Amino functional alkoxysilanes

Endpoint summary

Description of key information

No repeated dose toxicity studies are available for the registered substance, trimethoxy(methyl)silane and its reaction products with 3-aminopropyltriethoxysilane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane (EC 701 -410 -9 ), therefore data for its constituents have been used to address this endpoint and the data for the major constituent trimethoxy(methyl)silane (CAS 1185-55-3), present at 50 -70%. Since MTMS (and other alkyl alkoxysilanes) is the most prominent substance to which workers will be exposed during normal handling and use of the registered substance, and it gave the lowest NOAEC/Ls in repeated dose toxicity studies on relevant constituents, it is appropriate to use these data on MTMS to represent the repeated dose toxicity of the overall mixture as a worst-case for interim risk characterisation.

 

A 90-day oral repeated dose toxicity study (OECD Test Guideline 408) has been proposed with the registered substance. After approval by ECHA the test will be conducted and the risk characterisation updated using results from the new test data.

 

Block A

Trimethoxy(methyl)silane (CAS 1185-55-3) – data used for DNEL derivation for interim risk characterisation

 

A 90-day whole-body inhalation repeated dose toxicity study (Dow Corning Corporation, 2007) was conducted according to OECD Test Guideline 413 and in compliance with GLP, in which trimethoxy(methyl)silane (CAS 1185-55-3) was administered to rats six hours per day, five days per week. A NOAEC of 100 ppm (560 mg/m3) was determined based on an increased incidence of grossly observed urinary bladder calculi along with kidney dilation at the 400 ppm exposure concentration.

In the key oral Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with trimethoxy(methyl)silane (CAS 1185-55-3), conducted according to OECD Test Guideline 422 and in compliance with GLP (Dow Corning Corporation, 2005), the NOAEL for systemic effects was 50 mg/kg bw/day, based on findings in a number of organs including the liver and thymus gland.

 

Other constituents of the registered substance are aminofunctional substances. Their data relevant to the assessment of the registered substance have been included to aid selection for NOAEL/NOAEC selection for risk characterisation.

 

Block B

3-Aminopropyl(triethoxy)silane (CAS 919-30-2)

 

In a 90-day oral (gavage) repeated dose toxicity study with 3-aminopropyl(triethoxy)silane (CAS 919-30-2) (WIL Research Laboratories, 2001), conducted according to OECD Test Guideline 408 and in compliance with GLP, the NOAEL value was 200 mg/kg bw/day, based on mortality, clinical findings and liver effects seen at the highest dose of 600 mg/kg bw/day.

 

3-(Trimethoxysilyl)propylamine (CAS 13822-56-5)

 

In a 90-day oral (gavage) repeated dose toxicity study with 3-(trimethoxysilyl)propylamine (CAS 13822-56-5), conducted according to OECD Test Guideline 408 and in compliance with GLP, the NOAEL for systemic toxicity was 300 mg/kg bw/day, based on the mortalities observed at 1000 mg/kg bw/day dose, although the mortalities are likely to have resulted from local effects rather than systemic toxicity (Charles River, 2018).

 

Trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine (EC No. 701 -408 -8)

 

In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP for trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine (EC 701 -408 -8) the NOAEL was 75 mg/kg bw/day based on treatment-related lower motor activity observed in main and recovery females at 250 mg/kg bw/day in comparison with that in females in the other groups. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19.11.2003 to 19.05.2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

other: USEPA OPPTS 870.3650

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: No data
- Age at study initiation: Nine weeks
- Weight at study initiation: Males: 294.2-351.5; Females: 200.2-260.2 g
- Fasting period before study: None
- Housing: individually housed in suspended wire-mesh cages (pregnant rats in shoebox cages)
- Diet (e.g. ad libitum): Ad libitum (except during FOB)
- Water (e.g. ad libitum): Ad libitum (except during FOB)
- Acclimation period: Six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2-22.5
- Humidity (%): 36.0-62.0
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 09.02.2004 To: 19.04.2005

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Conducted over nitrogen atmopshere. Test substance was placed into a volumetric flask and corn oil added to achieve the desired volume. The weight of the test substance added to the flask was used to calculate nominal dose solution concentrations. Dosing solutions were prepared at least once every two weeks consistent with the previously determined 15-day stability. The concentration, homogeneity and stability of the test substance in vehicle for at least 15 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): No data
- Concentration in vehicle: Various
- Amount of vehicle (if gavage): Up to 3 ml/kg
- Lot/batch no. (if required): 122K0131
- Purity: Considered 100%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of methyltrimethoxysilane (MTMS) in corn oil dosing solutions was determined prior to the beginning of the definitive study.

Duration of treatment / exposure:

Toxicity group females and males were treated for 28 and 29 days, respectively. Reproductive group females were treated for 14 days prior to the mating period, during the mating period, and then up to and including post partum day 3, for a total of up to 51 days.

Frequency of treatment:

Daily, seven days/week

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

corn oil

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a range-finding study
- Rationale for animal assignment (if not random): Weight stratified randomisation process
- Rationale for selecting satellite groups: According to OECD TG 422
- Post-exposure recovery period in satellite groups: None

Observations and examinations performed and frequency:

Mortality/Morbidity: Animals were observed at least twice daily in their cages for moribundity and mortality throughout the in-life phase of the study.

Daily Observations: General clinical examinations were made at lease once a day and were conducted immediately after dosing. The examinations included, but were not limited to, changes in the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system functions, motor activity and behavior patterns. Findings were recorded for individual animals. General clinical examinations were not performed on days when detailed physical examinations were performed.

Detailed Physical Examinations: All animals received a detailed physical examination once before the first dose administration (to allow for within-subject comparisons), and weekly thereafter. Examinations were made outside the home cage in a standard arena at approximately the same time each day. Observations were detailed and carefully recorded. Examinations included, but were not limited to, changes in skin, fur eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity. Changes in gait, posture and response to handling as well as the presence of clonic or tonic movement, stereotypies, difficult or prolonged parturition or bizarre behavior were recorded. The presence or absence of findings was recorded for individual animals.

Body weights and food consumption were recorded weekly. Additional body weights for reproductive group females were obtained on gestational day 0, 7, 14, and 20, and within 24 hours of parturition, and on postnatal day 4. Individual food consumption was determined for each group following group specific schedules.

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 day of scheduled termination.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: Males and toxicity phase females
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On day of scheduled termination
- Animals fasted: Yes
- How many animals: Males and toxicity phase females
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the start of dosing and during the  last week of dosing. 
- Dose groups that were examined: adult males and  all toxicity group females
- Battery of functions tested: Unusual body movements, abnormal behaviour and/or posture, and resistence to removal, palperal closure, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, reactivity to handling, level of ambulatory activity including rearing, responsiveness to sharp noise, touch, tail pinch, gait evaluation and quantity of urine and fecal pellets voided, behavior, skin or hair coat, respiration, muscle movements, eyes, urine or feces, soiling, general abnormalities and posture, rectal temperature, hindlimb/forelimb grip strength and landing foot splay. Motor activity assessment involved obtaining baseline motor activity data prior to the first dose administration to assist in evaluating potential treatment-related effects by allowing each rat to act as its own control.

Sacrifice and pathology:

Males and toxicity group females were sacrificed after they had been  treated for 28 days.  Reproductive group females and pups were sacrificed  
on day 4 postpartum.  Complete necropsies were performed on the males and the toxicity group females and selected organs were weighed (adrenals, brain, heart, kidneys, liver, lungs, spleen, thymus, uterus, ovaries, testes, prostate, seminal vesicles, and epididymides). Microscopic examination was performed on protocol-specified tissues from all animals in the control and high dose group males and toxicity group females. Microscopic examination of the liver, thyroid, jejunum and duodenum was extended to males and toxicity group females in the 50 and 250 mg/kg dose groups. In addition, the adrenal gland from these middle dose groups was evaluated for the toxicity group females.

Statistics:

All data analyses were carried out using SAS version 8.2. Statistically significant probabilities were reported for /-values of <0.05, <0.02 and <0.01.

Body weight, body weight changes, organ weight, organ to body weight ratios, food consumption, hematology data, clinical chemistry and prothrombin times were analyzed using a one-way Analysis of Variance (ANOVA) if the data satisfied the requirements of normality of the residuals and homogeneity of variance as determined using the Shapiro-Wilk test for normality and Levene’s test for homogeneity of variance. If the data did not satisfy the parametric requirements, a Kruskal-Wallis test was used. If the ANOVA or Kruskal-Wallis test was significant, pair-wise comparisons of the dosed groups to control were made using the Dunnett’s Test or a Wilcoxon test, respectively.

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs included transient inactivity or salivation following dosing. 

Mortality:

mortality observed, treatment-related

Description (incidence):

There were two unscheduled deaths (one female each at 0 and 50 mg/kg  bw/day); both were related to dosing errors. 

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decreases in body weight gain and food consumption were noted for 1000 mg/kg bw/day group males. 

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

A marked increase in prothrombin time was observed for males at 250 and 1000 mg/kg bw/day whereas females were unaffected.  Exposure was also associated with increased blood platelet concentration for males and females, and increased red blood cell concentration in males at 1000  mg/kg bw/day.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

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):

Increased liver weight was observed for male and female animals at 250 and 1000 mg/kg bw/day.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Exposure to MTMS was associated with organ weight and/or histomorphological changes in males (liver, thymus, thyroid, duodenum, jejunum) and females (liver, thyroid, duodenum, jejunum, and adrenal gland) at dose levels at or above 250 mg/kg bw/day. 

