Bis(trimethoxysilylpropyl)amine

Endpoint summary

Description of key information

Oral (OECD 407), rat: NOAEL (systemic) = 1000 mg/kg bw (male/female), NOAEL (local) = 300 mg/kg bw (male/female)
Inhalation: No data available.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Jun - 11 Oct 2013

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)

Version / remarks:

(2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese guideline (MHLW No. 2, 5 and 7, 2011, 2012)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Hino Breeding Center, Japan
- Age at study initiation: 5 weeks
- Weight at study initiation: 142.4 - 177 g (males), 123 - 143.5 g (females)
- Housing: animals were housed individually in hanging stainless steel cages with wire-mesh floor.
- Diet (e.g. ad libitum): pelleted diet (MF, Oriental Yeast), ad libitum
- Water (e.g. ad libitum): chlorinated water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.3 - 23.5
- Humidity (%): 51.4 - 65.2
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 04 Jun 2013 To: 26 Jul 2013

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed and dissolved in olive oil to a prepare 50% (w/v) formulation. A part of the 50% (w/v) formulation was taken and diluted with olive oil to prepare formulations of 15 and 5% (w/v). Since the 50 and 15% (w/v) dosing formulation were confirmed to be stable for 168 h after preparation at a cold place, the formulations were prepared as the preservation period was within 168 h. The stability of the 5% (w/v) formulation was confirmed to be 4 h, therefore, the 5% (w/v) formulation was prepared each morning prior to dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Because there is information that the test substance reacts with water, a vehicle for dosing formulation was investigated with olive oil. As a result, the test substance dissolved in olive oil at 20.0% (w/v). After one day of storage at room temperature, there were no colour changes in the test substance solution. In addition, olive oil has been generally used as a vehicle in general toxicity studies and historical control data based on olive oil as vehicle is available. Therefore, olive oil was selected as vehicle.
- Lot/batch no.: 242003
- Concentration in vehicle: 5, 15 and 50% (w/v)
- Amount of vehicle (if gavage): 2 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stabilities of the 50, 15 and 5% (w/v) formulations in the cold place were confirmed by gas chromatography (GC) in CERI Hita. The test substance in the 50 and 15% (w/v) formulations were judged as stable for 168 h under the storage conditions, because those concentrations after storage were within 100 ± 10% (101 and 101%, respectively) of those immediately after preparation. The test substance in the 5% (w/v) formulation was judged as stable for 4 h under the storage condition, because that concentration after 4 h storage was within 100 ± 10 (101%) of that immediately after preparation. However, the test substance in the 5% (w/v) formulation was judged as unstable for 24 h under the storage condition, because that concentration after storage was without 100 ± 10% (89.8%) of that immediately after preparation. Concentration analysis for the 50, 15 and 5% (w/v) formulations was performed by GC in CERI Hita immediately after the first preparation. The concentrations of the formulations were confirmed to be within 100 ± 10% of each nominal concentration (50% (w/v): 102%; 15% (w/v): 103% and 5% (w/v): 98.8%) and the preparation procedure was judged to be appropriate.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

300 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 (28 day main study)
5 (14 day recovery study)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose selection was based on the results of the 7-day range finding study. In this study groups of three Crl:CD(SD) rats of each sex and dose were administered the test material dissolved in olive oil at 0, 25, 250, 500 and 1000 mg/kg bw/day via oral gavage for 7 consecutive days. General clinical observations and body weight measurements were performed in the dosing period. Necropsy and organ weights measurements at the liver, kidney and spleen were performed on the next day after the last dosing. Since no toxic effects were observed in all treatment groups, the high dose of 1000 mg/kg bw/day which was the highest dose according to OECD TG 407 was selected for the 28-day study.
- Rationale for selecting satellite groups: Recovery groups were set for the 0 and 1000 mg/kg bw/day groups to investigate a reversibility of possible toxic effects for 14 days after the 28 day test material administration.
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During the dosing period, all animals were observed their general clinical signs twice daily (before and after dosing). During the recovery period, observation was performed once a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed once before dosing. Thereafter, animals were examined once a week during the dosing and recovery periods on a blind test basis.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded one day prior to dosing and on days 1, 3, 8, 12, 17, 21, 26 and 28 of the dosing period and on days 1, 5, 10 and 14 of the recovery period and on the necropsy days.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from all animals on the last day of dosing for the main groups and on the last withdrawal day for the recovery groups.
- Anaesthetic used for blood collection: Yes (isoflurane anesthesia)
- Animals fasted: Yes (overnight fasting)
- How many animals: all animals
- Parameters examined: red blood cell count (RBC), haemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count (Platelet), reticulocyte count ratio (Reticulo), white blood cell count (WBC), differential leucocyte counts (neutrophils (Neutro), lymphocytes (Lymph), basophils (Baso), eosinophils (Eosino), monocytes (Mono), large unstained cells (LUC)), prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from all animals on the last day of dosing for the main groups and on the last withdrawal day for the recovery groups.
- Animals fasted: Yes (overnight fasting)
- How many animals: all animals
- Parameters examined: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (gamma-GTP), total cholesterol (T-Cho), triglyceride (TG), blood urea nitrogen (BUN), creatinine, total protein (T-protein), albumin, A/G ratio, glucose, total bilirubin (T-bil), total bile acids (TBA), inorganic phosphorus (IP), calcium (Ca), sodium (Na), potassium (K), chloride (Cl)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected in individual metabolic cages from the afternoon of last day of the dosing period for the main groups and of the recovery period for the recovery groups to each next morning.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: urine volume, colour, turbidity, urine osmotic pressure, pH, protein, glucose, occult blood, urinary sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Animals were examined on week 4 (on days 27 and 28) of the dosing period. Reflex tests and measurements of grip strength were performed on a blind test basis. In the recovery period, the examinations were not performed since no abnormalities related to the test substance were observed in either sex of the high dose groups.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals were subjected to a detailed gross necropsy after blood sampling and bleeding from the ventral aorta on the next day after last administration for the main groups and on the next day after last withdrawal day for the recovery groups. External surface of the body, all orifices, subcutis, cranial, thoracic, abdominal and pelvic cavities and their content were observed. For females, vaginal smears were collected before gross necropsy and the stages of estrous cycle were determined.

HISTOPATHOLOGY: Yes
- Histopathological examinations included: trachea, lungs, submandibular gland, forestomach, glandular stomach, duodenum-ileum, cecum-rectum, pancreas, liver, heart, kidneys, urinary bladder, testes, epididymides, prostate, coagulating gland, seminal vesicle, ovaries, uterus, vagina, cerebrum, cerebellum, pons, spinal cord, sciatic nerve, bone marrow, axillar lymph nodes, mesenteric lymph nodes, spleen, thymus, pituitary gland, thyroid, parathyroid, adrenas, eye ball, skeletal muscle, bone, mammary gland

Other examinations:

ORGAN WEIGHTS
- The weights of the following organs were determined: liver, heart, kidneys, testes, epididymides, prostate, seminal vesicles, ovaries, uterus, brain, spleen, thymus, thyroid, adrenals

Statistics:

Data regarding body weights, food consumption, grip strength and locomotor activity counts during the dosing period, and parameters of haematological and clinical chemistry examinations, urine volume, urine osmotic pressure, organ weights and body weights on the necropsy day of the main groups were analysed by the Bartlett´s test for homogeneity of variances. When the variances were homogeneous at a significant level of 5%, the Dunnett´s test was used. The frequencies of defecation and urination during the dosing period were analysed by the nonparametric Dunnett´s test. Data regarding body weights, food consumption during the recovery period and parameters of haematological and clinical chemistry examination, urine volume, urine osmotic pressure, organ weights and body weights on the necropsy day of the recovery groups were analysed by the F-test for variance ratio. When there were no significant differences at a significant level of 5% in this analysis, the Student´s t-test was used. When there was a significant difference, the Aspin-Welch t-test was used. The frequencies of defecation and urination during the recovery group were analysed by the Mann-Whitney U-test.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

statistically significant decrease in males of the 1000 mg/kg bw/day dose group (recovery period) (non-adverse)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

statistically significant decrease on day 3 in females of the 1000 mg/kg bw/day dose group (non-adverse)

