Triethoxypropylsilane

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Cross-reference

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Remarks:

.

Adequacy of study:

key study

Study period:

16 Oct 2019 - 08 Dec 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

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, Raleigh, NC, US
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation: males: 211-274 g, females: 168-220 g
- Fasting period before study: no
- Housing: Group housed (2–3 animals of the same sex and same dosing group together) in polycarbonate, solid-bottom cages containing appropriate bedding material
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet 5002 meal, ad libitum
- Water: municipal tap water (treated by reverse osmosis and ultraviolet irradiation), ad libitum
- Acclimation period: at least 10 days

DETAILS OF FOOD AND WATER QUALITY:
Results of analysis for nutritional components and environmental contaminants were provided by the supplier and are on file at the Testing Facility. It was considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water was performed, and results of these analyses are on file at the Testing Facility. It was considered that there are no known contaminants in the water that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 30 -70
- Air changes (per hr): not provided
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: inhalation (aerosol plus vapor)

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 7.9-L stainless-steel, conventional nose-only exposure systems (CNOS) for Groups 2 (500 mg/m³) and 3 (1250 mg/m³) and 11.0-L conventional nose-only systems for Groups 1 (control) and 4 (2500 mg/m³).

- Method of holding animals in test chamber: polycarbonate tube

- Source and rate of air: HEPA- and charcoal-filtered, temperature- and humidity-controlled supply air source

- System of generating aerosols: The test substance was aerosolized using a single jet Collison nebulizer (BGI, Inc.; Waltham, MA) for Exposure Systems 2 (500 mg/m³) and 3 (1250 mg/m³) and a three jet Collison nebulizer for Exposure System 4 (2500 mg/m³). Compressed air (generation air) from the facility source was supplied to the nebulizer to effect aerosolization of the test substance and was controlled using a Coilhose regulator (Model No. 8802K).
Test substance was delivered to each nebulizer via an appropriately sized syringe and a Razel syringe pump (Model A-99, Razel Scientific Instruments Inc.; Stamford, Connecticut). The aerosol/vapor mixture from the outlet of each nebulizer was directed through 22-mm respiratory tubing to a “T”-fitting mounted at the inlet of each CNOS. Supply air was metered to the “T”-fitting using a Gilmont rotameter-type flowmeter (Model No: 127 MM) for System 2 (500 mg/m³) and System 3 (1250 mg/m³) and a Dwyer rotameter-type flowmeter (Model No: DR4141M) for System 4 (2500 mg/m³). Supply air was mixed with test substance aerosol/vapor mixture at the “T”-fitting prior to entering the nose-only system.

- Temperature, humidity, air flow rate: Temperature and relative humidity within each exposure system was monitored using a Vaisala display (Model No.: HMT120, Vaisala; Helsinki, Finland) supplied with a Vaisala transmitter probe (Model No. HMP110). Airflow rates for each nose-only system was monitored by measuring the pressure drop between the ports of a venturi tube using a Dwyer Magnehelic® Indicating Transmitter pressure gauge. Each gauge was calibrated for conversion from pressure to airflow in liters per minute through the use of a Dry Test Meter (Model DTM-200A, Elster American Meter Co.; Nebraska City, NE). Temperature, relative humidity, and airflow rate in each exposure system were continually monitored and recorded at approximately 60-minute intervals during each exposure. Daily means for exposure system temperature, relative humidity, and airflow rate over the study are presented in the following table:

Group: 1 2 3 4
Exposure System: 1 2 3 4
Exposure Concentration (mg/m³): 0 500 1250 2500
Temperature (°C): 22.3 22.6 22.7 23.1
Standard Deviation: 0.68 0.40 0.37 0.39

Relative Humidity (%): 49.6 49.5 48.3 46.3
Standard Deviation: 3.53 3.47 3.51 3.25