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects on liver, thymus, thyroid, duodenum, jejunum and adrenals

Critical effects observed:

not specified

Toxic Response/Effects by Dose Level:

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

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

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

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

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

Ancanthocytosis (incidence 4*/10)

Increased blood platelet concentration: 21%*

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

Conclusions:

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

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1999-10-11 to 2000-03-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Labs, Raleigh, NC, USA
- Age at study initiation: 7 wk
- Weight at study initiation: 213-279 g (m); 150-207 g (f)
- Housing: 1/suspended wire mesh cage
- Diet: standard diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70.2-73.3 deg F
- Humidity (%): 35.2-58.5
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 1999-10-11 To: 2000-01-11

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: vehicle dispensed daily; mixed with magnetic stirrer

Dose volume: 10ml/kg bw/day

VEHICLE
- Justification for use and choice of vehicle: peanut oil sparged with nitrogen
- Concentration in vehicle: 0, 7, 20, 60 mg/ml
- Lot/batch no. : NQ0052 and NG0346

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

atomic absorption spectroscopy

Duration of treatment / exposure:

91 or 92 consecutive days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

70 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: Not applicable. All animals were euthanized and necropsied upon completion of the treatment period; no recovery or other satellite groups were included.
- Dose selection rationale: In a 14 -day range-finding study (see EPSR Repeated dose toxicity: oral 7.5.149 Momentive), the TS was administered in peanut oil by gavage to three groups of rats (5/sex) at 70, 200 and 600 mg/kg bw/day. A concurrent control group received the vehicle on a comparable regimen. All animals were dosed at a volume of 10 mL/kg. The animals were observed twice daily for mortality and moribundity. Clinical exams were performed daily and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded weekly. Complete necropsies were performed on all animals and selected organs were weighed. Selected tissues were examined microscopically from all animals. Survival was unaffected by gamma-aminopropyltriethoxysilane administration. No test-article related findings were observed at the macroscopic or microscopic examinations. Organ weights were unaffected by treatment. Clinical signs included one or more instances of rales and/or labored respiration in the 600 mg/kg bw/day group. These findings were noted in two males and two females during the last three days of dosing prior to necropsy. There were no statistically significant differences in body weights, body weight gains, or food consumption. However, there were reductions in body weight gains and food consumption in the 600 mg/kg bw/day males during weeks 0 to 1 and 1 to 2. The no-observed-adverse-effect level (NOAEL) was 200 mg/kg bw/day. Dose levels of 70, 200 and 600 mg/kg bw/day were selected for the 90-day oral study.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
Food intake was calculated as g/animal/day for the corresponding body weight intervals. When food consumption could not be measured for a given interval (due to spillage, weighing error, etc.), the appropriate interval was footnoted as "NA" (Not Applicable) on the individual tables.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and during wk 12
- Dose groups that were examined: . Ocular examinations were conducted on all animals. All ocular examinations were conducted using an indirect ophthalmoscope, preceded by pupillary dilation with an appropriate mydriatic agent
HAEMATOLOGY: Yes
- Time schedule for collection of blood: wks 4, 13
- Animals fasted: Yes, overnight
Blood samples for general clinical pathology evaluations were collected from a lateral tail vein at both time points. Blood samples for assessment of coagulation parameters were collected from the vena cava at the time of necropsy. The following hematology parameters were evaluated: total leukocyte count (white cell), erythrocyte count (red cells), hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, prothrombin time, activated partial thromboplastin time (APTT; terminal evaluation only), reticulocyte count (percent and absolute), differential leukocyte count (percent and absolute: neutrophil, lymphocyte, monocyte, eosinophil and basophil).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: wks 4, 13
The following serum chemistry parameters were evaluated: alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, blood urea nitrogen, total protein, total bilirubin, creatinine, Ca, Na, K, Cl, P, glucose, albumin, globulin, albumin/globulin ratio, and total cholesterol.

URINALYSIS: Yes
- Metabolism cages used for collection of urine: Yes
Urine samples were collected via metabolism chambers following the eighth exposure of female rats and following the seventh exposure of male rats. Urine volume was measured using calibrated test tubes, and urine color and turbidity were visually assessed. Urinalysis parameters were urine osmolality, pH, protein, glucose, ketone, bilirubin, blood and urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Vaginal smears for determination of the stage of estrus were obtained from all surviving females once daily beginning 21 days prior to the scheduled necropsy. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P]) beginning 21 days prior to the scheduled necropsy. The final vaginal smear for each female was collected on the day of necropsy.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete necropsy was conducted on all animals. The necropsy included, but was not limited to, examination of the external surface, all orifices and the cranial, abdominal and pelvic cavities and their viscera.

HISTOPATHOLOGY: Yes
At the time of necropsy, the following tissues and organs were collected and preserved in 10% neutral buffered formalin: adrenals (2), aorta, bone with marrow (femur, sternebrae), bone marrow smear (from femur), brain (forebrain, midbrain, hindbrain), coagulating gland, eyes with optic nerve (2; preserved in Davidson's solution), gastrointestinal tract (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum), heart, kidneys (2), liver (sections of two lobes), lungs (including bronchi, fixed by inflation with fixation), lymph node (mesenteric, submandibular), mammary gland (females only), ovaries with oviducts (2), pancreas, peripheral nerve (sciatic), pituitary, prostate, salivary glands (submaxillary, 2), seminal vesicles (2), skeletal muscle (vastus medialis), skin, spinal cord (cervical, midthoracic, lumbar), spleen, testis with epididymis (1) and vas deferens, thymus, thyroid (with parathyroids if present (2)), trachea, urinary bladder, uterus with vagina and cervix, and all gross lesions (when possible). Bone marrow smears were obtained from all animals not found dead, but were not placed in 10% neutral buffered formalin. The right testis/epididymis from all males at the scheduled necropsy and both testes/epididymides from those males found dead were preserved in Bouin's solution and prepared for microscopic examination using PAS/hematoxylin staining. The left testis/epididymis from all males euthanized at the scheduled necropsy were prepared for sperm analysis as described below. The following organs from animals euthanized at the scheduled necropsy were weighed: adrenals, brain, epididymides (weighed separately; total and cauda), kidneys, liver, ovaries (with oviducts), pituitary, prostate, seminal vesicles with coagulating glands (with accessory fluids), testes (weighed separately), and thyroid (fixed weight). Organ to final body weight and organ to brain weight ratios were calculated. The tissues noted above from all animals found dead or euthanized in extremis and from all animals in the control and 600 mg/kg/day groups euthanized at the scheduled necropsy, as well as the lungs, liver, and kidneys from all animals in the 70 and 200 mg/kg/day groups were examined microscopically.

In addition, PAS/hematoxylin-stained sections of the right testis and epididymis from all males were examined microscopically at the scheduled necropsy. Spermatogenic analysis was conducted according to the following protocol. For motility/viability assessment, immediately following euthanasia, the reproductive tract of each male was exposed via a ventral mid-line incision. The right epididymis was excised and weighed separately. An incision was made in the distal region of the cauda epididymis. The cauda was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37oC) with 10 mg/ml Bovine Serum Albumin (BSA). A sample of the diluted sperm was then loaded into a 100 µm cannula for determination of motility. As sperm motility can be affected by temperature shock, all cannulas, diluents and slides were pre-warmed and maintained at approximately 37oC. Motility eterminations were performed under constant temperature using the Hamilton-Thorne HTM-IVOS Version 10 computer-assisted sperm analysis (CASA) system. At least 200 (if possible) motile and nonmotile spermatozoa/animal were analyzed. A sample of sperm for morphology assessment was obtained from the right cauda epididymis of each male. Sperm morphology was evaluated using a modification of the wet-mount technique described by Linder et al., 1992. Abnormal forms of sperm (double heads, double tails, micro- or megacephalic, etc.) were recorded from a differential count of 200 spermatozoa/animal. For enumeration of epididymal and testicular sperm numbers and sperm production rates, the left testis and epididymis from each male at the scheduled necropsy were weighed and frozen, then homogenized and evaluated for sperm production rate using the method described by Blazak et al., 1985. Analyses were performed using the Hamilton-Thorne CASA system.

Statistics:

All analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5% comparing the test article-treated groups to the control group by sex. All means were presented with standard deviations (S.D.) and the number of sampling units (N) used to calculate the means. Statistical analyses were not conducted if the number of animals was two or less. All statistical tests were performed using appropriate computing devices or programs. Body weight, body weight change, food consumption, clinical pathology, absolute and relative organ weight data and epididymal and testicular sperm numbers and sperm production rates were subjected to a one-way analysis of variance (ANOVA), followed by Dunnett's test if the ANOVA revealed statistical significance (p<0.05). The percentage of motile spermatozoa and the percentage of sperm with normal morphology were analyzed by the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences, followed by the Mann-Whitney U-Test comparing the control and test article-treated groups if the ANOVA revealed statistical significance (p<0.05). Clinical laboratory values for leukocytes that occur at a low incidence (i.e., monocytes, eosinophils and basophils) were not subjected to statistical analysis.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The animals that died had laboured breathing/gasping, partially closed eyes, pallor, hypothermia, dermal atonia and/or tremor. The predominant clinical sign in surviving animals was rales (rattling/crackling sound during breathing) in the high dose group. Also at this dose, wet and/or dried material on various parts of the body and abnormal excreta were observed. These signs were observed sporadically at lower doses. (The deaths of one control male, one 200 mg/kg bw/day female and two 600 mg/kg bw/day males were due to dosing error or not considered treatment-related.)

Mortality:

mortality observed, treatment-related

Description (incidence):

Deaths of 1 male and 8 females at the top dose (600 mg/kg bw/day) were believed to be treatment-related.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No clear treatment-related effects were reported on: body weight gain, food consumption, oculopathy, haematology parameters, oestrous cycle data or spermatogenic endpoints. 

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased mean aspartate aminotransferase (AST) values for males at wk 13 and alanine aminotransferase (ALT) for both sexes at wks 4 and 13 (greatest at wk 13), were associated (in males) with liver weight increases and cellular changes seen on microscopic examination.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Transient or sporadic variations in serum urea nitrogen and sodium were not considered biologically significant.    

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased liver weights in high dose males (non statistically significant increase in mean liver:brain weight; statistically significant increase in liver:body weight compared to controls) correlated with increased ALT and AST and hepatocellular vacuolation evident on microscopic examination. Increased adrenal weight (absolute, adrenal:brain, adrenal:body weight) in 600 mg/kg bw/day males was not associated with macroscopic, microscopic, haematologic or clinical chemistry findings, and was considered spurious.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

For one male and most females that died prior to scheduled necropsy, macroscopic changes included distension of various parts of the gut. The only macroscopic change at scheduled necropsy was an increased incidence of gaseous distension of the gut, primarily of the cecum, in 7/12 males and 2/7 females in the high dose group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic examination revealed changes to the liver of 600 mg/kg bw/day males consisting of centrilobular to generalized hepatocellular vacuolation. No other test article-related microscopic changes were observed.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Based on mortality, clinical signs and liver effects at 600 mg/kg bw/day.