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):

males: statistically significant increase in ratio of monocytes (1000 mg/kg bw/day), prolongation of PT (100 mg/kg bw/day); females: statistically significant shortenings of activated partial thromboplastin time (100 and 1000 mg/kg bw/day) (non-adverse)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

females: statistically significant decrease of AST (all dose groups), statistically significant increase of gamma-GTP (300 mg/kg bw/day); statistically significant decrease of total bilirubin (1000 mg/kg bw/day, recovery group) (non-adverse)

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

females: statistically significant increase of locomotor activity counts (100 mg/mg bw/day), statistically significant decrease of locomotor activity (300 mg/kg bw/day) (non-adverse)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

females: statistically significant decrease in relative weights of brain/adrenals (100 mg/kg bw/day) (non-adverse)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

edema (submucosal layer), slight hyperplasia of the surface epithelial cells (fundic mucosa (males) and pyloric mucosa (females)) and slight necrosis (pyloric mucosa (males)) in the 1000 mg/kg bw/day groups (including recovery) (adverse, local effects)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

edema (submucosal layer), slight hyperplasia of the surface epithelial cells (fundic mucosa (males) and pyloric mucosa (females)) and slight necrosis (pyloric mucosa (males)) in the 1000 mg/kg bw/day groups (including recovery) (adverse, local effects)

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
In males, no abnormalities were observed in the 1000 or 100 mg/kg bw/day dose groups. Loss of hair and exudate in the neck were observed in one male of the 300 mg/kg bw/day dose group. In females, no abnormalities were observed in the control or any treatment group. No abnormalities were observed in either sex of the control or the 1000 mg/kg bw/day recovery groups.

BODY WEIGHT AND WEIGHT GAIN
No statistically significant changes were noted in either sex of any treatment groups. In males, statistically significant decrease or decreased tendency were observed throughout the recovery period in the 1000 mg/kg bw/day dose group.

FOOD CONSUMPTION AND COMPOUND INTAKE
In males, no statistically significant changes were noted in any treatment groups. In females, a statistically significant decrease was found on day 3 in the 1000 mg/kg bw/day dose group. No abnormalities were noted in the 100 and 300 mg/kg bw/day dose groups. No statistically significant changes were noted in either sex of the 1000 mg/kg bw/day dose group of the recovery group.

HAEMATOLOGY
In males, a statistically significant increase in ratio of monocytes was observed in the 1000 mg/kg bw/day dose group. A statistically significant prolongation of prothrombin time (PT) was observed in the 100 mg/kg bw/day dose group. No abnormalities were observed in any parameters in the 300 mg/kg bw/day dose group. The change of PT observed in the 100 mg/kg bw/day dose group was considered to be incidental, since no abnormalities were observed in the 1000 and 300 mg/kg bw/day dose groups and no dose-dependency were observed. In females, statistically significant shortenings of activated partial thromboplastin time were observed in the 1000 and 100 mg/kg bw/day dose groups. No abnormalities were observed in any parameters in the 300 mg/kg bw/day dose group. In the recovery group, a statistically significant increase of platelet count was found in males in the 1000 mg/kg bw/day dose group. In females, no significant changes were noted in the 1000 mg/kg bw/day dose group.

CLINICAL CHEMISTRY
In males, no significant changes were noted in any treatment groups. In females, statistically significant decreases of aspartate aminotransferase were observed in all treatment groups. A statistically increase of gamma-glutamyl transpeptidase was found in the 300 mg/kg bw/day dose group. This change was considered to be incidental since no abnormalities were observed in the 1000 mg/kg bw/day dose group and no dose-dependency was found. In the recovery group, no significant changes were noted in the 1000 mg/kg bw/day dose group in males. In females, a statistically significant decrease of total bilirubin was found in the 1000 mg/kg bw/day dose group.

URINALYSIS
No statistically significant changes in urine volume or urine osmotic pressure were noted in either sex or any treatment groups. No abnormalities were observed in the other examined parameters in either sex of the control or any dose groups. In the recovery group, no statistically significant changes in urine volume or urine osmotic pressure were noted in either sex of the 1000 mg/kg bw/day dose group. Moreover, no abnormalities were observed in the other examined parameters in either sex of the control or 1000 mg/kg bw/day groups.

NEUROBEHAVIOUR
In males, no statistically changes in grip strength or locomotor activity counts were noted in any treatment groups. No abnormalities were observed in the control or any treatment groups in the reflex test. In females, no statistically significant changes in locomotor activity counts were noted in the 1000 mg/kg bw/day. In the 100 mg/kg bw/day does group, locomotor activity counts of interval between 10 and 20 min and total score showed statistically significant increase. In the 300 mg/kg bw/day dose group, locomotor activity counts of interval between 40 and 50 min showed statistically significant decrease. These changes in the 100 and 300 mg/kg bw/day dose groups were considered to be incidental since these changes were not observed in the 1000 mg/kg bw/day dose group and dose-dependency was not observed. In grip strength, no significant changes were observed in any treatment groups. In reflex test, no abnormalities were observed in the control or any treatment groups. No neurobehavioural examinations were performed in animals of the recovery group since no effects of the test substance were noted in week 4 during the dosing period.

ORGAN WEIGHTS
In males, no significant changes were noted in any treatment groups. In females, no significant changes were noted in the 1000 and 300 mg/kg bw/day dose groups. In the 100 mg/kg bw/day dose group, statistically significant decreases in relative weights of the brain and adrenals were found. These changes were considered to be incidental since no abnormalities were observed in the 1000 and 300 mg/kg bw/day dose groups and no dose-dependency were found. In the recovery group, statistically significant decreases in relative and absolute weights of the spleen were found in the 1000 mg/kg bw/day dose group. In females, statistically significant increases in relative and absolute weights of the thyroid were found in the 1000 mg/kg bw/day dose group.

GROSS PATHOLOGY
In males, staining around nose and mouth, thickening of wall of the esophagus, roughening of the mucosa in the forestomach, bilateral enlargement of the mediastinal lymph node, enlargement of the hepatic lymph node, edematous change of the thymus, hydrothorax of the thoracic cavity, unilateral dark reddish change and bilateral enlargement at the adrenal were observed in one animal of the 1000 mg/kg bw/day dose group. Gritty and brownish substance in the lumen of the stomach was observed in two animals, gritty and brownish substance in the lumen of the cecum was recorded in another animal of the 1000 mg/kg bw/day dose group. In the 300 mg/kg bw/day dose group, erosion in the cervical skin and bilateral enlargement of the submandibular lymph node were observed in one animal in which loss of hair and exudate in the neck was observed in the general clinical signs observations. The erosion in the cervical skin observed in the 300 mg/kg bw/day dose group was considered to be incidental and occurred at the retention of the administration, and the bilateral enlargement of the submandibular lymph node was considered as the secondary change occurred from erosion in the cervical skin. In females, gritty and brownish substance in the lumen of the cecum was observed in two animals of the 1000 mg/kg bw/day dose group. Gritty and brownish substance in the lumen of the stomach was observed in one animal of the 1000 mg/kg bw/day dose group. In the 100 mg/kg bw/day dose group, cyst in the vagina of one animal was observed. No abnormalities were observed in the control or the 300 mg/kg bw/day dose groups. In the recovery group, no abnormalities were recorded in the control group. In males, blackish region of the mucosa and thickening of wall in the glandular stomach and solid and brownish substance in the lumen of the stomach were observed in one animal of the 1000 mg/kg bw/day dose group. In females, thickening of wall of the glandular stomach and solid and brownish substance in the lumen of the stomach were observed in one animal of the 1000 mg/kg bw/day dose group.