Airflow Rate (LPM): 40 34 28 39
Standard Deviation: 1.3 1.8 0.7 0.8



- Treatment of exhaust air: All nose-only system exhaust passed through a Solberg canister filter (Solberg Manufacturing, Inc.; Itasca, IL) 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: Exposure atmospheres will be sampled and total test substance concentrations (aerosol plus vapor) will be calculated by analyzing an aliquot of combined impinger contents using gas chromatography with flame ionization detection (GC/FID).
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Due to the mixture of liquid and vapor phases in the exposure atmosphere, samples of the exposure atmospheres were collected using a sampling train consisting of two 30-mL midget impingers connected in series. Samples were collected from each test substance exposure system at approximately 60-minute intervals during each exposure and once weekly for the control exposure system. Sample flow through each sample train was controlled using a needle valve connected to the facility vacuum source. The sample flow rate was measured prior to each sample using a mini-BUCK Calibrator (A.P Buck Inc.; Orlando, FL).
Each midget impinger contained approximately 10 mL of isopropanol as the trapping solvent. Following sample collection, additional isopropanol was not added to the impingers, and solvent from each impinger in the sampling train was combined. A 2-mL vial was filled with the combined trapping solution, capped, and was then analyzed using gas chromatograph equipped with flame ionization detector. Liquid sample injection (~2 µL) onto the chromatography column occurred via manual injection into a split/splitless injector, the chromatograph was displayed and the area under the sample peak was calculated and stored. The concentration, in micrograms per milliliter (µg/mL), was calculated using the ln-quadratic equation based on the GC calibration curve. Using the sample concentration in µg/mL, the exposure concentration in mg/m³ was calculated by multiplying the calculated concentration by the solution volume recovered from the impingers and dividing by the sample volume.

Exposure Atmosphere Concentration (mg/m³) = (A x B x 1000)/(C x D)
Where: A = Sample Concentration (µg/mL), B = Solution Volume (mL), 1000 = Conversion Factor, C= Sample Flow Rate (cc/min), D = Sample Time (min)

Mean analyzed concentrations (total test substance, aerosol plus vapor) for each exposure system for each sampling day are presented in Table 3.

Duration of treatment / exposure:

13 weeks (minimum of 65 exposures per animal)

Frequency of treatment:

6 hours per day, 5 days per week

Dose / conc.:

500 mg/m³ air (nominal)

Remarks:

mean analyzed exposure concentration: 514 mg/m³

Dose / conc.:

1 250 mg/m³ air (nominal)

Remarks:

mean analyzed exposure concentration: 1246 mg/m³

Dose / conc.:

2 500 mg/m³ air (nominal)

Remarks:

mean analyzed exposure concentration: 2467 mg/m³

No. of animals per sex per dose:

10 (main group)
5 (recovery group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The exposure concentrations were selected by the Sponsor Representative in consultation with the Study Director based on a previous 2-week inhalation study in which rats were exposed at 500, 1300 and 2500 mg/m³ of triethoxy(propyl)silane. In that study, there were no substance-related clinical observations or macroscopic findings. Nonadverse microscopic findings included minimal to mild hyaline droplet accumulation in the kidney of the 1300 and 2500 mg/m³ group males. No test substance-related microscopic findings were noted in the respiratory tract of either sex. Based on minimal or lack of test substance-related findings in the range-finding study and the need to produce a stable exposure atmosphere, the Sponsor elected to use exposure concentrations of 500, 1250, and 2500 mg/m³ for this study.
- Fasting period before blood sampling for clinical biochemistry: at least 8 h (no more than 24 h)
- Rationale for selecting satellite groups: To evaluate the recovery, persistence, or progression of any effects following a 4-week recovery
period.
- Post-exposure recovery period in satellite groups: 29 days

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality; 0.5-2 h post-exposure for health condition; at least once daily on non-exposure/recovery days


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: within 4 days of receipt, one week prior to randomization (± 2 days), on the day of randomization, on Day 1 (prior to exposure), weekly (± 2 days) during the study period, on the day of scheduled necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: within 4 days of receipt, one week prior to randomization (± 2 days), on the day of randomization, on Day 1 (prior to exposure), weekly (± 2 days) during the study period, on the day prior to the first day of scheduled necropsies, on the day of scheduled necropsy

FOOD CONSUMPTION: YES
- Food consumption was recorded once weekly (+/- 2 days) throughout the study period, beginning on Day 1