Dose descriptor:

LOAEL

Effect level:

600 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

clinical signs

histopathology: non-neoplastic

mortality

Critical effects observed:

not specified

Table 1: Summary of mortalities

	Dose (mg/kg bw/day)
	Control		70		200		600
	male	female	male	female	male	female	male	female
died	1/15*	0/15	0/15	0/15	0/15	1/15*	1/15 2/15*	8/15
week of study	4*		-		-	9*	2*, 7	1-6

* dosing error

Conclusions:

In a well-reported 90-day oral repeated dose toxicity study, conducted according to OECD Test Guideline 408 and in accordance with GLP, identified a NOAEL value of 200 mg/kg bw/day in male and female rats based on mortality, clinical observations and liver effects at 600 mg/kg bw/day.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

4th December 2017 to 1st March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

19998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Remarks:

(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: 120 to 184 g.
- Fasting period before study: no
- Housing: polycarbonate cages (Makrolon type IV, height 18 cm) with appropriate bedding (Lignocel S 8-15, JRS), up to 5 animals of same sex and dose per cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
Food was Pelleted rodent diet (SM R/M-Z) from SSNIFF® Spezialdiäten GmbH, Soest, Germany. Water was municipal tap water and water analyses was performed periodically.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23°C
- Humidity (%): 37-59%
- Air changes (per hr): Ten or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 Feb 2018 To: 16 May 2018

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Dehydrated and deacidified, specific gravity 0.92

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

The dosing formulations were prepared daily as a solution and dosed within 1 hour after adding vehicle to the test item. As aspiration is a known hazard with the oral dosing of this type of test item (aminosilane), the feeding tube was thoroughly cleaned before administering the test item to the animals and the total volume was dispensed before the feeding tube was withdrawn. The first day of dosing was designated as Day 1.
The dosing formulations were stirred continuously during dose administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): none given in study report
- Concentration in vehicle: 20, 60 and 200 mg/mL for 100, 300 and 1000 mg/kg bw/day
Adjustment was made for specific gravity of the vehicle (0.92) and test item (1.0140). No correction was made for the purity/composition of the test item.
- Amount of vehicle (if gavage): Dosing volume: 5 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Doses were collected for concentration (all groups) and Homogeneity (low and high dose groups) in Week 1, Week 2 and Week 13.
Concentration Analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. In Week 2, additional duplicate top, middle, and bottom samples of Group 2, using pre-weighed test item, were measured. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. In Week 2, additional duplicate top, middle, and bottom samples of Group 2, using pre-weighed test item, were measured. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ±10%.
Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (Test Facility Study No. 20136855) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Test Facility Study No. 20136855.

Duration of treatment / exposure:

90 days (minimum)

Frequency of treatment:

Once daily, seven days a week.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

10 animals per sex per group in each main group, with an additional 5 animals per sex in recovery groups (vehicle and 1000 mg/kg bw groups).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: limit dose
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: none given in study report
- Post-exposure recovery period in satellite groups: two weeks
- Section schedule rationale (if not random): Random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION
- Food consumption was quantitatively measured weekly starting on Day 1 and continuing weekly throughout the Dosing and Recovery Periods.

WATER CONSUMPTION
- Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during pre-treatment in all animals and at end of dosing period in vehicle and high dose animals.
- Dose groups that were examined: The eyes were examined using an ophthalmoscope after application of a mydriatic agent (Tropicol 5 mg/mL solution, THEA Pharma, Wetteren, Belgium) during pre-treatment in all Main Study and Recovery animals, and at the end of the Dosing Period in Week 13 in all Group 1 (vehicle) and 4 (high dose) Main study and Recovery animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment (all animals) end of recovery (vehicle and high dose animals only)
- Anaesthetic used for blood collection: yes - isoflurane
- Animals fasted: Yes
- How many animals: 10 per group (end of treatment); 5 per group (end of recovery)
- Parameters checked in Table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment (all animals) end of recovery (vehicle and high dose animals only)
- Animals fasted: Yes / No / Not specified
- How many animals: 10 per group (end of treatment); 5 per group (end of recovery)
- Parameters checked in Table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once, during week 12-13
- Dose groups that were examined: all groups, 5 animals per sex per group
- Battery of functions tested: hearing ability / pupillary reflex / static righting reflex / fore- and hind-limb grip strength / locomotor activity

IMMUNOLOGY: No

OTHER: Oestrous cycle determination: Daily vaginal lavage was performed for all females from Week 11 (Day 71) up to and including Week 13 (Day 91).
Oestrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (see Table 3)

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analysed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 (low dose) vs. Group 1 (vehicle)
Group 3 (mid dose) vs. Group 1 (vehicle)
Group 4 (high dose) vs. Group 1 (vehicle)
Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).
Non-Parametric
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.
Incidence
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Slight to (incidentally) moderate rales were noted for males and females treated at 100 mg/kg/day and higher, with a dose-related increase in incidence (i.e. few animals affected on some occasions at the low dose level to most or all animals affected on many occasions at the high dose level).
In addition, hunched posture, laboured or deep respiration, gasping, squeaking, piloerection, diarrhoea and/or ptosis were noted on a few occasions in individual males and females at 1000 mg/kg/day.
In females at 1000 mg/kg/day, these signs were no longer present after cessation of treatment, whereas laboured respiration, rales, piloerection and ptosis were still observed in males on some occasions during the recovery period.
Salivation seen after dosing among males and females treated at 100 mg/kg/day and higher was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.

Mortality:

mortality observed, treatment-related

Description (incidence):

Six premature deaths from the 1000 mg/kg/day dose group between Days 62 and 87. One male was found dead (Animal No. 45) and one male (Animal No. 42) and four females (Animal Nos. 92, 96, 98, and 100) were euthanized in extremis. All animals had respiratory clinical observations prior to death (rales, gasping, abdomen distended with gas, and/or deep/laboured breathing). Other clinical signs noted prior to death included hunched posture, piloerection, diarrhoea, abdomen filled with gas and/or lean appearance. Except for animal No. 92, a severe body weight loss up to 22% was observed in all found dead or euthanized animals. All but one (Animal No. 92, euthanized in extremis) had distention of the gastrointestinal tract with gas at necropsy. Due to the suspicion of gavage-related reflux, the caudal nasal cavities and nasopharynx were evaluated in these animals, in addition to the protocol tissue list.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights and body weight gain were decreased with statistical significance for males at 1000 mg/kg/day from Weeks 5 and 7 onwards, respectively, and this persisted until the end of the 2-week recovery period (body weight gain was 0.84x of controls at the end of treatment and 0.83x at the end of recovery).

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of treatment, increased neutrophil count (1.73x and 2.50x for males and females, respectively) and/or monocyte count (males, 2.00x of controls) were noted at 1000 mg/kg/day.
At the end of the 2-week recovery period, neutrophil count was still increased in both sexes, but without reaching statistical significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of treatment, alanine aminotransferase (ALAT) and aspartate aminotransferase activity (ASAT) were slightly increased in males at 1000 mg/kg/day (2.02x and 1.36x of controls, respectively).
In addition, decreased total protein (0.96x and 0.92x in males and females, respectively) and albumin levels (0.92x in females), and increased bile acids (1.66x in females) were noted. Sodium and chloride concentrations were increased in all treated male groups, however lacking a dose relationship (1.06x, 1.06x and 1.04x for sodium and 1.07x, 1.05x and 1.03x for chloride at dose levels of 100, 300 and 1000 mg/kg/day, respectively). In females, this was only noted for sodium (1.01x for all dose levels).
At the end of the 2-week recovery period, all above mentioned parameters were similar between animals treated at 1000 mg/kg/day and controls.
It should be noted that high alanine aminotransferase and aspartate aminotransferase activity were noted for two individual females at 1000 mg/kg/day (Animal Nos. 94 and 95).
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Urinalysis findings:

not examined

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):

Test item-related lower liver weights were noted in females at the end of the treatment period in the 1000 mg/kg/day group as shown in Table 4.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings after treatment were noted in the larynx, nasal cavity, nasopharynx, stomach, large intestine, mesenteric lymph node of males and females and Harderian gland in males and are summarized in Table 5 to Table 12.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

An increased incidence in irregular oestrous cycle was noted in a few females at 1000 mg/kg/day and an acyclic oestrous cycle was noted in one female each at 300 and 1000 mg/kg/day. These changes are considered to be adverse although they may be secondary to reduced body weights secondary to local effects.

Details on results:

Microscopic effects in larynx and caudal nasal cavity that were observed at all dose levels are consistent with gavage related reflux. Changes were noted during microscopic examination of the large intestines and stomach, and in mesenteric lymph node and harderian glands. Microscopic changes were noted in the stomach from 300 mg/kg bw/day and in the cecum at 1000 mg/kg bw/day.
Following the 2-week treatment free period there was partial recovery of the clinical signs and microscopic findings in the larynx, stomach, and large intestines; no recovery of the mesenteric lymph node; and full recovery of the Harderian gland.
The adverse histologic changes in the upper airways noted starting at 100 mg/kg/day were considered by the study author to be strongly suggestive of a pathogenesis that is related to a combination of the irritant nature of the test item, the administration procedure, and the test species. While this may be important for dose selection for future rat studies, this may not necessarily be relevant to other species.
Based on these results, the no-observed-adverse-effect level (NOAEL) is considered to be 100 mg/kg/day.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Changes in oestrous cyclicity observed at 1000 and 3000 mg/kg bw/day.

Dose descriptor:

LOAEL

Remarks:

Local effects

Effect level:

>= 0 - < 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

no

Table 4 Mean Percent Liver Differences from respective Control Groups

	Females
	Main Study			Recovery
Dose level (mg/kg/day)	100	300	1000	1000
BODY WEIGHT	-1	0	-3	0
LIVER
Absolute	1	-2	-14^**	-4
Relative to body weight	2	-2	-12^**	-4

*: P<0.05, **: P<0.01

Table 5 Summary Test Item-Related Microscopic Findings in the Larynx, Nasopharynx and Nasal Cavity – Scheduled Euthanasia Animals (Day 91-92)

	Males				Females
Dose level mg/kg bw/day	0	100	3000	1000	0	100	300	1000
Larynx^a	10	10	10	8	10	10	10	8
Erosion/ulceration	0	0	5	4	0	1	0	0
Minimal	-	-	4	1	-	1	-	-
Slight	-	-	1	3	-	-	-	-
Metaplasia squamous	0	0	1	8	0	0	1	8
Minimal	-	-	1	2	-	-	-	2
Slight	-	-	-	2	-	-	1	3
Moderate	-	-	-	4	-	-	-	3
Inflammation acute	0	0	3	1	0	1	0	0
Minimal	-	-	2	1	-	1	-	-
Slight	-	-	1	-	-	-	-	-
Inflammation, chronic	0	0	0	4	0	0	0	3
Minimal	-	-	-	2	-	-	-	-
Slight	-	-	-	2	-	-	-	3
Hyperplasia, epithelial	0	0	4	1	0	0	0	2
Minimal	-	-	3	-	-	-	-	-
Slight	-	-	1	1	-	-	-	2
Pharynx, nasal^ab	0	3	3	0	0	6	3	0
Squamous metaplasia	-	0	0	-	-	0	1	-
Minimal		-	-			-	1
Exudate	-	1	1	-	-	2	1	-
Minimal		-	-			1	1
Slight		1	1			1	-
Erosion/ulceration	-	0	2	-	-	1	0	-
Slight		-	1			1	-
Moderate		-	1			-	-
Nasal Cavity^ab	0	3	3	0	0	6	3	0
Turbinate fusion	-	2	2	-	-	6	2	-
Minimal		-	-			-	2
Slight		1	1			5	-
Moderate		1	1			1	-
Exudate	-	1	2	-	-	2	2	-
Minimal		-	-			1	-
Slight		1	2			1	2
Erosion/ulceration	-	0	2	-	-	3	1
Minimal		-	-			1	-
Slight		-	2			2	1

^a = Number of tissues examined from each group.