HISTOPATHOLOGY: NON-NEOPLASTIC
In males, hypertrophy of the acinar cells in the submandibular gland, severe ulcer related to the macroscopic findings in the esophagus, slight hyperplasia of the squamous epithelium in the limiting ridge and severe ulcer related to the macroscopic findings in the forestomach, slight edema in the submucosal layer, slight hyperplasia of the surface epithelial cells in the fundic mucosa and slight necrosis of the pyloric mucosa at the glandular stomach, single cell necrosis of the hepatocyte in the liver, focal myocardial necrosis in the heart, degeneration and necrosis of the tubular epithelium in the kidney, decreased hematopoietic cells in the bone marrow, germinal center development in the spleen, cell infiltration in the capsule and septa and phagocytosis of the degenerate lymphocytes related to the macroscopic findings in the thymus and unilateral hemorrhage and necrosis related to the macroscopic findings in the adrenals were observed in one animal of the 1000 mg/kg bw/day dose group. Solitary cyst in the medulla in the kidney, lymphocyte infiltration in the dorsolateral prostate, focal myocarditis in the heart and Rathke´s pouch remnant in the pituitary gland were observed in one animal each in the 1000 mg/kg bw/day dose group. In the 300 mg/kg bw/day dose group, unilateral pelvic dilatation and subcapsular solitary cyst in the medulla in the kidney, ulcer in the skin and germinal center development and increased plasmacytes in the submandibular lymph node related to the macroscopic findings in the skin and submandibular gland were observed in one animal. In the 100 mg/kg bw/day dose group, focal necrosis of the hepatocytes in the liver was observed in one animal. The changes in the kidney observed in the 300 mg/kg bw/day dose group and observed in the liver of the 100 mg/kg bw/day dose group were considered to be incidental and not related to the administration of the test substance, since those changes were historically observed in the control animals of the laboratory and no similar changes were observed in the 1000 mg/kg bw/day dose group. In females, degeneration and necrosis of the tubular epithelium and subcapsular solitary cyst in the kidney and decreased hematopoietic cells in the bone marrow were observed in one female of the 1000 mg/kg bw/day dose group. Slight hyperplasia of the surface epithelial cells in the pyloric mucosa in the glandular stomach was observed in one female. In the 100 mg/kg bw/darey dose group, mineralization of cortico-medullary junction in the kidney and squamous epithelial cyst in the vagina were observed in one animal. No abnormalities were observed in the 300 mg/kg bw/day dose group. The changes observed in the 100 mg/kg bw/day dose group were considered to be incidental and not related to the administration of the test substance, since the changes were historically observed in the control animals of the laboratory and no dose-dependency was found as no similar changes were observed in the 300 and 1000 mg/kg bw/day dose groups. In the control group, mineralization of the cortico-medullary junction in the kidney and focal necrosis of the hepatocytes in the liver were observed in one animal each. In the recovery group, slight hyperplasia of the surface of epithelial cells in the pyloric mucosa and slight focal necrosis of the fundic mucosa related to the macroscopic findings in the glandular stomach were observed in one male animal of the 1000 mg/kg bw/day dose group. No abnormalities were observed in the control group. In females, slight hyperplasia of the surface epithelial cells in the pyloric mucosa related to the macroscopic findings in the glandular stomach was observed in one animal. Subcapsular solitary cyst in the kidney was observed in one animal of the control group.

Dose descriptor:

NOAEL

Remarks:

(local)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: necrosis of the pyloric/fundic mucosa, hyperplasia of the surface epithelial cells in the pyloric/fundic mucosa, edema in the submucosal layer (males), hyperplasia of the surface epithelial cells in the pyloric mucosa (females)

Dose descriptor:

NOAEL

Remarks:

(systemic)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed with respect to general/detailed clinical observations, haematological/clinical chemistry/neurobehaviour examination, body weights or urinalysis

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

no

Table 1: Summary of functional observations (motor activity)

Sex		Males				Females
Dose (mg/kg bw/day)		0	100	300	1000	0	100	300	1000
No. of animals		10	5	5	10	10	5	5	10
Motor activity (interval)	0-10 (mn)	213 ± 49	215 ± 44	224 ± 72	215 ± 49	217 ± 49	254 ± 30	265 ± 23	250 ± 52
	10-20 (min)	108 ± 46	101 ± 14	138 ± 64	104 ± 71	153 ± 65	237 ± 7*	186 ± 62	180 ± 51
	20-30 (min)	74 ± 42	59 ± 37	89 ± 39	51 ± 40	107 ± 52	164 ± 26	130 ± 74	103 ± 70
	30-40 (min)	77 ± 54	63 ± 41	68 ± 49	55 ± 41	97 ± 53	106 ± 57	91 ± 51	83 ± 67
	40-50 (min)	40 ± 42	39 ± 28	18 ± 13	31 ±45	74 ± 60	131 ± 78	4 ± 7*	56 ± 53
	50-60 (mIn)	34 ± 37	13 ± 17	15 ± 19	17 ± 25	54 ± 46	68 ± 41	57 ± 81	46 ± 54
	Total	546 ± 196	489 ± 118	552 ± 150	473 ± 200	702 ± 202	959 ± 99*	733 ± 241	718 ± 168

*: p<0.05

Table 2: Summary of haematological parameters (males)

Item	Sex	Male groups				Recovery groups
	Dose (mg/kg bw/day)	0	100	300	1000	0	1000
Mono (%)		2.50 ± 0.27	2.64 ± 1.11	2.48 ± 0.69	3.74* ± 0.48	2.82 ± 0.63	3.02 ± 0.60
PT (sec)		15.4 ± 1.20	18.68* ± 0.98	15.48 ± 1.18	15.56 ± 1.15	17.26 ± 1.52	19.42 ± 4.56
Platelat (x10^4/µl)		137.34 ± 7.75	129.94 ± 9.41	141.18 ± 22.00	115.06 ± 15.69	109.44 ± 5.66	125.14** ± 3.49

*: p<0.05

**: p<0.01

Table 3: Summary of haematological parameters (females)

Item	Sex	Female groups				Recovery groups
	Dose (mg/kg bw/day)	0	100	300	1000	0	1000
APTT (sec)		19.90 ± 0.62	16.98* ± 2.00	18.48 ± 1.50	17.18* ± 1.00	17.66 ± 2.71	17.44 ± 1.88

*: p<0.05

Table 4: Summary of clinical chemistry parameters (females)

Item	Sex	Female groups				Recovery groups
	Dose (mg/kg bw/day)	0	100	300	1000	0	1000
AST (IU/l)		95.6 ± 6.5	76.2** ± 7.9	75.4** ± 5.9	72.4** ± 7.4	71.2 ± 5.9	74.8 ± 15.0
gamma-GTP (IU/l)		0.56 ± 0.17	0.72 ± 0.41	1.02* ± 0.16	0.82 ± 0.13	0.52 ± 0.27	0.64 ± 0.22
T-Bil (mg/dl)		0.064 ± 0.013	0.058 ± 0.008	0.052 ± 0.008	0.056 ± 0.011	0.076 ± 0.005	0.060** ± 0.007

*: p<0.05

**: p<0.01

Table 5: Summary of absolute organ weights (males)

Item	Sex	Male groups				Recovery groups
	Dose (mg/kg bw/day)	0	100	300	1000	0	1000
Spleen (g)		0.722 ± 0.089	0.650 ± 0.157	0.712 ± 0.154	0.602 ± 0.147	0.888 ± 0.033	0.698* ± 0.102
Final body weight (g)		345.04 ± 45.59	335.56 ± 33.27	341.76 ± 40.21	329.42 ± 47.50	424.46 ± 20.98	391.42* ± 22.89

*: p<0.05

Table 6: Summary of absolute organ weights (females)

Item	Sex	Female groups				Recovery groups
	Dose (mg/kg bw/day)	0	100	300	1000	0	1000
Thyroid (g)		14.22 ± 2.66	14.88 ± 1.70	13.36 ± 0.74	13.30 ± 1.68	13.12 ± 1.47	17.24* ± 2.64
Final body weight (g)		196.52 ± 21.66	210.92 ± 12.75	202.70 ± 11.87	202.44 ± 12.52	236.56 ± 14.91	230.58 ± 17.05

*: p<0.05

Table 7: Summary of relative organ weights (males)

Item	Sex	Female groups				Recovery groups
	Dose (mg/kg bw/day)	0	100	300	1000	0	1000
Spleen (g/100 g)		0.208 ± 0.024	0.192 ± 0.026	0.210 ± 0.050	0.180 ± 0.035	0.208 ± 0.004	0.178* ± 0.020
Final body weight (g)		345.04 ± 45.59	335.56 ± 33.27	341.78 ± 40.21	329.42 ± 47.50	424.46 ± 20.98	391.42* ± 22.89

*: p<0.05

Table 8: Summary of relative organ weights (females)

Item	Sex	Female groups				Recovery groups
	Dose (mg/kg bw/day)	0	100	300	1000	0	1000
Brain (g/100 g)		0.966 ± 0.057	0.888* ± 0.026	0.960 ± 0.055	0.920 ± 0.045	0.826 ± 0.047	0.828 ± 0.018
Thyroid (mg/100 g)		7.28 ± 1.28	7.06 ± 0.66	6.60 ± 0.66	6.60 ± 1.00	5.54 ± 0.54	7.54* ± 1.46
Adrenals (mg/100 g)		31.58 ± 4.16	25.18* ± 1.12	28.98 ± 4.81	29.96 ± 0.76	25.50 ± 2.90	26.74 ± 3.17
Final body weight (g)		196.52 ± 21.66	210.92 ± 12.75	202.70 ± 11.87	202.44 ± 12.52	236.56 ± 14.91	230.58 ± 17.05

