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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1999-10-11 to 2000-03-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to an appropriate guideline, in compliance with GLP and was considered to be reliability 1 (reliable without restrictions). Read across to the registered substance is considered scientifically justified; the read across is considered to be reliability 2.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
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

Test material

Constituent 1
Reference substance name:
3-aminopropyltriethoxysilane
EC Number:
213-048-4
EC Name:
3-aminopropyltriethoxysilane
Cas Number:
919-30-2
IUPAC Name:
3-(triethoxysilyl)propan-1-amine

Test animals

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

Administration / exposure

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
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 70, 200, and 600 mg/kg bw/day
Basis:
other: 2-wk range finding study (WIL-242147)
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.
Positive control:
None

Examinations

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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
(Adapted from SIAR, 2003)

CLINICAL SIGNS AND MORTALITY (see also table 1, below)
Deaths of 1 male and 8 females at the top dose (600 mg/kg bw/day) were believed treatment-related. These animals 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.)

BODY WEIGHT, WEIGHT GAIN AND FOOD CONSUMPTION; OPHTHALMOSCOPIC EXAMINATION AND HAEMATOLOGY
No clear treatment-related effects were reported on: body weight gain, food consumption, oculopathy, haematology parameters, oestrous cycle data or spermatogenic endpoints. 

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

ORGAN WEIGHTS
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 PATHOLOGY
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.

HISTOPATHOLOGY:
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.


Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
600 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: mortality, clinical signs, liver effects

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:
A well reported 90-day oral study, conducted in the main according to the current guideline and in accordance with GLP, identified a NOAEL value of 200 mg/kg bw/day in male and female rats. Mortality, clinical observations and liver effects were evident at 600 mg/kg bw/day