^b = Tissues examined only from a select number of test item-treated animals per group.

Table 6 Test Item-Related Microscopic Findings in the Larynx – Recovery Euthanasia Animals (Day 105)

	Males				Females
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Larynx^a	10			5	10			3
Metaplasia squamous	0			4	0			1
Minimal	-			1	-			1
Slight	-			3	-			-
Inflammation, chronic	0			2	0			0
Minimal	-			2	-			-

^a = Number of tissues examined from each group.

Table 7 Test Item-Related Microscopic Findings in the Stomach – Scheduled Euthanasia Animals (Day 91-92)

	Males				Females
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Stomach^a	10	10	10	8	10	10	10	8
Erosion/ulceration	0	0	0	3	0	0	1	0
Minimal	-	-	-	3	-	-	1	-
Basophilia, mucosa	0	0	0	5	0	0	0	4
Minimal	-	-	-	2	-	-	-	2
Slight	-	-	-	3	-	-	-	2
Infiltrate, inflammatory cell	3	0	0	6	0	0	1	2
Minimal	3	-	-	2	-	-	-	-
Slight	-	-	-	4	-	-	1	2
Hemorrhage	1	0	0	4	0	0	0	1
Minimal	1	-	-	3	-	-	-	1
Slight	-	-	-	1	-	-	-	-

^a = Number of tissues examined from each group.

Table 8 Test Item-Related Microscopic Findings in the Stomach – Recovery Euthanasia Animals (Day 105)

	Males				Females
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Stomach^a	5	-	-	5	5	-	-	3
Basophilia, mucosa	0			0	0			1
Minimal	-			-	-			1
Infiltrate, inflammatory cell	0			1	1			0
Minimal	-			-	1			-
Slight	-			1	-			-

^a = Number of tissues examined from each group.

Table 9 Test Item-Related Microscopic Findings in Large Intestine and Mesenteric Lymph Node – Scheduled Euthanasia Animals (Day 91-92)

	Males				Females
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Cecum^a	10	10	10	8	10	10	10	8
Basophilia, mucosal	0	10	10	8	0	7	7	8
Minimal	-	4	-	1	-	6	5	-
Slight	-	6	8	4	-	1	2	3
Moderate	-	-	2	3	-	-	-	5
Single cell necrosis, mucosa	0	0	0	8	0	0	0	6
Minimal	-	-	-	1	-	-	-	5
Slight	-	-	-	-	-	-	-	1
Colon^a	10	10	10	8	10	10	10	8
Basophilia, mucosal	0	0	0	0	0	0	0	1
Slight	-	-	-	-	-	-	-	1
Rectum^a	10	10	9	8	10	10	10	8
Basophilia, mucosal	0	0	2	6	0	0	1	8
Minimal	-	-	2	3	-	-	1	7
Slight	-	-	-	3	-	-	-	1
Mesenteric lymph node^a	10	10	10	8	10	10	10	8
Macrophage aggregate, increased	1	1	3	6	0	1	3	5
Minimal	1	1	3	2	-	1	3	4
Slight	-	-	-	4	-	-	-	-
Moderate	-	-	-	-	-	-	-	1

^a = Number of tissues examined from each group.

Table 10 Test Item-Related Microscopic Findings in Large Intestine and Mesenteric Lymph Node – Scheduled Euthanasia Animals (Day 105)

	Males				Females
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Cecum^a	5	-	-	5	5	-	-	3
Infiltration, inflammatory cell	0			0	0			2
Minimal	-			-	-			-
Slight	-			-	-			2
Colon^a	5	-	-	5	5	-	-	3
Basophilia, mucosal	0			3	0			1
Slight	-			3	-			1
Mesenteric lymph node^a	5	-	-	5	5	-	-	3
Macrophage aggregate, increased	4			5	1			2
Minimal	4			3	1			1
Slight	-			2	-			1

^a = Number of tissues examined from each group.

Table 11 Test Item-Related Microscopic Findings in the Harderian gland – Scheduled Euthanasia Animals (Day 91-92)

	Males				Females
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Harderian gland^a	10	10	10	8	10	10	10	8
Atrophy	1	2	0	4	0	1	0	1
Minimal	1	2	-	2	-	-	-	-
Slight	-	-	-	2	-	1	-	1

^a = Number of tissues examined from each group.

 

Table 12 Summary Test Item-Related Microscopic Findings in the Harderian gland– Scheduled Euthanasia Animals (Day 105)

	Males				Females
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Harderian gland^a	5	-	-	5	5	-	-	3
Atrophy	1			1	0			0
Minimal	1			1	-			-

^a = Number of tissues examined from each group.

Refer to the attached files for summary, individual and histopathology result tables.

Conclusions:

3-(Trimethoxysilyl)propylamine has been administered to rats once daily by oral gavage for 90 days, in a study conducted according to OECD TG 408 and in compliance with GLP (Charles River Laboratories, 2018). In this study the high dose of 1000 mg/kg/day was not well tolerated with unscheduled deaths in males and females at 1000 mg/kg bw/day and adverse clinical signs of respiratory distress with correlating microscopic findings affecting the larynx (starting at 300 mg/kg bw/day), nasal cavities and/or nasopharynx (starting at 100 mg/kg bw/day). Adverse microscopic findings were also noted in the stomach (starting at 300 mg/kg bw/day), and cecum (at 1000 mg/kg bw/day).
The clinical/microscopic signs of respiratory distress (due to reflux) and effects in the stomach (and cecum) were considered to be adverse but secondary to the local site-of-contact effects of the test item, rather than a systemic effect of the test item. Therefore, these findings are not considered in the derivation of the NOAEL. Based on these effects, the LOAEL for local effects is considered to be <100 mg/kg bw/day.
An increased incidence in irregular oestrous cycle was noted in a few females at 1000 mg/kg bw/day and an acyclic oestrous cycle was noted in one female each at 300 and 1000 mg/kg bw/day. These changes could have occurred due to the low and/or reduced body weight of those individual females at the end of the treatment period and are considered to be adverse.
Based on the adverse increased incidence in irregular oestrous cycle and/or acyclic oestrous cycles from 300 mg/kg bw/day onwards, the no-observed-adverse-effect level (NOAEL) was 100 mg/kg bw/day.
The conclusion that the oestrus cyclicity observations are adverse is conservative, however, the effects may be related to low and/or reduced body weight resulting from and therefore secondary to local effects. This will be clarified in the EOGRT test.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28th April 2017 to May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD 421, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents

Version / remarks:

2000

Deviations:

no

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: At room temperature, protected from moisture/water as well as heat and ignition sources
- Stability under test conditions: Not stable above 180°C (potential formaldehyde release). Stable under test conditions.
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- 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: The test item was dosed undiluted. The required amount of test item for daily dosing was transferred into a container and stored at room temperature.
- Preliminary purification step: No correction factor required
- Final dilution of a dissolved solid, stock liquid or gel: The test item was used undiluted.
- Final preparation of a solid: not applicable

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River,
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks old males and 13 weeks old females
- Weight at study initiation: 260 and 295g for males and 194 and 249g for females
- Fasting period before study: none
- Housing: On arrival and following the pre-test (females only) and pre-mating period, Main animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages. Recovery males and females were group housed during the entire study period. During the mating phase, Main males and females were cohabited on a 1:1 basis in Macrolon plastic cages. During the post-mating phase, Main males were housed in their home cage with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages. During the lactation phase, Main females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water.
- Diet: Pelleted rodent diet, ad libitum
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: The feed and water were analyzed by the supplier for nutritional components and environmental contaminants. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C (target) and 20 to 22°C (actual)
- Humidity (%): 40 to 70% (target) and 49 to 73% (actual)
- Air changes (per hr): 10x/hour
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was administered undiluted.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance administered neat. No analytical verification conducted as not needed.

Duration of treatment / exposure:

Main and Recovery Males were treated for 29 days. Females that delivered were treated for 50-63 days, i.e. 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver or had a total litter loss were treated for 41 or 53 days. Recovery females (not participating in the reproduction part of the study) were treated for 50 days.

Frequency of treatment:

7 days per week

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Test animals: 10 males and 10 females
Recovery groups: 5 males and 5 females for high dose and control groups

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 14-day oral dose range finder with oral administration of Reaction mass of trimethoxy(aminoalkyl)-silanes and modified alkylether oligomers in rats, and in an attempt to produce graded responses to the test item.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: Recovery groups were selected for low and high dose treatment groups.
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

not used

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.
- Cage side observations checked included: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: three times daily, shortly before, immediately after and 1 to 2 hours after dosing. During the recovery period, animals were observed at least once daily up to the day prior to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD CONSUMPTION:
- Food consumption was quantitatively measured weekly, except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

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: Blood was collected at the end of the treatment period on the day of scheduled necropsy. Blood of Recovery animals was collected at the end of the treatment period and on the day of scheduled necropsy at the end of the recovery period.
- Anaesthetic used for blood collection: Yes (isoflurane)
- 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: Blood was collected at the end of the treatment period on the day of scheduled necropsy. Blood of Recovery animals was collected at the end of the treatment period and on the day of scheduled necropsy at the end of the recovery period.
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of treatment and from all recovery F0-animals at the end of treatment and at the end of recovery.
- Metabolism cages used for collection of urine: not specified
- Animals fasted: Yes
- Parameters checked in table [No.1] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 of treatment
- Dose groups that were examined: Selected 5 main males and 5 main females, and all recovery males and females
- Battery of functions tested: sensory activity / grip strength / motor activity / hearing ability / pupillary reflex / static righting reflex

IMMUNOLOGY: NO

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table No 2)

HISTOPATHOLOGY: Yes (see table No 2)