*: p<0.05

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was conducted according to an appropriated OECD test guideline, with acceptable restrictions. The study was conducted in compliance with GLP.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see remark

Remarks:

There were positive skin reactions observed with the vehicle control, light paraffin oil. The mild to moderate microscopic skin lesions found for animals treated with the vehicle alone suggests that the vehicle and/or test procedure contributed to the skin irritation observed in the Silane-treated animals. Hence, no clear resulton both the local and the systemic, repeated dose toxicity hazard could be obtained from this study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

(1983)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Dutchland (Denver, PA)
- Age at study initiation: 17-19 weeks
- Weight at study initiation: 3531.0±163.44 g (control group males); 3535.6±138.82 g (low dose males); 3476.7±172.42 g (mid dose males); 3532.2±172.82 g (high dose males); 3498.6±204.20 g (control females); 3430.7±110.13 g (low dose females); 3452.6±130.63 g (mid dose females); 3493.2±213.12 g (high dose females)
- Housing: The rabbits were housed individually in stainless steel cages with wire floors. A layer of Deotized Animal Cage Board@ (Shepherd Specialty Papers, Inc., Kalamazoo, MI) was kept under each cage and changed daily.
- Diet: Agway Prolab Certified Rabbit Diet (Agway, Inc., St. Mary's, OH, and Waverly, NY) was available to the rabbits on a restricted basis during the acclimation period. The amount of food was increased gradually from 4 ounces/day/rabbit at the time of arrival to 12 ounces/day/rabbit 3 days prior to dosing and were maintained on 12 ounces/day until the first day of dosing. Food was available ad libitum from day 1 until the end of the study.
- Water: Water (Municipal Authority of Westmoreland County, Greensburg, PA) was administered ad libitum by an automatic watering system with demand control valves mounted on each rack.
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-22.2 (64-72°F)
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: from 12 April 1988 to 23 May 1988

Type of coverage:

occlusive

Vehicle:

paraffin oil

Remarks:

mineral oil

Details on exposure:

TEST SITE
- Area of exposure: dorsal area of the trunk
- % coverage: at least 10
- Type of wrap if used: Lycra/Spandex jacket that was lined with Vinyllite and held in place with velcro strips
- Time intervals for shavings or clipplings: Prior to the first dose and subsequently as needed (care was taken to avoid abrading the skin.)
- Other: Prior to the initiation of the study, the animals were sham-wrapped once in the manner used during the study to minimise the stress involved with the first application of the dose.

REMOVAL OF TEST SUBSTANCE
- Washing: Yes (The back of each animal was wiped with a dry cloth. The use of a damp cloth seemed unappropriated , since this might exacerbate the irritant properties. The test material reacts rapidly with water to form methanol.)
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied: 2 ml/kg bw
- Concentration (if solution): 0.5, 1, and 5% in vehicle
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): Probe studies indicate that the test article is severely irritating and must be diluted with and appropriate vehicle prior to application. The test material reacts rapidly with water to form methanol.
- Appearance: clear, oily viscous liquid
- Source: Fisher Scientific, Pittsburgh, PA
- CAS No.: 8012-95-1
- Lot/batch no.: 875079, 871981, and 870338
- Stability: stable at room temperature
- Storage conditions: room temperature
- Purity: > 99%

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Dosing solutions were analysed by a gas chromatographic procedure developed at the test facility. Prior to the start of the study, stability and homogeneity ofthe test item solutions in light paraffin oil were determined at nominal concentrations of 0.5, 3.0, and 5.0% test substance. The test substance concentrations in the dosing solutions used in the study were confirmed prior to administration of the solutions to the animals.
The results indicated that the distribution of the test item in the vehicle was uniform. Concentration verification analyses on formulations used for dosing showed analytical values ranging from 96.3-104.2% of the nominal concentration.

Duration of treatment / exposure:

6 h/day

Frequency of treatment:

5 days/week for 4 weeks

Dose / conc.:

8.4 mg/kg bw/day (nominal)

Remarks:

0.5%: nominal test material concentration in mineral oil

Dose / conc.:

16.8 mg/kg bw/day (nominal)

Remarks:

1%: nominal test material concentration in mineral oil

Dose / conc.:

84 mg/kg bw/day (nominal)

Remarks:

5%: nominal test material concentration in mineral oil

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses for the study were selected based on the results of the acute toxicity and primary irritancy studies (BRRC Project Report 45-49) and a series of repeated dose percutaneous dose-range finding studies conducted on the test item.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (twice daily for mortality)
- Cage side observations checked: signs of toxicity, with particular attention being paid to the treated area of skin

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: Careful examination was made prior to dosing each day for signs of irritation.
- Scoring system for skin irritation: Skin irritation signs were graded for erythema and edema according to the Draize scoring system.
The scoring system used for erythema was: 0 = no erythema; 1 = barely perceptible erythema; 2 = well defined erythema; 3 = moderate to severe erythema; 4 = severe erythema.
The scoring system used for edema was: 0 = no edema; 1 = barely perceptible edema; 2 = slight, well defined edema; 3 = moderate edema; 4 = severe edema

BODY WEIGHT: Yes
- Time schedule for examinations: immediately prior to the first dose (Day 1), prior to the 6th, 11th, and 16th doses (days 8, 15, and 22, respectively), and on day 28 prior to fasting; fasted body weights were also measured prior to sacrifice (day 29) to allow expression of organ weights relative to body weight

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes
- Time schedule: on days 1, 3, 5, 7, 8, 10, 12, 14, 15, 17, 19, 21, 22, 24, 26, and 28

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: prior to sacrifice by cardiac puncture
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: erythrocyte count, haemoglobin concentration, haematocrit, erythrocyte indices, total leukocyte count, differential leukocyte count, and platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to sacrifice by cardiac puncture
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: glucose, urea nitrogen, creatinine, total protein, albumin, globulin, serum haemoglobin, indirect bilirubin, total bilirubin, direct bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), gamma-glutamyl transferase (GGT), lactate dehydrogenase, alkaline phosphatase, creatinine phosphokinase, calcium, phosphorus, sodium, potassium, and chloride

URINALYSIS: Yes
- Time schedule for collection of urine: at necropsy, urine was taken from the urinary bladder for urinalysis measurements
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked: specific gravity, pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, microscopic elements, and volume (bladder contents)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete necropsy was performed on each animal.
- Organs checked: brain (cerebral cortex, cerebellar cortex, medulla/pons), eyes, pituitary, salivary gland, heart, aorta, thymic region, thyroid (with parathyroid), lungs (2 coronal sections including all lobes and mainstem bronchi), trachea, oesophagus, spinal cord (cervical, thoracic, lumbar), pancreas, liver (2 lobes) with gallbladder, kidneys, urinary bladder, testes, prostate, epididymis, ovaries, vagina, uterus (corpus and cervix), spleen, lymph nodes (mesenteric & non-mesenteric), skeletal muscle, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, adrenals, sciatic nerve, mammary gland (females), skin (treated and untreated), and sternum (including marrow)

HISTOPATHOLOGY: Yes
- How many animals: rabbits of the vehicle control and high dose groups and for treated and untreated skin from rabbits in the low and mid dose treatment groups
- Organs checked: brain, pituitary, heart, thymic region, thyroid, lungs, liver, kidneys, testes, epididymis, ovaries, uterus, spleen, lymph nodes, adrenals, skin (treated and untreated), sternum (including marrow)

ORGAN WEIGHTS. Yes
- Organs checked: liver, kidneys, adrenals, brain, and heart

Statistics:

Food consumption, body weight, and organ weight data were intercompared for the dose and control groups by use of Levene's test for homogeneity of variances, by analysis of variance, and by pooled variance t-tests. The t-tests were used, if the analysis of variance was significant, to delineate which groups differed from the control group. If Levene's test indicated heterogeneous variances, the groups were compared by an analysis of variance for unequal variances followed, if necessary, by separate variance t-tests. Medians and quartile deviations were calculated for non-parametric data. These data were statistically analysed by the Kruskal-Wallis test and, if necessary, by the Wilcoxon rank sum test as modified by Mann-Whitney. Frequency data were compared using Fisher's exact tests where appropriate. All statistical tests, except the frequency comparisons were performed using BMDP Statistical Software (Dixon, 1985). The frequency data tests are described in Biometry (Sokal, R. R. and Rohlf, F. J., W. 8. Freeman and Company: San Francisco, 1969). The fiducial limit of 0.05 was used as the critical level of significance for all tests.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Mild to moderate skin irritation in the low dose group and mild to marked irritation in mid and high dose groups. Since some effects were observed in the solvent controls, irritations seen in test gruops may partly have been caused by the solvent.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreased body weight gain was observed in both treated males and females as compared to the controls, but the effects were not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Decreased food consumption was observed in both treated males and females as compared to the controls, but the effects were not statistically significant.