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during the pretreatment period (to include unused replacement animals), near the end of the dosing period (Day 86/85 for males/females)
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No
- How many animals: all
- Parameters checked: Red blood cell count, Hemoglobin concentration, Hematocrit, Mean corpuscular volume, Red blood cell distribution width, Mean corpuscular hemoglobin concentration, Mean corpuscular hemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells (absolute), Other cells (as appropriate), Activated partial thromboplastin time, Fibrinogen, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day of scheduled necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Creatine kinase, Total bilirubin, Urea nitrogen, Creatinine, Calcium, Phosphorus, Total protein, Albumin, Globulin (calculated), Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride

URINALYSIS: Yes
- Time schedule for collection of urine: on day of scheduled necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Color, Appearance/Clarity, Specific gravity, pH, Volume, Proteine, Glucose, Bilirubin, Ketones, Blood

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: on day of scheduled necropsy
- Dose groups that were examined: all
- Number of animals: all
- Parameters checked: Alkaline phosphatase (ALP), Differential cell counts for Macrophages, Neutrophils, Lymphocytes, Eosinophils and Basophils; Lactate dehydrogenase (LDH), Total protein

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)
Main and Recovery Study animals were subjected to a complete necropsy examination, which included evaluation of the carcass; all external surfaces and orifices; the cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

ORGAN WEIGHTS: Yes (see table 1)
Paired organs were weighed together. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

HISTOPATHOLOGY: Yes (see table 1)
Tissues identified in table 1 for microscopic examination were evaluated from all animals in the control and high-dose groups at the terminal and recovery necropsies. Gross lesions and target tissue (kidneys in males only) were prepared from all animals in the low- and mid-dose groups at the terminal necropsy. In addition, the right kidney from all males at the terminal and recovery necropsies were examined and semi-quantitatively scored for the amount of positive alpha 2u-globulin staining.

Statistics:

Body weight, body weight gains, food consumption, hematology variables, coagulation variables, clinical chemistry variables, urinalysis variables, bronchoalveolar lavage fluid variables, organ weights, organ weight relative to body weight, and organ weight relative to brain weight were assessed using parametric/non-parametric methods. Levene's test was used to assess the homogeneity of group variances. The groups were then compared using an overall one-way ANOVA F-test if Levene's test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett's or Dunn's test respectively.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related clinical observations. All clinical observations in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necropsies.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant mean body weight gains were noted in the 500 mg/m³ group males and females during the exposure period when compared to the control group but differences were attributed to biological variability and were not considered related to test substance administration due to lack of a dose-response trend. In addition, the 2500 mg/m³ group females selected for the recovery necropsy showed a lower mean body weight when compared to the control group, but this remained consistent through each interval; therefore, this was not considered test substance-related, but rather due to biological variability.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Minimally lower mean food consumption was noted in the 500 and 1250 mg/m³ group males and females and 2500 mg/m³ group females generally throughout the exposure period when compared to the control group; however, these differences were not considered to be test substance-related and were attributed to biological variation. In addition, some statistically significant differences were observed when the control and 2500 mg/m³ groups were compared during the exposure and recovery periods; however, these differences were also not considered to be test substance-related and were attributed to biological variation.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated groups. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related changes in hematology and coagulation parameters at any exposure concentration.
All differences in hematology and coagulation parameters, regardless of statistical significance, were not considered test substance-related based on their absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related changes in clinical chemistry parameters at any exposure concentration.
Mildly increased alanine aminotransferase and aspartate aminotransferase were noted in one 2500 mg/m³-female. These changes were not considered to be related to test substance administration based on low incidence and the potential contribution of leakage of muscle enzymes secondary to animal handling.
Remaining differences in clinical chemistry parameters were not considered test substance-related based on their absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test substance-related changes in urinalysis parameters at any exposure concentration.
All differences in urinalysis parameters, regardless of statistical significance, were not considered test substance-related based on their absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test substance-related organ weight changes were noted in the terminal and recovery euthanasias. There were isolated organ weight values that were statistically different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these
values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to test substance exposure.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test substance-related gross findings were noted at the terminal or recovery euthanasias. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to test substance exposure.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Terminal Euthanasia (Day 92)
Test substance-related histologic changes were limited to an exposure-dependent hyaline droplet accumulation within the kidneys in the 500, 1250, and 2500 mg/m³ group males at the terminal euthanasia (Table 2). Test substance-related findings of increased globular deposits of immunohistochemistry (IHC) positive material (alpha 2µ-globulin) were noted in the 500, 1250, and 2500 mg/m³ group males (Table 2). These findings were similar to the hyaline droplet accumulation noted on hematoxylin and eosin stained slides.
No test substance-related findings were noted in the respiratory tract of either sex.