Other examinations:

Thyroid hormone:
Blood samples were processed for serum for possible analysis for the thyroid hormone parameters total thyroxine (T4) and/or thyroid-stimulating hormone (TSH). These serum samples were stored until (possible) analysis in a freezer (≤-75°C).
Samples for T4 of F0-males and PND 13-15 pups were analysed.
Samples for T4 of F0-females and PND 4 pups and samples for TSH of F0-males, F0-females and PND 13-15 pups were not analysed.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

no effects observed

Description (incidence and severity):

Rales were observed in several 75 and 250 mg/kg bw/day treated main and recovery animals predominantly in the first two weeks of treatment. In one female at 250 mg/kg bw/day the rales were accompanied by laboured respiration. Rales were also observed in a single 25 mg/kg bw/day treated male and female on one day in the first week of treatment. The rales were always observed temporarily, lasting between one observation to maximally three days.
Piloerection was observed in one 250 mg/kg bw/day treated female (no.94) for two days in the first week of mating.
No specific clinical signs were noted in the animals of all dose groups during the weekly arena observations.
During the treatment period, salivation was observed among animals of the 75 and 250 mg/kg bw/day dose group immediately after dosing on one or more occasions. Salivation was observed on a single occasion in a 25 mg/kg bw/day treated female. Dose response relationship was observed. Salivation observed in this study was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Other clinical signs noted during the treatment period, including alopecia, scales and/or scabs, occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the morning of Day 3 (prior to dosing) male no.42 (Group 4) was found moribund (gasping) and died shortly thereafter. Macroscopic examination of this animal revealed an enlarged mandibular lymph node (unilateral) foamy contents in the trachea, dark red foci in the thymus and reddish foci in the lungs. Based on the time of occurrence and the absence of similar signs in the other animals, it was considered an accidental event, rather than indicative of test item related toxicity.
No further mortality occurred during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Lower body weights and body weight gain were observed in main and recovery males at 250 mg/kg bw/day, achieving levels of statistical significance for body weights on days 15 and 22 of treatment and on all occasions during treatment for body weight gain when compared to controls. At the end of treatment, a body weight difference of only approximately 4% between controls and males at 250 mg/kg bw/day was noted.

During the recovery period, the difference in body weights persisted during the recovery period, achieving levels of statistical significance on all occasions. The body weight gain data indicated that growth of the recovery males ran parallel. The body weight gain (when compared to study day 1) in high dose recovery males at start of recovery, was statistically significantly lower than in control recovery males. The increase in difference in mean body weights between control and high dose males over one day (from end of treatment to start of recovery) was due to relatively high body weights of the control males that continued in the recovery period compared to those of the whole group. Since the body weight gain over the recovery period was comparable for the control and high dose males, no toxicological significance was attached to this finding.

Body weights and body weight gain in 25 and 75 mg/kg bw/day treated males were considered not to be affected by treatment.
Body weights and body weight gain in female rats were considered to have been unaffected by treatment.
Over the recovery period, body weights and body weight gain were also unaffected by cessation of treatment in female rats.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight in main and recovery male and recovery (not-mated) female rats was similar to the control level over the treatment period and recovery period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

At the end of the treatment period, haematological parameters of treated rats and recovery rats were considered not to have been affected by treatment.
The statistical significance for the mean number of lymphocytes in 75 mg/kg bw/day treated males and for the mean corpuscular haemoglobin concentration (MCHC) in 75 mg/kg bw/day treated females were fortuitous findings. In the absence of a treatment-related distribution or corroborative findings these changes were considered to be of no toxicological significance.
Coagulation parameters of treated rats were considered not to be different from controls at the end of treatment as well as after a subsequent fourteen-day recovery period.
The lower prothrombin time (PT) seen in 250 mg/kg bw/day treated males, achieving a level of statistical significance was considered not to be of toxicological relevance as the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

At the end of treatment, lower mean levels for bile acids were observed in 250 mg/kg bw/day treated males and recovery females, achieving a level of statistical significance in recovery females. The high dose animals did not recover from this difference and the levels of bile acids remained lower at the end of the subsequent fourteen-day treatment free period, achieving a level of statistical significance in 250 mg/kg bw/day treated males.
In main females, the mean levels for bile acids did not indicate an effect by treatment and were comparable between the treated and control animals at the end of treatment. However, it should be noted that a greater individual variation was observed and the mean level for bile acids was approximately two times higher than in the recovery females. This was likely to be related to the difference in physiological status between the primiparous and nulliparous females.
The statistical significances for the mean level for inorganic phosphate in 75 and 250 mg/kg bw/day treated males and for the mean level of calcium in 250 mg/kg bw/day treated main females occurred by chance. As the changes in these parameters were minimal (<10%) and the values for these parameters remained within the historical range for rats of this strain and age, these findings were considered to be of no toxicological significance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis parameters of treated rats were considered not to be different from controls at the end of treatment as well as after a subsequent fourteen-day recovery period.
The pH value of the urine in 75 mg/kg bw/day treated (main) females achieving a level of statistical significance when compared to controls, was considered to have arisen as a result of slightly low control value and in the absence of a treatment-related distribution considered to be of no toxicological significance.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

The functional observation parameters, hearing ability, pupillary reflex, static righting reflex and grip strength, were considered not to be affected by treatment.
The group mean data for motor activity, comprising total movements and ambulation, showed a large variation between the groups in both males and females.
Whereas the group mean values for total movements and ambulation in the 25 and 75 mg/kg bw/day treated main and recovery males were approximately 20% lower than controls, these group mean values in the 250 mg/kg bw/day treated males were similar to that of controls. In the absence of clear dose response relationship and since the motor activity data of individual animals of all groups were within the historical control range for male rats of this strain, age and used in this type of studies, it was concluded that the variation in motor activity between groups was not treatment related. Moreover, a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period was observed in all groups.
In lactating (main) females, the motor activity was higher in the 25 and 75 mg/kg bw/day treated animals, whereas a marked decrease was observed in the 250 mg/kg bw/day treated females in comparison with controls. The increases in group mean total movements were 19% and 16% and for ambulation were 36% and 27% for the 25 and 75 mg/kg bw/day treated females, respectively. In the 250 mg/kg bw/day treated females, the group mean values were 43% lower for total movements, achieving a level of statistical significance, and 40% lower for the ambulation, when compared to controls.
The motor activity in not-mated (recovery) females at 250 mg/kg bw/day was in general higher than in lactating females, but a similar difference between control and high dose females was observed with a 30% lower value for total movements and a 40% lower value for ambulation in the latter animals, but not achieving a level of statistical significance in comparison with controls.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

At the end of treatment, a dose related increase for the splenic weight was observed in Main male rats, achieving a level of statistical significance for the relative spleen weight in high dose males when compared to controls. In Main females, a dose related decrease was observed for the adrenal glands, achieving levels of statistical significance for the absolute and relative adrenal gland weight in high dose females when compared to controls.
No test item-related alterations were observed in the other organ weights at the end of treatment.
At the end of recovery, in the males a difference in absolute liver weights and relative weights of the brain and seminal vesicles was observed between control and high dose males, achieving levels of statistical significance. These differences were considered to be related to the differences in terminal body weight and considered not related to treatment and of no toxicological significance.
In high dose females at the end of recovery, minimal higher weights of the heart, liver and kidneys were observed, achieving levels of statistical significance for absolute and relative heart weight and relative liver and kidney weights when compared to controls. Since all these organ weights remained within the historical control range for female rats of this strain and age and no treatment related effects were observed in main females at the end of treatment no toxicological significance was attached to these findings

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations.
All of the recorded macroscopic findings at the end of treatment in main males and females and at the end of recovery in recovery males and females were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone
At the end of treatment, the serum levels of T4 in F0-males were comparable between the treated and control animals and considered not to be affected by treatment.
At the end of the subsequent fourteen-day recovery period, a difference in levels of T4 between the high dose males and controls was observed, achieving a level of statistical significance. The difference was considered to have arisen as a result of slightly high control value and since the T4 values of the high dose males were well within the historical control range for rats of this strain and age no toxicological significance was attached to this finding.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (nominal)

System:

central nervous system

Organ:

not specified

Treatment related:

yes

Conclusions:

In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test with trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine the NOAEL for systemic effects was concluded to be 75 mg/kg bw/day based on a treatment-related lower motor activity observed in main and recovery females at 250 mg/kg bw/day in comparison with that in females in the other groups. 

Reference 5

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2001.06.11 to 2001.07.16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

other: HanBrl:WIST (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd.
- Age at study initiation: 6 weeks
- Weight at study initiation: males 143-160g females 112-130g
- Housing: five per cage in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (Lignocel)
- Diet: Pelleted standard Provimi Kliba rat maintenance diet (batch 07/00) ad libitum
- Water: Community tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 11 June to 16 July 2001

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Prepared daily, Y-15167 weighed into a tared glass beaker and vehicle added. Mixture prepared using a magnetic stirrer and used at room temperature.

VEHICLE
- Concentration in vehicle: 0, 10, 40, 200 mg/mL
- Amount of vehicle (if gavage): 5mL/kg
- Lot/batch no.: 9.3.01 SCO / 7.3.01 SCO

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (after 2 hours) were determined at teh start of the study. Concentration and homogeneity also determined during Week 3 (according to NMR method)

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of non-GLP 5-day dose range finding study
- Rationale for animal assignment: random (computer-generated algorithm)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily days 1-3, once daily thereafter

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 4
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table No. 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 4
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table No.2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 4
- Dose groups that were examined: All
- Battery of functions tested: sensory activity, grip strength, motor activity

Sacrifice and pathology:

ORGANS WEIGHTS: Yes (see table 3)
GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4)

Statistics:

The following statistIcal methods were used to analyze the body weight, organ weights and ralios. as well as clinical laboratory data:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied insteadof the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Student's t-test was applied to grip strength and locomotor activity.
• Fisher's exact-test was applied to the macroscopic findings.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

A male at 1000 mg/kg/day was found dead on Day 4. The cause of death was considered to be a dosing error.

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):

effects observed, treatment-related

Description (incidence and severity):

Reduced locomotor activity was noted at 1000 mg/kg/day, most clearly seen in males.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Necrotic tracheitis was noted in the 1000 mg/kg/day male which died on Day 4 and was considered consistent with a dosing error.