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:

no effects observed

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):

A treatment related decrease in absolute and relative liver weights was observed in mid and high dose males as compared to the controls. However, the effects were not statistically significant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related gross lesions were observed only in the treated skin of both males and females of all treatment and control groups, with higher severity and/or incidence of severity tending to the treatment groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related microscopic lesions were observed only in the treated skin of both males and females of all treatment and control groups, with higher severity and/or incidence of severity tending to the treatmen groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No animals died during the study.
Treatment-related clinical signs of toxicity were restricted to the site of application of test material or vehicle. Erythema and oedema were observed in some animals from most groups including controls. These lesions tended to occur earlier and were more severe in the high and mid dose groups. Erythema was observed for all males in the high dose group with maximum severity scores of well defined (2 males) and moderately severe (3 males). Erythema was also observed for all animals in the mid dose group with maximum severity scores of barely perceptible (4 males) and moderately severe (1 male). Erythema, scored as barely perceptible or well defined, was observed for 2 males in the control group and 1 male in the low dose group. Oedema was observed for all males in the high dose group with maximum severity scores of well defined (1 male) and moderate (4 males). Oedema, scored principally as well defined, was observed for 1 male from each of the control and mid dose groups. Oedema was not observed for males in the low dose group. Findings of erythema and/or oedema occurred principally during the 3rd or 4th week of dosing for the 2 males in the control group, from days 5-13 for the male in the low dose group, and in all weeks of dosing for males in the mid and high dose treatment groups.
Erythema was scored principally as well defined for females in the high dose group and barely perceptible for females in the control, low dose, and mid dose groups. All females in the high dose group had erythema starting in the first week of dosing. The incidence of erythema for the control (1 animal), low dose (1 animal), and mid dose (3 animals) groups observed near the end of the first week of dosing increased to 4 or 5 females per group at the end of the last week of dosing. Oedema (barely perceptible to moderate severity) was observed for all females in the high dose group after the first week of dosing. Oedema, scored principally as barely perceptible, was observed for 2 females from the mid dose group after the first week of dosing and 2 additional females each from this group and the low dose group at the end of the dosing period. Oedema (barely perceptible and/or well defined) was observed for 3 females in the control group during the 3rd or 4th weeks of the study.
Other treatment-related clinical signs of toxicity (in addition to erythema and oedema) were limited to the skin at the application site and included exfoliation, ecchymosis, excoriation, encrustation, ulceration, and fissuring for both males and females. Lesions were observed predominantly in the mid and high dose groups with more animals in the high dose groups affected than in the other groups. Exfoliation and ecchymosis were observed in approximately equal incidence for all groups of females including controls. The incidence of all lesions in males and all other lesions in females appeared to show a dose response. Pustule and abscess were observed for 1 low dose female. No other treatment-related clinical signs of toxicity were observed in this study.

BODY WEIGHT AND WEIGHT GAIN
Statistically significant alterations in absolute body weights and body weight gain were not observed in male or female rabbits administered 8.4, 16.8, or 84.0 mg/kg bw/day of the test material. However, decreased mean body weight gain compared to controls or mean body weight losses were observed for males in the mid dose and high dose groups during the study. Mean body weight loss was observed for males in the mid dose group during the first week of the study and for males in the high dose group during the first and second weeks of the study compared to a mean body weight gain of 78.5 g and 166.2 g for controls for the first and second week of treatment, respectively. Mean body weight gains during the remainder of the study for the mid and high dose groups compared to controls were decreased: 38% to 57% (mid dose males) and 86% to 89% (high dose males).
Mean body weight losses were observed throughout the study for females in the control and high dose groups. The maximum loss for both groups occurred during the first week of the study with mean losses of 89.3 g for controls and 204.4 g for the high dose treatment group. Mean body weight losses during the 4 week exposure period were 49.5 g for controls and 161.0 g for the high dose females.

FOOD CONSUMPTION
Statistically significant alterations in food consumption were not observed in male or female rabbits administered 8.4, 16.8, or 84.0 mg/kg bw/day of the test material. However, mean food consumptions were decreased compared to the control group for males in the high dose treatment group (15% to 28%) during the 4 weeks of exposure and for males in the mid dose treatment group (4% to 18%) during days 1-15. Additionally, mean food consumption was decreased for females from the high dose treatment group compared to the control group (4% to 17%) during the days 1-22.

HAEMATOLOGY
There were no statistically significant differences between the values for any of the haematoligic parameters in treated rabbits compared with those of the control rabbits, nor were there any biologically important trends.

CLINICAL CHEMISTRY
The only statistically significant change in serum chemistry parameters was observed in 8.4 and 84 mg/kg bw/ dosed male rabbits where SDH values were lower than in control rabbits. This coupled with somewhat lower values, albeit not statistically significant, for both AST and ALT in the high dose male rabbits suggests a possible effect on synthesis of liver enzymes. However, no such trend was observed either in the high dose female rabbits, or a dose relationship in the lower dose male rabbits. Therefore, this alteration is not likely to be biologically important. Liver necrosis would result in increased rather than decreased liver enzyme values.

URINALYSIS
No biologically or statistically significant changes were observed in the urine parameters for either male or female rabbits.

ORGAN WEIGHTS
Statistically significant alterations in organ weights were not observed for male or female rabbits administered 8.4, 16.8, or 84.0 mg/kg bw/day of the test item. However, a dose-related decrease in mean absolute liver weight compared to the control group was observed for males in the mid dose (-16%) and high dose (-21%) treatment groups. Dose-related decreases in mean liver weights expressed relative to body weight or brain weight were also observed for males in the mid dose (-9% and -12%, respectively) and high dose (-14% and -15%, respectively) groups. An apparent dose-related decrease in mean liver weight was also observed for males in the low dose group. However, this decrease was a result of an exceptionally small liver in one animal (approximately 50% of control mean) and was not considered to be related to treatment based on the lack of effect on body weight, supporting clinical chemistry findings or gross or microscopic findings in the liver, and the lack of similar effects on liver weight for the four remaining males in this group (mean liver weight for the remaining animals = 2% greater than control mean).

GROSS PATHOLOGY AND HISTOPATHOLOGY
Treatment-related gross and microscopic lesions were observed only in the treated skin of both males and females. Gross lesions were observed on the treated and untreated skin of animals from all groups. Treatment-related gross skin lesions were limited to the treatment area. Gross lesions observed included exfoliation, erythema, surface crust, haemorrhage, and/or fissuring. The severity and/or incidence for several of the gross lesions tended to be greater in the treated groups than in control groups. Microscopic lesions indicative of both chronic and acute inflammation were observed in all treatment groups (including controls). Acanthosis, ulceration, folliculitis, and dermatitis for males, and oedema, congestion, haemorrhage, and hyperkeratosis for both males and females occurred in approximately equal frequency for control and treated animals. A dose-related increase in the incidence of ulceration was observed for females in the mid dose (3 animals) and high dose (4 animals) groups and the incidence of folliculitis for females was greatest in the mid and high dose groups (3 animals each). The severity of epidermitis and ulceration for males and acanthosis, epidermitis, ulceration, dermatitis, and dermal fibrosis for females increased in a dose-related manner. The range of severity for these lesions was mild to moderate for the control and low dose animals and mild to marked for the mid and high dose animals. Microscopic lesions were also observed in untreated skin of rabbits from all dosage groups. These lesions were less frequent and generally less severe than those observed at the application site.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 84 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The effect level is based on systemic toxicity. No systemic toxicity was observed in any of the test doses. All effects seen in the treatment groups are based on local skin irritation and consequently indisposition of the animals.

