to A-1120 for 9 doses over an 11 day period.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
11 days
Frequency of treatment:
9 applications
Dose / conc.:
257.5 mg/kg bw/day (nominal)
Dose / conc.:
772.5 mg/kg bw/day (nominal)
Dose / conc.:
1 545 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20/sex/control and high dose
10/sex/ mid and low dose
Control animals:
other: filtered water
Dose descriptor:
NOAEL
Effect level:
>= 1 545 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No signs of systemic toxicity.
Critical effects observed:
no

No clinical signs of toxicity except local effects on skin. Barely perceptible erythema was observed occasionally in males of the 772.5 and 1545 mg/kg bw/day groups and in females of the 1545 mg/kg bw/day group during the first week of treatment. Exfoliation and/or excoriation, were observed during the treatment period in males and females of the 772.5 and 1545 mg/kg bw/day groups. One female of the 257.5 mg/kg bw/day group also showed excoriation during the treatment period. During the 19 -day recovery period, exfoliation and/or excoriation were observed in males and females of the 1545 mg/kg bw/day group. No skin reactions were observed after day 17.

Decreases in food consumption were observed in males of the 772.5 and 1545 mg/kg bw/day during the treatment period. Some body weight decreases were observed but there was no clear dose-response. Slight increases in water consumption were observed in females of the 772.5 and 1545 mg/kg bw/day groups during the study. However, the increases were not considered to be toxicologically significant due to the lack of any associated changes in clinical pathology parameters and the small magnitude of the changes. There were no treatment-related effects on haematology, clinical chemistry, isoenzymes or urinalysis at the end of the treatment period. There was no effect on haematological parameters at the end of the recovery period.

There were slight increases in adrenal weights in the mid and high dose groups, but in light of the lack of pathological findings these were not considered to be toxicologically significant.

Various signs of skin irritation were observed. Exfoliation and excoriation were noted at the end of the treatment period of males in the mid and high dose groups, and females of all groups. Microscopic findings in these groups were hyperkeratosis, acanthosis, epidermitis and dermatitis. Ulceration and dermal fibrosis were observed occasionally in the same treated groups. Residual effects, as indicated by minimal hyperkeratosis and dermatitis were observed in males and females of the high dose group at the end of the 19 -day recovery period.

Conclusions:
Treatment of rats to N-(3-(trimethoxysilyl)propyl)ethylenediamine for nine cutaneous applications during an 11-day period produced transient clinical, necropsy and microscopic observations indicative of mild to moderate skin irritation in males treated with 772.5 and 1545 mg/kg bw/day and females treated with 257.5 mg/kg bw/day and above. There was no clear indication of systemic toxicity. Therefore the NOAEL for systemic effects is at least 1545 mg/kg bw/day in rats.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed

Additional information

In the key study (Charles River, 2017), which is a 90-day repeated dose toxicity study conducted to OECD TG 413 and in compliance with GLP, exposure of Sprague-Dawley rats to an aerosol of N-[3-(trimethoxysilyl)propyl] ethylenediamine via nose-only inhalation for 6 hours per day on a 5-day basis for 13 weeks at target exposure concentrations of 5, 15, and 45 mg/m3 resulted in non-adverse test substance-related effects at 5 and 15 mg/m3. Adverse test substance-related clinical observations, lower body weights and body weight gains, macroscopic findings in the lungs, increased lung weights, and microscopic findings in the nasal cavity, larynx, trachea, and lungs were noted in the 45 mg/m3 group males and females at the end of the treatment period. At the recovery necropsy, many of the effects of test substance exposure, including higher lung weights, persisted in the nasal cavity, larynx, trachea, and lungs in the 45 mg/m3 group and were noted at generally a lower incidence and severity than that seen at the primary necropsy. Therefore, the NOAEC for the local effects on the lungs and respiratory tract was 15 mg/m3. There were no adverse systemic effects observed at any concentration.

The supporting study for this endpoint is an oral test conducted according to OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test), and to GLP (Dow Corning Corporation, 2002b) concluded a NOAEL of 500 mg/kg bw/day as no treatment-related adverse effects were observed at any dose. The earlier seven-day dose range-finding study (Dow Corning Corporation, 2002a), not conducted to GLP, also found the NOAEL to be at least 500 mg/kg bw/day.

 

A nine-day dermal study is also available (Bushy Run Research Center, 1993) which supports the finding of low repeated dose systemic toxicity. In this study, treatment of rats for nine cutaneous applications during an 11-day period produced transient clinical, necropsy and microscopic observations indicative of mild to moderate skin irritation in males treated with 772.5 and 1545 mg/kg bw/day and females treated with 257.5 mg/kg bw/day and above. Skin effects included barely perceptible erythema exfoliation and excoriation. Therefore the NOAEL for systemic effects is at least 1545 mg/kg bw/day in rats.

Justification for classification or non-classification

Based on the 90-day aerosol inhalation toxicity data, N-(3-(trimethoxysilyl)propyl)ethylenediamine requires classification for local effects on the respiratory tract following exposure to aerosols.

 

Based on the available information on the registered substance no classification is proposed for oral or dermal repeated exposure, in accordance with Regulation (EC) No 1272/2008.