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reduced motor activity was noted at 1000 mg/kg bw/day

Critical effects observed:

not specified

Conclusions:

Oral administration to Wistar rats at 0, 50, 200 or 1000 mg/kg bw/day for 28 days resulted in no test item-related mortality (a sinlge male died from suspected dosing error), no clinical signs during daily observations or weekly behavioural observations, no findingsduring functional observation battery observations, no effects upon haematology or clinical chemistry parameters, no changes in absolute or relative organ weights and no macro- or microscopic changes.
Treatment-related changes were noted for locomotor activity which was slightly reduced at 1000 mg/kg/day.
Based on the results of this study the NOAEL was concluded to be 200 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 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

Remarks:

Study included to support DNEL risk assessment.

Adequacy of study:

key study

Study period:

2007

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)

GLP compliance:

yes

Species:

rat

Strain:

other: Crl: CD® (SD) IGS BR VAF/Plus®

Sex:

male/female

Details on test animals or test system and environmental conditions:

Seventy-eight female and seventy-eight male rats were obtained from Charles River Laboratories, Kingston, NY. Animals were 10 weeks old at experimental start.
Animal Receipt and Quarantine/Acclimation: Upon receipt, animal resource personnel inspected each animal. Animals judged to be in good health and suitable as test animals were quarantined/acclimated for a minimum of five days. The attending veterinarian examined all animals before release from quarantine/acclimation and documented the general state of animal health.
Animal Housing: Animals were individually housed in suspended wire-mesh cages during the course of the study. The cages were elevated above Bed-O’Cobs bedding and subjected to routine cleaning consistent with good housekeeping practices. Prior to exposure, animals were acclimated according to the following schedule:
Three days prior to exposure: Animals were placed in the exposure caging for 2.8 – 3.0 hours.
Two days prior to exposure: Animals were placed in the exposure caging for 6.0 hours.
One day prior to exposure: Animals were placed in the exposure caging for 6.0 – 6.1 hours in the inhalation chambers.
Following each exposure, the test article treated animals were housed in a separate animal room from the control animals.

Environmental Enrichment: Animals were given Gnaw PucksTM and Cozee PadsTM in their home cages for environmental enrichment.

Environmental Conditions: Animals were housed in environmentally controlled animal rooms. With the exception of the deviations noted in Table 13 environmental conditions were within 18 – 26°C for temperature, 30 – 70% relative humidity, and 10 – 15 air changes per hour. Lighting was controlled to provide a 12-hour fluorescent-light/dark cycle. Temperature and humidity were recorded approximately every fifteen minutes using a HOBO® data logger (Onset Computer, Bourne, MA; software: BoxCar® Pro 4.3.1.1.). In addition, twice a day on weekdays and once a day during weekends and holidays these values were manually recorded.

Basal Diet: Certified Rodent Diet #5002, PMI Nutritional International Inc., St. Louis, MO, was offered ad libitum except while animals were in the inhalation exposure cages and during the fasting period prior to terminal sacrifice. Manufacturer’s periodic analyses of the certified feed for the presence of heavy metals and pesticides was reviewed by the study director to ensure that none are present in concentrations that would be expected to affect the outcome of the study.

Drinking Water: Municipal water, further purified by reverse osmosis (RO) was available ad libitum except while animals were in the inhalation exposure cages. Water collected from the RO system was monitored on a semi-annual basis to determine compliance with the EPA drinking water standards. The most recent analysis was reviewed by the study director to ensure that there were no contaminants known to be present in water, at levels expected to interfere with the integrity of the study.

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

Vapour Generation: Generation of test article vapor was performed using heated stainless steel J-tubes containing stainless steel beads. Test article was metered from reservoirs into J-tubes using either a Fluid Metering Incorporated (FMI®) pump or a Harvard model syringe pump equipped with Hamilton brand glass syringes. Compressed air flowed through the J-tube at a predetermined controlled rate. The carrier/vapour mixture passed from the J-tube directed to the inlet port at the top of the exposure chamber. Just prior to entering the exposure chamber, the carrier/vapour mixture was combined with chamber supply (dilution) air where it was diluted to the target chamber concentration as it entered the exposure chamber.

Inhalation Chambers: Exposures were conducted in 2000-liter stainless steel and glass Rochester-style inhalation chambers and stainless steel exposure caging (four layers of 20 animal compartments). The animal cage position assignment within each chamber was rotated daily. Chamber temperature, relative humidity, and airflow were monitored and recorded during the acclimation period similar to that anticipated for the exposure periods. Vapor generation systems were not in operation during acclimation. The chambers were targeted for 12 – 15 chamber volume air changes per hour and environmental conditions of 20 ± 3°C and 50 ± 20%RH. Chamber airflow, temperature and relative humidity were monitored continuously and values recorded approximately once every 30 minutes during exposures. Chamber oxygen content was monitored, and manually recorded, once during the first day of exposure to ensure that under applied experimental conditions the oxygen content was above the minimum 19% acceptable limit.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test Atmosphere Monitoring: The test atmosphere from each chamber was sampled by an automated sampling system. The system was designed such that test atmosphere was continuously pulled from the chamber and delivered to the analyzer through individual chamber sample lines and a stream selector valve. The sample lines were continuously purged with fresh chamber atmosphere during the entire exposure period.
Chamber atmosphere was analyzed using a gas chromatograph equipped with a flame ionization detector (GC/FID) to determine the actual chamber concentration of test article. In addition, given the potential hydrolysis of methyltrimethoxysilane in humid air, the methyl alcohol concentration within each chamber was also determined. The concentration of test article in the chamber atmosphere during the exposure period was evaluated a minimum of once every 60 minutes. Methyl alcohol (a known hydrolysis product of methyltrimethoxysilane) concentrations were measured from each exposure chamber a minimum of seven times during each day’s exposure period. A continuously purged sample line was used to transfer chamber atmosphere to the GC/FID for analysis.
The GC/FID methods were established prior to the experimental start date. Standard curves relating test article vapor concentration and methanol concentration to GC/FID response were established before the first exposure and then again as necessary. Preparation of calibration curves involved bag standards at five different levels that bracketed the expected range of test article or methyl alcohol chamber concentrations. The peak area attributed to the test article or methyl alcohol was plotted against the nominal bag standard concentration. Linear regression analysis of these data was performed to define the parameters of the calibration curve. Acceptance criteria for the GC/FID calibration curve included linear regression correlation coefficient of > 0.98 and < 10% difference between the prepared bag standard concentration and the calculated bag standard concentration derived from the linear regression equation of the calibration curve.
Each calibration curve was verified prior to the exposure period by analysis of a bag standard. The bag standard actual concentration derived from the calibration curve needed to be within 10% of the bag standard nominal concentration for the calibration curve to be considered acceptable for use.
The mean daily actual measured methyltrimethoxysilane vapor concentration was compared with the daily-calculated nominal concentration as a quality control mechanism to evaluate exposure system performance. A difference of ≤15% was considered acceptable for the 25 ppm targeted exposure level, and ≤10% at all other target levels.
Homogeneity of test atmosphere within each chamber was evaluated once prior to initiation of animal exposures. Acceptance criteria required the mean values for each chamber zone not exceed 10% difference from the reference zone (approximate sampling location used during animal exposures).
The stability of methyltrimethoxysilane vapor in a gas bag and sample line loss was evaluated using prepared bag standards. Stability was considered acceptable over the time the measured concentration did not exceed 10% difference from the original concentration measured immediately following preparation.
Exposure chambers were leak tested prior to the first exposure to ensure proper operation. Acceptance criteria required that the mean chamber airflow measured at the inlet not be more than 10% different from the airflow measured on the exhaust side.

Duration of treatment / exposure:

Exposure levels and treatment regimen: Test article was administered by whole-body vapour inhalation. Animals were positioned in the chambers and the exposure period was defined as a 6-hr/day exposure at the target concentration. Animals were removed from the chambers after a second T99 had expired. The T99 period was considered as the time required for the chamber to reach 99% of the target concentration or clearance of 99% of the achieved concentration following generation of test article. Based on chamber flow rate and size, the T99 period was calculated to be 23 minutes for all chambers. For practical purposes, the T99 2 minutes was used for this study.
The target exposure levels were: 0, 25, 100, 400 and 1600 ppm trimethoxy(methyl)silane in air. These exposure levels were determined based on results from a previously conducted 14-day range finding study (Tobin, 2007). Exposures were conducted at approximately the same time each day, five days per week over the course of thirteen weeks.

Frequency of treatment:

6 hours/day, 5 days/week at target concentrations

Dose / conc.:

25 ppm

Remarks:

Ca. 0.14 mg/L

Dose / conc.:

100 ppm

Remarks:

Ca. 0.56 mg/L

Dose / conc.:

400 ppm

Remarks:

Ca. 2.2 mg/L

Dose / conc.:

1 600 ppm

Remarks:

Ca. 8.9 mg/L

No. of animals per sex per dose:

10/sex/dose level and 10 additional animals/sex/control and high dose groups for a 28-day recovery period

Control animals:

yes, concurrent vehicle

Details on study design:

This study was conducted following the general principals of the OECD Guidelines for Testing Chemicals, Subchronic Inhalation Toxicity 90-Day Study, No. 413, adopted May 12, 1981. The study was conducted to evaluate the toxic effects of, and subsequent recovery from, whole-body vapor inhalation exposure of methyltrimethoxysilane. Five (5) groups of 10 male and 10 female Sprague-Dawley rats were exposed to target methyltrimethoxysilane exposure concentrations of 0 (control), 25, 100, 400 and 1600 ppm, 5 days per week for thirteen weeks. Additional satellite groups of 10 males and 10 females were included in the 0 and 1600 ppm target groups for evaluation of a 28-day post exposure recovery period. Exposures terminated on study 90 with non-recovery animals euthanized on study day 91. Recovery group animals remained for 28-days post exposure and were euthanized on study day 119.

Observations and examinations performed and frequency:

Mortality/Morbidity/Moribundity: All animals were observed in their cages for mortality, morbidity, and moribundity at least twice daily on weekdays, and once daily during weekends and holidays.
Clinical Observations: General clinical observations were made at least once a day, beginning on the first day of exposure, at approximately the same time each day. The health condition of the animals was recorded. Clinical observations included, but were not limited to, changes in the skin, fur, eyes, and mucous membranes, respiratory system, circulatory system, autonomic and central nervous systems, motor activity, and behavior patterns. General clinical observations were performed on all animals on the day of, and prior to, their scheduled necropsy.

Parameters Measured
Individual Body Weights: Individual body weights were recorded for randomization, weekly throughout the duration of the study, then again prior to sacrifice on the day of scheduled termination.
Individual Food Consumption: Feeder weights were recorded weekly throughout the duration of the study.
Ophthalmic Examinations: Examinations were performed on all animals prior to group assignment. Animals with findings noted during this initial examination were not used on study. Additional examinations were conducted during the final week of exposure prior to 90-day terminal sacrifice, and during the final week of the 28-day post exposure recovery period. An indirect ophthalmoscope (following dilation using mydriatic eye drops) was used for all examinations.