Dose descriptor:

NOAEL

Remarks:

local

Remarks on result:

not determinable because of methodological limitations

Remarks:

It cannot clearly be determined to what extend the skin irritating effects are the result of the test substance treatment because skin irritation was also observed in the controls

Critical effects observed:

no

Mild to moderate skin irritation was seen at the lowest dosage level, 8.4 mg/kg bw/day. Mild to marked irritation was noted at dosage levels of 16.8 mg/kg/day and above. Since some effects were observed with the silane-free solvent control animals, the irritation seen in the test groups may in part have been caused by the solvent.  

Tab. 1: Summary of Skin irritation data

	control								8.4 mg/kg bw (0.5%)
Day	Erythema				Oedema				Erythema				Oedema
	males		females		males		females		males		females		males		females
	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range
1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
2	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
3	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
4	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
5	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
6	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
7	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0
8	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0
9	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
10	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
11	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
12	0.2±0.4	0-1	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
13	0.2±0.4	0-1	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
14	0.2±0.4	0-1	0.4±0.9	0-2	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
15	0.2±0.4	0-1	0.4±0.9	0-2	0.0±0.0	0-0	0.4±0.9	0-2	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0
16	0.2±0.4	0-1	0.4±0.9	0-2	0.0±0.0	0-0	0.4±0.9	0-2	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0
17	0.2±0.4	0-1	0.4±0.9	0-2	0.0±0.0	0-0	0.4±0.9	0-2	0.0±0.0	0-0	0.4±0.5	0-1	0.0±0.0	0-0	0.0±0.0	0-0
18	0.2±0.5	0-1	0.6±0.9	0-2	0.0±0.0	0-0	0.4±0.9	0-2	0.0±0.0	0-0	0.4±0.5	0-1	0.0±0.0	0-0	0.0±0.0	0-0
19	0.4±0.5	0-1	0.6±0.9	0-2	0.0±0.0	0-0	0.4±0.9	0-2	0.0±0.0	0-0	0.4±0.5	0-1	0.0±0.0	0-0	0.0±0.0	0-0
20	0.2±0.4	0-1	0.6±0.9	0-2	0.0±0.0	0-0	0.4±0.9	0-2	0.0±0.0	0-0	0.4±0.5	0-1	0.0±0.0	0-0	0.0±0.0	0-0
21	0.2±0.4	0-1	0.4±0.5	0-1	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.4±0.5	0-1	0.0±0.0	0-0	0.0±0.0	0-0
22	0.2±0.4	0-1	0.4±0.5	0-1	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.4±0.5	0-1	0.0±0.0	0-0	0.0±0.0	0-0
23	0.2±0.4	0-1	0.4±0.5	0-1	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.4±0.5	0-1	0.0±0.0	0-0	0.0±0.0	0-0
24	0.2±0.4	0-1	0.8±0.8	0-2	0.0±0.0	0-0	0.4±0.9	0-2	0.0±0.0	0-0	0.8±0.4	0-1	0.0±0.0	0-0	0.2±0.4	0-1
25	0.2±0.4	0-1	0.8±0.8	0-2	0.2±0.4	0-1	0.4±0.9	0-2	0.0±0.0	0-0	0.8±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0
26	0.4±0.9	0-2	0.6±0.5	0-1	0.4±0.9	0-2	0.4±0.9	0-2	0.0±0.0	0-0	0.8±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0
27	0.4±0.9	0-2	0.6±0.5	0-1	0.4±0.9	0-2	0.4±0.9	0-2	0.0±0.0	0-0	0.8±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0
28	0.4±0.9	0-2	0.6±0.5	0-1	0.4±0.9	0-2	0.6±0.9	0-2	0.0±0.0	0-0	0.8±0.4	0-1	0.0±0.0	0-0	0.6±0.5	0-1
29	0.4±0.9	0-2	0.6±0.5	0-1	0.4±0.9	0-2	0.6±0.9	0-2	0.0±0.0	0-0	0.8±0.4	0-1	0.0±0.0	0-0	1.2±1.1	0-3

Conclusions:

A dermal repeated dose toxicity conducted according to the OECD TG 410 and in compliance with GLP with bis(trimethoxysilylpropyl)amine (CAS 82985-35-1) is available, although the results were disregarded as there were positive skin reactions observed with the vehicle control, light paraffin oil. The mild to moderate microscopic skin lesions found for animals treated with the vehicle alone suggests that the vehicle and/or test procedure contributed to the skin irritation observed in the Silane-treated animals. Bis(trimethoxysilylpropyl)amine (CAS 82985-35-1) was tested on both male and female New Zealand White rabbits to determine specific target organ toxicity after repeated skin contact. Solutions of differing concentrations of the test item (0.5, 1, and 5%, referring to 8.4, 16.8, and 84.0 mg/kg bw) in a light paraffin oil were applied to the clipped dorsal trunk skin under an impervious covering for 6 hours/day, 5 days/week for 4 weeks. No mortality occurred throughout the study period. Decreased body weight gain and decreased food consumption were observed in both treated males and females as compared to the controls, but the effects were not statistically significant. A treatment related decrease in absolute and relative liver weights was observed in mid and high dose males as compared to the controls. However, the effects were not statistically significant. Mild to moderate skin irritation was seen at the lowest dosage level, 8.4 mg/kg/day, whereas mild to marked irritation was noted at dosage levels of 16.8 mg/kg/day and above. Treatment-related gross and microscopic lesions were observed only in the treated skin of both males and females of all treatment and control groups, with higher severity and/or incidence of severity tending to the treatment groups. The predominant finding was hyperkeratosis. However, degenerative effects to the skin were not observed. Since some findings were also observed with the solvent control animals, the irritation seen in the test groups may in part have been caused by the solvent. Among this, light paraffin oil is known to be irritating to the skin (Nessel et al. (1999) Toxicological Sciences 49:48-55). The study provided evidence for cumulative irritation from repeated skin contact with the test item, but there was no evidence of systemic toxicity. The other effects observed in this study (decreases in food consumption, body weight gain, and liver weight for males in the mid and high dose groups) were not indicative of a consistent systemic effect of the test material and may have been partially related to debility resulting from skin irritation. Therefore, no clear result on both the local and the systemic repeated dose toxicity hazard could be obtained from this study..

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see remark

Remarks:

There were positive skin reactions observed with the vehicle control, light paraffin oil. The mild to moderate microscopic skin lesions found for animals treated with the vehicle alone suggests that the vehicle and/or test procedure contributed to the skin irritation observed in the Silane-treated animals. Hence, no clear resulton both the local and the systemic, repeated dose toxicity hazard could be obtained from this study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Version / remarks:

(1983)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Dutchland (Denver, PA)
- Age at study initiation: 17-19 weeks
- Weight at study initiation: 3531.0±163.44 g (control group males); 3535.6±138.82 g (low dose males); 3476.7±172.42 g (mid dose males); 3532.2±172.82 g (high dose males); 3498.6±204.20 g (control females); 3430.7±110.13 g (low dose females); 3452.6±130.63 g (mid dose females); 3493.2±213.12 g (high dose females)
- Housing: The rabbits were housed individually in stainless steel cages with wire floors. A layer of Deotized Animal Cage Board@ (Shepherd Specialty Papers, Inc., Kalamazoo, MI) was kept under each cage and changed daily.
- Diet: Agway Prolab Certified Rabbit Diet (Agway, Inc., St. Mary's, OH, and Waverly, NY) was available to the rabbits on a restricted basis during the acclimation period. The amount of food was increased gradually from 4 ounces/day/rabbit at the time of arrival to 12 ounces/day/rabbit 3 days prior to dosing and were maintained on 12 ounces/day until the first day of dosing. Food was available ad libitum from day 1 until the end of the study.
- Water: Water (Municipal Authority of Westmoreland County, Greensburg, PA) was administered ad libitum by an automatic watering system with demand control valves mounted on each rack.
- Acclimation period: approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.8-22.2 (64-72°F)
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: from 12 April 1988 to 23 May 1988

Type of coverage:

occlusive

Vehicle:

paraffin oil

Remarks:

mineral oil

Details on exposure:

TEST SITE
- Area of exposure: dorsal area of the trunk
- % coverage: at least 10
- Type of wrap if used: Lycra/Spandex jacket that was lined with Vinyllite and held in place with velcro strips
- Time intervals for shavings or clipplings: Prior to the first dose and subsequently as needed (care was taken to avoid abrading the skin.)
- Other: Prior to the initiation of the study, the animals were sham-wrapped once in the manner used during the study to minimise the stress involved with the first application of the dose.