In the kidney, tubular hyaline droplet accumulation was characterized by an increased number and prominence of round, distinctly eosinophilic, variably sized, and slightly refractile droplets within the cytoplasm of tubular epithelial cells within the cortex and outer stripe of the outer medulla (proximal convoluted tubules). This change was diffusely distributed and present in all of the test substance-treated male groups. There was an absence of associated inflammation or tubular degeneration/necrosis. While the presence of minimal hyaline droplets in the S2 segment of proximal convoluted tubules is a normal finding in male rats (Frazier et al., 2012), the
exposure concentration-related increased incidence and severity of hyaline droplet accumulation was considered an effect of test substance exposure.
Hyaline droplets represent low molecular weight protein accumulation within lysosomes due to disturbance of the normal balance of tubular reabsorption and hydrolysis as a result of either increased filtered protein load or decreased catabolism. These droplets may also represent chemical protein complexes. The hyaline droplet accumulation observed here is comparable to alpha 2u-globulin nephropathy; a condition specific to male rats. Special or immunohistochemical staining procedures were necessary for confirmation of alpha 2u-globulin nephropathy in this study and were performed. The findings of increased globular deposits of IHC positive material were similar to hyaline droplet accumulation noted on hematoxylin and eosin stained slides. The absence of associated inflammation or tubular degeneration/necrosis indicate that the hyaline droplet accumulation and increased globular alpha 2u-globulin present in the current study was a nonadverse effect of test substance exposure.
All other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to test substance exposure.

Recovery Euthanasia (Day 120)
Test substance-related histologic changes were noted in a single 2500 mg/m³ group male and globular deposits of IHC positive material (alpha 2u-globulin) were noted in 3 of the 2500 mg/m³ group males, suggesting that the changes in the kidney were largely reversible. As described previously, these changes were considered nonadverse.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

All differences in bronchoalveolar lavage fluid (BALF) parameters, regardless of statistical significance, were not considered test substance-related based on their absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.

Key result

Dose descriptor:

NOAEC

Effect level:

>= 2 500 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

other: IHC positive material (alpha 2u-globulin) in all dose groups

Key result

Dose descriptor:

NOEC

Effect level:

>= 2 500 mg/m³ air (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No effects observed.

Key result

Critical effects observed:

no

Table 2: Summary of Microscopic Findings (Kidney) – Terminal Euthanasia (Day 92)

	Males				Females
Target Exposure Concentration (mg/m³)	0	500	1250	2500	0	500	1250	2500
No. Animals per Group	10	10	10	10	10	10	10	10
Kidney (No. examined)	(10)	(10)	(10)	(10)	(10)	(0)	(0)	(10)
Accumulation, hyaline droplet Minimal Mild	0   0 0	4   4 0	10   3 7	10   0 10	0	-	-	0
Increased (globular) alpha 2u-globulin Minimal Mild	0   0 0	4   3 1	6   3 3	9   3 6	-	-	-	-

- = no noteworthy findings.

Table 3: Study Mean Analyzed Exposure Concentrations

Exposure System:		1		2		3		4
Target Concentration (mg/m^3):		0		500		1250		2500
Mean Concentration (mg/m^3):		0		514		1246		2467
Standard Deviation:		0.0		25.9		50.6		82.4
Number of Sampling Days:		13		66		66		66

Conclusions:

In a repeated nose-only inhalation study for the test substance triethoxy(propyl)silane (CAS 2550-02-9) conducted according to OECD 413 and to GLP (reliability score 1) the NOEC was considered to be ≥2500 mg/m³ in female rats and the NOAEC was considered to be ≥2500 mg/m³ in male rats.