Sacrifice and pathology:

Clinical Pathology: Food was removed from all animals on the evening prior to scheduled termination. Clinical pathology assessments were made on all surviving animals from which adequate samples were collected.
Blood samples were collected for hematological and clinical chemistry evaluations from all animals on the day of scheduled euthanasia as a terminal procedure. While under Isoflurane® anesthesia, a syringe and needle were used to collect blood samples from the abdominal vena cava and distributed to three sample collection tubes containing either sodium citrate, EDTA, or no anticoagulant.

Hematology: Blood samples for the following hematology tests were collected into test tubes containing EDTA. Hematology samples were analyzed within 24 hours of collection. Analysis was performed using the Cell-Dyn 3700TM (Abbott Diagnostics, Dallas, TX).
Erythrocyte count Hemoglobin
Erythrocyte Indices (MCV, MCH, MCHC) Hematocrit
Leukocyte counts (total and differential) Platelet count
Blood samples for coagulation assessment (prothrombin time) were placed into test tubes containing sodium citrate, centrifuged within two hours of collection and the plasma separated for testing. Samples were maintained at room temperature (18-24 °C) or refrigerated (2-8 °C) prior to analysis. Analysis was conducted within 24 hours of collection. Analysis was preformed using the ACL 100TM (Beckman Coulter, Fullerton, CA).

The following serum chemistries were determined:
Alanine aminotransferase Glucose
Albumin Phosphorus
Alkaline phosphate Potassium
Aspartate aminotransferase Sodium
Calcium Total bilirubin
Cholesterol Total protein
Chloride Urea nitrogen
Creatinine
Blood samples were placed into test tubes without anticoagulant, allowed to clot and centrifuged within two hours of collection and the serum separated for testing. For bilirubin and total protein analysis, the serum samples were frozen at  -20C, until analysis. Samples were analyzed using the Cobas Integra 400 plusTM (Roche Diagnostics, Indianapolis, IN).

Gross Pathology: All animals were subjected to a full gross necropsy which included examination of the external body surface, all orifices, as well as the cranial, thoracic and abdominal cavities including contents.

Histomorphology: Eyes were preserved in Davidson’s solution until processing with the testes and epididymides preserved in Bouin’s solution for 24 – 36 hours then transferred into 70% ethanol until processing. All other tissues were placed in 10% Neutral Buffered Formalin for preservation. Tissue processing included routine procedures including embedding in paraffin, sectioning and staining with hematoxylin and eosin for evaluation.
Histomorphological examination was conducted on all tissues collected from both the recovery and non-recovery animals in groups 1 and 5. Following initial review, additional selected tissues including urinary bladder, kidney, liver, lung and prostate were evaluated for groups 2, 3, and 4.

Other examinations:

The following organs were weighted: adrenals, kidneys, liver, lung, testes, ovaries, brain, epididymides, seminal vesicles and prostate.

Statistics:

Daily mean inhalation exposure concentrations and environmental conditions, along with standard deviations, were calculated using Microsoft Office Excel. Mean and standard deviation were calculated for body weights, changes in body weights, food consumption, organ weights, hematology and clinical chemistry values, using ProvantisTM, version 6.5. Environmental conditions of animal rooms were monitored and recorded using a HOBO® data logger (Onset Computer, Bourne, MA; software: BoxCar® Pro 4.3.1.1.)
All data analysis was carried out using SAS version 9.1.3. Statistically significant probabilities were reported for p-values of < 0.05, <0.02, and < 0.01.
Data from the recovery groups was analyzed separately from the 90 day groups. Body weight, changes in body weight, food consumption data, organ weight, organ to body weight ratios, hematology data, clinical chemistry and prothrombin times were analyzed using a one-way Analysis of Variance (ANOVA) if the data satisfied the requirements of normality of the residuals and homogeneity of variance as determined using the Shapiro-Wilk test for normality and Levene’s test for homogeneity of variance. If the data did not satisfy the parametric requirements, a Kruskal-Wallis test was used. If the ANOVA or Kruskal-Wallis test was significant, pair-wise comparisons of the exposed groups to control were made using the Dunnett’s Test or a Wilcoxon test, respectively.
For variables with multiple measurements across time (body weight and food consumption), a repeated measurements ANOVA was performed to determine if there was a time by exposure group interaction.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test article-related clinical signs reported for all surviving animals were limited to groups 4 and 5 and primarily included soiling of the urogenital and abdominal regions of both sexes.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two animals were found dead (one group 4 male on study day 25; one group 5 male on study day 72) and one animal (control group male on study day 65) was sacrificed moribund prior to terminal sacrifice. There were no abnormal clinical or gross pathological findings for the group 4 male. Test article-related clinical signs for the group 5 male included decreased activity, soiling around muzzle, abdomen and urogenital regions with gross pathological findings including dilation of kidneys and urinary bladder with calculus in bladder.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights trended lower than controls over the exposure period for group 5 males (~6%) and groups 4 and 5 females (~5%). Statistically significant decreases were noted during week one for group 3 and week two for group 5 only. A statistically significant decrease in mean body weight was measured in the group 5 recovery group females beginning exposure week four. This difference persisted through the completion of exposures and into week one of the post exposure recovery period. Although not statistically significant, body weights remained decreased from controls for males (~4%) and females (~6%) during the recovery period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no differences in food consumption for either sex, in any of the 90-day exposure groups. Weekly comparison of recovery group food consumption yielded statistical differences during weeks 6, 7, 8 and 10 for group 5 males and weeks 4 and 5 for group 5 females. Food consumption was similar to controls for both sexes in group 5 during each week of the 28-day post exposure period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There was no test article-related ophthalmic finding at the end of the 90-day exposure period.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Relative to body weight, female adrenal glands were statistically increased (27%) for group 5. There was no histological correlate and the finding was not present in males or in recovery group females. In males, group 4 kidney weight was increased, but a comparable effect was not seen in group 5. In females, absolute adrenal gland weights were statistically increased in group 4 (18%) and group 5 (25%).

Also in males, the weights of testes and epididymides were statistically decreased in recovery group rats exposed to 1600 ppm. This finding correlated histologically with two recovery group males showing marked testicular seminiferous tubule degeneration and corresponding epididymal oligospermia (one unilateral, one bilateral). In regular study (90-day) rats, seminiferous tubule degeneration was observed only in one control and one low-exposure (25 ppm) rats. These findings were considered common spontaneous findings in young Sprague-Dawley rats and not test article-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test article-related gross necropsy findings were primarily limited to the group 4 and 5 animals and included moderate dilation of the kidney, decreased soft testes, and calculi in the urinary bladder.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histomorphologic changes included minimal to moderate urinary bladder hyperplasia and inflammation in all group 5 males and 9/10 females. Kidney changes were characterized by hyperplasia of the pelvic epithelium and/or granulomatous inflammation. The one group 5 male animal found dead on study day 72, demonstrated an apparent urinary obstruction possibly leading to acute uremia, with calcification of the aorta and pulmonary hemorrhagic edema as secondary effects. Additional changes included prostatic inflammation in moderate or severe degrees in two 1600 ppm exposure group 5 rats.

Following the 28-day recovery period, calculi were observed in the group 5 males only. Minimal to moderate hyperplasia of urinary bladder epithelium persisted in most rats, and exposure-related urinary bladder calculi were observed in several. Chronic or granulomatous inflammation in the renal pelvis was observed in several female rats. In male rats, there was no histomorphological evidence of a residual effect on the kidneys after the recovery period. In females, the incidence of pelvic epithelial hyperplasia and inflammation was modestly increased over controls. There were no indications of a residual effect on the prostate gland following the recovery period. No animals had more than mild inflammation of the prostate gland, and the incidence of inflammation was higher in control animals.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

Average measured methyltrimethoxysilane exposure concentrations included below the limit of quantitation (BLQ), 25 +/- 0.8, 99 +/- 3.2, 398 +/- 12.8, and 1612 +/- 35.6 ppm for groups 1 through 5, respectively. Calculated nominal concentrations included 23 +/- 0.4, 94 +/- 1.6, 383 +/- 10.4, and 1570 +/- 48.9 ppm for groups 2 through 5, respectively. Mean measured methyl alcohol concentrations were below the limit of calibration (8.7 ppm) for all exposure groups except group 5 which was 19 +/- 3.7 ppm.

Dose descriptor:

NOAEC

Effect level:

0.56 other: mg/l

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Dose descriptor:

LOAEC

Effect level:

2.2 other: mg/l

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the increased incidence of grossly observed urinary bladder calculi along with the kidney dilation at the 400 ppm exposure level.

Critical effects observed:

not specified

Toxic Response/Effects by Dose Level:

25 ppm (ca. 0.14 mg/L):

No findings attributed to test article   

100 ppm (ca. 0.56 mg/L):

No findings attributed to test article   

400 ppm (ca. 2.2 mg/L): 

Clinical signs:

- Increased incidence of abdominal and urogenital soiling Gross pathology:

- Calculi in the urinary bladder (2 males) Histomorphologic changes: 

- Urinary bladder epithelial hyperplasia in males and females 

Organ weights: 

Female adrenal weight increase (absolute):  18%*   

1600 ppm (ca. 8.9 mg/L):

Clinical signs:

- Increased incidence of abdominal and urogenital soiling Gross pathology: - Calculi in urinary bladder of 4 males and 1 female, persisting through 28-day recovery

- Kidney dilation   

Organ weights:

Female adrenal weight increase (absolute/relative):

Absolute:  25%*         Relative:  27%*

Female kidney weight increase (relative):

Relative:  12%*

Absolute adrenal weights were significantly increased compared to controls for group 4 (18%) and 5 (25%) females.  Adrenal and kidney to  

body weight ratios were also significantly increased compared to controls  for group 5 (27% and 12%, respectively) females.