REMOVAL OF TEST SUBSTANCE
- Washing: Yes (The back of each animal was wiped with a dry cloth. The use of a damp cloth seemed unappropriated , since this might exacerbate the irritant properties. The test material reacts rapidly with water to form methanol.)
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied: 2 ml/kg bw
- Concentration (if solution): 0.5, 1, and 5% in vehicle
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): Probe studies indicate that the test article is severely irritating and must be diluted with and appropriate vehicle prior to application. The test material reacts rapidly with water to form methanol.
- Appearance: clear, oily viscous liquid
- Source: Fisher Scientific, Pittsburgh, PA
- CAS No.: 8012-95-1
- Lot/batch no.: 875079, 871981, and 870338
- Stability: stable at room temperature
- Storage conditions: room temperature
- Purity: > 99%

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Dosing solutions were analysed by a gas chromatographic procedure developed at the test facility. Prior to the start of the study, stability and homogeneity ofthe test item solutions in light paraffin oil were determined at nominal concentrations of 0.5, 3.0, and 5.0% test substance. The test substance concentrations in the dosing solutions used in the study were confirmed prior to administration of the solutions to the animals.
The results indicated that the distribution of the test item in the vehicle was uniform. Concentration verification analyses on formulations used for dosing showed analytical values ranging from 96.3-104.2% of the nominal concentration.

Duration of treatment / exposure:

6 h/day

Frequency of treatment:

5 days/week for 4 weeks

Dose / conc.:

8.4 mg/kg bw/day (nominal)

Remarks:

0.5%: nominal test material concentration in mineral oil

Dose / conc.:

16.8 mg/kg bw/day (nominal)

Remarks:

1%: nominal test material concentration in mineral oil

Dose / conc.:

84 mg/kg bw/day (nominal)

Remarks:

5%: nominal test material concentration in mineral oil

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses for the study were selected based on the results of the acute toxicity and primary irritancy studies (BRRC Project Report 45-49) and a series of repeated dose percutaneous dose-range finding studies conducted on the test item.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (twice daily for mortality)
- Cage side observations checked: signs of toxicity, with particular attention being paid to the treated area of skin

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: Careful examination was made prior to dosing each day for signs of irritation.
- Scoring system for skin irritation: Skin irritation signs were graded for erythema and edema according to the Draize scoring system.
The scoring system used for erythema was: 0 = no erythema; 1 = barely perceptible erythema; 2 = well defined erythema; 3 = moderate to severe erythema; 4 = severe erythema.
The scoring system used for edema was: 0 = no edema; 1 = barely perceptible edema; 2 = slight, well defined edema; 3 = moderate edema; 4 = severe edema

BODY WEIGHT: Yes
- Time schedule for examinations: immediately prior to the first dose (Day 1), prior to the 6th, 11th, and 16th doses (days 8, 15, and 22, respectively), and on day 28 prior to fasting; fasted body weights were also measured prior to sacrifice (day 29) to allow expression of organ weights relative to body weight

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes
- Time schedule: on days 1, 3, 5, 7, 8, 10, 12, 14, 15, 17, 19, 21, 22, 24, 26, and 28

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: prior to sacrifice by cardiac puncture
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: erythrocyte count, haemoglobin concentration, haematocrit, erythrocyte indices, total leukocyte count, differential leukocyte count, and platelet count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to sacrifice by cardiac puncture
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: glucose, urea nitrogen, creatinine, total protein, albumin, globulin, serum haemoglobin, indirect bilirubin, total bilirubin, direct bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), gamma-glutamyl transferase (GGT), lactate dehydrogenase, alkaline phosphatase, creatinine phosphokinase, calcium, phosphorus, sodium, potassium, and chloride

URINALYSIS: Yes
- Time schedule for collection of urine: at necropsy, urine was taken from the urinary bladder for urinalysis measurements
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked: specific gravity, pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, microscopic elements, and volume (bladder contents)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete necropsy was performed on each animal.
- Organs checked: brain (cerebral cortex, cerebellar cortex, medulla/pons), eyes, pituitary, salivary gland, heart, aorta, thymic region, thyroid (with parathyroid), lungs (2 coronal sections including all lobes and mainstem bronchi), trachea, oesophagus, spinal cord (cervical, thoracic, lumbar), pancreas, liver (2 lobes) with gallbladder, kidneys, urinary bladder, testes, prostate, epididymis, ovaries, vagina, uterus (corpus and cervix), spleen, lymph nodes (mesenteric & non-mesenteric), skeletal muscle, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, adrenals, sciatic nerve, mammary gland (females), skin (treated and untreated), and sternum (including marrow)

HISTOPATHOLOGY: Yes
- How many animals: rabbits of the vehicle control and high dose groups and for treated and untreated skin from rabbits in the low and mid dose treatment groups
- Organs checked: brain, pituitary, heart, thymic region, thyroid, lungs, liver, kidneys, testes, epididymis, ovaries, uterus, spleen, lymph nodes, adrenals, skin (treated and untreated), sternum (including marrow)

ORGAN WEIGHTS. Yes
- Organs checked: liver, kidneys, adrenals, brain, and heart

Statistics:

Food consumption, body weight, and organ weight data were intercompared for the dose and control groups by use of Levene's test for homogeneity of variances, by analysis of variance, and by pooled variance t-tests. The t-tests were used, if the analysis of variance was significant, to delineate which groups differed from the control group. If Levene's test indicated heterogeneous variances, the groups were compared by an analysis of variance for unequal variances followed, if necessary, by separate variance t-tests. Medians and quartile deviations were calculated for non-parametric data. These data were statistically analysed by the Kruskal-Wallis test and, if necessary, by the Wilcoxon rank sum test as modified by Mann-Whitney. Frequency data were compared using Fisher's exact tests where appropriate. All statistical tests, except the frequency comparisons were performed using BMDP Statistical Software (Dixon, 1985). The frequency data tests are described in Biometry (Sokal, R. R. and Rohlf, F. J., W. 8. Freeman and Company: San Francisco, 1969). The fiducial limit of 0.05 was used as the critical level of significance for all tests.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Mild to moderate skin irritation in the low dose group and mild to marked irritation in mid and high dose groups. Since some effects were observed in the solvent controls, irritations seen in test gruops may partly have been caused by the solvent.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreased body weight gain was observed in both treated males and females as compared to the controls, but the effects were not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Decreased food consumption was observed in both treated males and females as compared to the controls, but the effects were not statistically significant.

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:

no effects observed

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):

A treatment related decrease in absolute and relative liver weights was observed in mid and high dose males as compared to the controls. However, the effects were not statistically significant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related gross lesions were observed only in the treated skin of both males and females of all treatment and control groups, with higher severity and/or incidence of severity tending to the treatment groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related microscopic lesions were observed only in the treated skin of both males and females of all treatment and control groups, with higher severity and/or incidence of severity tending to the treatmen groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No animals died during the study.
Treatment-related clinical signs of toxicity were restricted to the site of application of test material or vehicle. Erythema and oedema were observed in some animals from most groups including controls. These lesions tended to occur earlier and were more severe in the high and mid dose groups. Erythema was observed for all males in the high dose group with maximum severity scores of well defined (2 males) and moderately severe (3 males). Erythema was also observed for all animals in the mid dose group with maximum severity scores of barely perceptible (4 males) and moderately severe (1 male). Erythema, scored as barely perceptible or well defined, was observed for 2 males in the control group and 1 male in the low dose group. Oedema was observed for all males in the high dose group with maximum severity scores of well defined (1 male) and moderate (4 males). Oedema, scored principally as well defined, was observed for 1 male from each of the control and mid dose groups. Oedema was not observed for males in the low dose group. Findings of erythema and/or oedema occurred principally during the 3rd or 4th week of dosing for the 2 males in the control group, from days 5-13 for the male in the low dose group, and in all weeks of dosing for males in the mid and high dose treatment groups.
Erythema was scored principally as well defined for females in the high dose group and barely perceptible for females in the control, low dose, and mid dose groups. All females in the high dose group had erythema starting in the first week of dosing. The incidence of erythema for the control (1 animal), low dose (1 animal), and mid dose (3 animals) groups observed near the end of the first week of dosing increased to 4 or 5 females per group at the end of the last week of dosing. Oedema (barely perceptible to moderate severity) was observed for all females in the high dose group after the first week of dosing. Oedema, scored principally as barely perceptible, was observed for 2 females from the mid dose group after the first week of dosing and 2 additional females each from this group and the low dose group at the end of the dosing period. Oedema (barely perceptible and/or well defined) was observed for 3 females in the control group during the 3rd or 4th weeks of the study.
Other treatment-related clinical signs of toxicity (in addition to erythema and oedema) were limited to the skin at the application site and included exfoliation, ecchymosis, excoriation, encrustation, ulceration, and fissuring for both males and females. Lesions were observed predominantly in the mid and high dose groups with more animals in the high dose groups affected than in the other groups. Exfoliation and ecchymosis were observed in approximately equal incidence for all groups of females including controls. The incidence of all lesions in males and all other lesions in females appeared to show a dose response. Pustule and abscess were observed for 1 low dose female. No other treatment-related clinical signs of toxicity were observed in this study.