 

Histomorphologic changes:

- Kidney:  hyperplasia of the pelvic epithelium and granulomatous inflammation for males - Urinary bladder:  epithelial hyperplasia in males and females - Prostate:  slight increase in severity of inflammation * Statistically significant findings:

"        Body weight:  absolute and percent gain was within normal limits for  all test groups "        Food/water consumption: within normal limits for all test groups "        Description, severity, time of onset and duration of clinical signs: The increased incidence of abdominal and urogenital soiling at the 400  

and 1600 ppm exposure levels was observed beginning on exposure day 11 and persisting on and off throughout the completion entire 90-day  

exposure duration.. "        

Ophthalmologic findings incidence and severity: No test article attributed findings "        Hematological findings incidence and severity: No test article attributed findings "        Clinical biochemistry findings incidence and severity: No test article attributed findings "        Mortality and time to death:  One (1) 400 ppm group 4 male on study day  25, One (1) 1600 ppm group 5 male on study day 72. "        Gross pathology incidence and severity: increased incidence of urinary bladder calculi and dilation of the kidneys as described above. "        Organ weight changes: Kidney and adrenal glands related to treatment as specified above. "        Histopathology incidence and severity: Urinary bladder epithelial  hyperplasia in both sexes at 400 and 1600 ppm and hyperplasia of the  

pelvic epithelium along with granulomatous inflammation for 1600 ppm male  kidneys. Prostatic inflammation seen in all groups with a slight  

increase in severity at the 1600 ppm level.

Conclusions:

Based on the increased incidence of grossly observed urinary bladder calculi along with the kidney dilation at the 400 ppm exposure level, the No Observable Adverse Effect Level (NOAEL) for methyltrimethoxysilane vapor administered six hours per day, five days per week for a 90-day interval via whole-body inhalation exposure to male and female Sprague-Dawley rats, was 100 ppm.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

560 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Trimethoxy(methyl)silane and its reaction products with 3-aminopropyltriethoxysilane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane is a UVCB substance. The identity and concentration range of the constituents have been determined by GC analysis. A detailed description of the composition can be found in Section 1.2 of the Chemical Safety Report.

 

No repeated dose toxicity studies are available for the registered substance, trimethoxy(methyl)silane and its reaction products with 3-aminopropyltriethoxysilane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane, therefore data for its constituents have been used to address this endpoint and the data for the major constituent trimethoxy(methyl)silane (CAS 1185-55-3), present at 50 -70%, have been used as key to allow interim risk characterisation. A 90-day oral repeated dose toxicity study (OECD Test Guideline 408) has been proposed with the registered substance.

 

Block A

Trimethoxy(methyl)silane (CAS 1185-55-3) – data used for DNEL derivation for interim risk characterisation

 

In the key 90-day whole-body inhalation repeated dose toxicity study (Dow Corning Corporation, 2007), conducted according to OECD Test Guideline 413 and in compliance with GLP, in which trimethoxy(methyl)silane vapour (CAS 1185-55-3) was administered to male and female Sprague-Dawley rats at concentrations of 25, 100, 400 and 1600 ppm (equivalent to 0.14, 0.56, 2.2, 8.9 mg/l) six hours per day, five days per week. An increased incidence of grossly observed urinary bladder calculi along with the kidney dilation were observed at the 400 ppm exposure level. The NOAEC for trimethoxy(methyl)silane vapour was concluded to be 100 ppm (equivalent to 560 mg/m3).

 

In the key oral Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test with trimethoxy(methyl)silane (CAS 1185-55-3), conducted according to OECD Test Guideline 422 and in compliance with GLP (Dow Corning Corporation, 2005), exposure to trimethoxy(methyl)silane was associated with organ weight and/or histomorphological changes in males (liver, thymus, thyroid, duodenum, jejunum, and red blood cell) and females (liver, thyroid, duodenum, jejunum, and adrenal gland) at dose levels at or above 250 mg/kg bw/day. A marked increase in prothrombin time was observed for males at 250 and 1000 mg/kg bw/day whereas females were unaffected. Exposure was also associated with increased blood platelet concentration for males and females at 1000 mg/kg bw/day. Based on these data the NOAEL for systemic toxicity was concluded to be 50 mg/kg bw/day.

 

 

Block B

3-Aminopropyl(triethoxy)silane (CAS 919-30-2)

 

A 90-day oral repeated dose toxicity study, conducted according to OECD Test Guideline 408 and in compliance with GLP (WIL Research Laboratories, 2001), is available for 3-aminopropyl(triethoxy)silane (CAS 919-30-2) which is a minor constituent of the registered substance, part of Block B, and also shares the same silanol hydrolysis product as the other amino alkoxysilane constituents in Block B, 3-(trimethoxysilyl)propylamine (CAS 13822-56-5). Block B constituents are typically present at 0-10 % in the mixture. In the study, the NOAEL value was concluded to be 200 mg/kg bw/day in male and female rats based on mortality, clinical observations and liver effects at 600 mg/kg bw/day.

 

3-(Trimethoxysilyl)propylamine (CAS 13822-56-5)

 

A 90-day oral (gavage) repeated dose toxicity study in male and female rats, conducted according to OECD Test Guideline 408 and in compliance with GLP, is available for 3-(trimethoxysilyl)propylamine (CAS 13822-56-5), which is a minor constituent of the registered substance part of Block B and also shares the same silanol hydrolysis product as the other amino alkoxysilane constituents in Block B. The study authors concluded a LOAEL for local effects of <100 mg/kg bw/day. The study reviewer concluded a conservative NOAEL for systemic toxicity of 300 mg/kg bw/day, based on the mortalities observed at 1000 mg/kg bw/day dose, although the mortalities are likely to have resulted from local effects rather than systemic toxicity (Charles River, 2018).

Trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine (EC No. 701 -408 -8)

 

In the key combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD Test Guideline 422 and in compliance with GLP for trimethoxy(methyl)silane and its reaction products with [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and 3-(trimethoxysilyl)propylamine, undiluted test material was administered orally by gavage for a minimum of 28 days to 10 male and 10 female Wistar Han rats per dose at three dose levels of 25, 75 and 250 mg/kg bw/day (Charles River Laboratories, 2018). A control group was also included that received water. A 14-day recovery period was included for 5 additional male and 5 female rats from the high and low dose groups. The recovery animals (used to study the potential reversibility of possible adverse effects) were not mated and consequently were not used for the assessment of reproduction/ developmental toxicity.

No toxicologically significant clinical signs were observed in male and female rats treated up to 250 mg/kg bw/day. The motor activity in male and female rats showed a large variation between the groups. Despite the fact that the motor activity recorded in the animals of all dose groups was within the historical control range, there was no indication of a treatment related change in males, but it could not be ruled out that the marked decrease in group mean values for total movements and ambulations in both main and recovery females at 250 mg/kg bw/day was a treatment related effect. Any corroborating evidence from other possibly related parameters was not observed in the current study, e.g. no other behavioural changes, no effects on grip strength and absence of histopathological alterations in nerve and muscle tissue in these females. Nevertheless, in case the changes in motor activity in females at 250 mg/kg bw/day were treatment related the magnitude of the decrease of approximately 40% lower group mean values for total movements and ambulations should be an indication of an adverse effect.

Slightly reduced body weight gain was observed in males at 250 mg/kg bw/day during treatment, resulting in approximately 4% lower group mean body weight compared to that of controls. During the recovery period, the body weight gain was similar between these two groups and the difference in body weight continued until termination of the recovery males. Based on the magnitude of the changes in body weight the effect of treatment was considered to be non-adverse.

At the end of treatment, lower mean levels for bile acids were observed in 250 mg/kg bw/day treated males and recovery females. These high dose animals had not recovered from this difference and the levels of bile acids remained lower at the end of the subsequent fourteen-day treatment free period in the recovery animals.

At the end of treatment, a dose related increase in splenic weight (in males) and decrease in adrenal weight (in females) was observed. The changes in weight of these organs in high dose animals were still within the historical control range and there were no microscopic correlates for these organ weight changes. In addition, the splenic weights in control and high dose recovery males and the adrenal weights in control and high dose recovery females were comparable at the end of the fourteen-day treatment free period. Therefore, these changes in organ weights at the end of treatment were considered to be non-adverse after treatment up to 250 mg/kg bw/day. 

No treatment-related changes were noted in the other parameters investigated in this study (i.e. food consumption, haematology, coagulation and (male) T4 thyroid hormone levels, macroscopic and microscopic examination) at the end of treatment.

Furthermore, the 14-day recovery period after cessation of treatment did not induce changes between the 250 mg/kg bw/day treated males and females compared to their concurrent controls in any of the parameters investigated.

Based on a treatment-related lower motor activity observed in main and recovery females after treatment at 250 mg/kg bw/day, a NOAEL of 75 mg/kg bw/day was concluded for systemic toxicity.  

 

This source substance is the methoxy analogue of the registered methoxy/ethoxy substance (target). The repeated dose toxicity study for this analogue are included to cover Blocks C to G, and unspecified minor constituents, for which there are no repeated dose toxicity data. The only difference between the target and source substances is one of the three starting materials. The source substance has a starting material, 3-aminopropyl(trimethoxy) silane [237-511-5, 13822-56-5] and the target has the starting material that is the triethoxy analogue, 3-aminopropyl(triethoxy)silane [213-048-4, 919-30-2]. Therefore, the target substance has additional ethoxy constituents. However, whether ethoxy or methoxy, the target and source substance constituents hydrolyse to the same respective silanol hydrolysis products. The only difference is the non-silicon hydrolysis product; the target releases methanol and ethanol, whereas the source substance releases methanol only. The small molecule alkoxysilanes of Block A, Block B and those of Block U1 have similar physicochemical properties as the starting materials. Together they constitute about 60-70% of the substance. Blocks C-G and U2 constitute approximately 20-30% of the substances. These are oligomers formed by combination of the monomer starting materials. Under acidic conditions (pH <4) as experienced in the stomach and relevant to oral studies, the hydrolysis of the source and target substances is rapid and will form the same hydrolysis products. 

 

Block D

N-ethyl-3-trimethoxysilyl-2-methyl-propanamine

 

A 28-day oral repeated dose toxicity study, conducted according to OECD Test Guideline 407 and in compliance with GLP, is also available with N-ethyl-3-trimethoxysilyl-2-methyl-propanamine (CAS 227085-51-0) (RCC, 2001e). The only treatment-related effect noted in the study was slightly reduced locomotor activity at the high dose of 1000 mg/kg bw/day. Therefore, the concluded NOAEL was 200 mg/kg bw/day.

Data on this substance are relevant as its silanol hydrolysis product is an analogue of the hydrolysis product of constituents trisilyl(alkylamine), octa(ethoxy/methoxy)-methyl- in Block D. Therefore, data on this substance are included as supporting data for completeness.

Justification for classification or non-classification

The available data indicate that trimethoxy(methyl)silane and its reaction products with 3-aminopropyltriethoxysilane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane does not need to be classified for specific target organ toxicity following repeated exposure by either the oral or inhalation routes according to Regulation (EC) No 1272/2008.