BODY WEIGHT AND WEIGHT GAIN
Statistically significant alterations in absolute body weights and body weight gain were not observed in male or female rabbits administered 8.4, 16.8, or 84.0 mg/kg bw/day of the test material. However, decreased mean body weight gain compared to controls or mean body weight losses were observed for males in the mid dose and high dose groups during the study. Mean body weight loss was observed for males in the mid dose group during the first week of the study and for males in the high dose group during the first and second weeks of the study compared to a mean body weight gain of 78.5 g and 166.2 g for controls for the first and second week of treatment, respectively. Mean body weight gains during the remainder of the study for the mid and high dose groups compared to controls were decreased: 38% to 57% (mid dose males) and 86% to 89% (high dose males).
Mean body weight losses were observed throughout the study for females in the control and high dose groups. The maximum loss for both groups occurred during the first week of the study with mean losses of 89.3 g for controls and 204.4 g for the high dose treatment group. Mean body weight losses during the 4 week exposure period were 49.5 g for controls and 161.0 g for the high dose females.

FOOD CONSUMPTION
Statistically significant alterations in food consumption were not observed in male or female rabbits administered 8.4, 16.8, or 84.0 mg/kg bw/day of the test material. However, mean food consumptions were decreased compared to the control group for males in the high dose treatment group (15% to 28%) during the 4 weeks of exposure and for males in the mid dose treatment group (4% to 18%) during days 1-15. Additionally, mean food consumption was decreased for females from the high dose treatment group compared to the control group (4% to 17%) during the days 1-22.

HAEMATOLOGY
There were no statistically significant differences between the values for any of the haematoligic parameters in treated rabbits compared with those of the control rabbits, nor were there any biologically important trends.

CLINICAL CHEMISTRY
The only statistically significant change in serum chemistry parameters was observed in 8.4 and 84 mg/kg bw/ dosed male rabbits where SDH values were lower than in control rabbits. This coupled with somewhat lower values, albeit not statistically significant, for both AST and ALT in the high dose male rabbits suggests a possible effect on synthesis of liver enzymes. However, no such trend was observed either in the high dose female rabbits, or a dose relationship in the lower dose male rabbits. Therefore, this alteration is not likely to be biologically important. Liver necrosis would result in increased rather than decreased liver enzyme values.

URINALYSIS
No biologically or statistically significant changes were observed in the urine parameters for either male or female rabbits.

ORGAN WEIGHTS
Statistically significant alterations in organ weights were not observed for male or female rabbits administered 8.4, 16.8, or 84.0 mg/kg bw/day of the test item. However, a dose-related decrease in mean absolute liver weight compared to the control group was observed for males in the mid dose (-16%) and high dose (-21%) treatment groups. Dose-related decreases in mean liver weights expressed relative to body weight or brain weight were also observed for males in the mid dose (-9% and -12%, respectively) and high dose (-14% and -15%, respectively) groups. An apparent dose-related decrease in mean liver weight was also observed for males in the low dose group. However, this decrease was a result of an exceptionally small liver in one animal (approximately 50% of control mean) and was not considered to be related to treatment based on the lack of effect on body weight, supporting clinical chemistry findings or gross or microscopic findings in the liver, and the lack of similar effects on liver weight for the four remaining males in this group (mean liver weight for the remaining animals = 2% greater than control mean).

GROSS PATHOLOGY AND HISTOPATHOLOGY
Treatment-related gross and microscopic lesions were observed only in the treated skin of both males and females. Gross lesions were observed on the treated and untreated skin of animals from all groups. Treatment-related gross skin lesions were limited to the treatment area. Gross lesions observed included exfoliation, erythema, surface crust, haemorrhage, and/or fissuring. The severity and/or incidence for several of the gross lesions tended to be greater in the treated groups than in control groups. Microscopic lesions indicative of both chronic and acute inflammation were observed in all treatment groups (including controls). Acanthosis, ulceration, folliculitis, and dermatitis for males, and oedema, congestion, haemorrhage, and hyperkeratosis for both males and females occurred in approximately equal frequency for control and treated animals. A dose-related increase in the incidence of ulceration was observed for females in the mid dose (3 animals) and high dose (4 animals) groups and the incidence of folliculitis for females was greatest in the mid and high dose groups (3 animals each). The severity of epidermitis and ulceration for males and acanthosis, epidermitis, ulceration, dermatitis, and dermal fibrosis for females increased in a dose-related manner. The range of severity for these lesions was mild to moderate for the control and low dose animals and mild to marked for the mid and high dose animals. Microscopic lesions were also observed in untreated skin of rabbits from all dosage groups. These lesions were less frequent and generally less severe than those observed at the application site.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

>= 84 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The effect level is based on systemic toxicity. No systemic toxicity was observed in any of the test doses. All effects seen in the treatment groups are based on local skin irritation and consequently indisposition of the animals.

Dose descriptor:

NOAEL

Remarks:

local

Remarks on result:

not determinable because of methodological limitations

Remarks:

It cannot clearly be determined to what extend the skin irritating effects are the result of the test substance treatment because skin irritation was also observed in the controls

Critical effects observed:

no

Mild to moderate skin irritation was seen at the lowest dosage level, 8.4 mg/kg bw/day. Mild to marked irritation was noted at dosage levels of 16.8 mg/kg/day and above. Since some effects were observed with the silane-free solvent control animals, the irritation seen in the test groups may in part have been caused by the solvent.  

Tab. 1: Summary of Skin irritation data

	control								8.4 mg/kg bw (0.5%)
Day	Erythema				Oedema				Erythema				Oedema
	males		females		males		females		males		females		males		females
	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range
1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
2	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
3	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
4	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
5	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
6	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
7	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0
8	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0
9	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
10	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
11	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
12	0.2±0.4	0-1	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
13	0.2±0.4	0-1	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
14	0.2±0.4	0-1	0.4±0.9	0-2	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0	0.0±0.0	0-0
15	0.2±0.4	0-1	0.4±0.9	0-2	0.0±0.0	0-0	0.4±0.9	0-2	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0
16	0.2±0.4	0-1	0.4±0.9	0-2	0.0±0.0	0-0	0.4±0.9	0-2	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0
17	0.2±0.4	0-1	0.4±0.9	0-2	0.0±0.0	0-0	0.4±0.9	0-2	0.0±0.0	0-0	0.4±0.5	0-1	0.0±0.0	0-0	0.0±0.0	0-0
18	0.2±0.5	0-1	0.6±0.9	0-2	0.0±0.0	0-0	0.4±0.9	0-2	0.0±0.0	0-0	0.4±0.5	0-1	0.0±0.0	0-0	0.0±0.0	0-0
19	0.4±0.5	0-1	0.6±0.9	0-2	0.0±0.0	0-0	0.4±0.9	0-2	0.0±0.0	0-0	0.4±0.5	0-1	0.0±0.0	0-0	0.0±0.0	0-0
20	0.2±0.4	0-1	0.6±0.9	0-2	0.0±0.0	0-0	0.4±0.9	0-2	0.0±0.0	0-0	0.4±0.5	0-1	0.0±0.0	0-0	0.0±0.0	0-0
21	0.2±0.4	0-1	0.4±0.5	0-1	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.4±0.5	0-1	0.0±0.0	0-0	0.0±0.0	0-0
22	0.2±0.4	0-1	0.4±0.5	0-1	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.4±0.5	0-1	0.0±0.0	0-0	0.0±0.0	0-0
23	0.2±0.4	0-1	0.4±0.5	0-1	0.0±0.0	0-0	0.2±0.4	0-1	0.0±0.0	0-0	0.4±0.5	0-1	0.0±0.0	0-0	0.0±0.0	0-0
24	0.2±0.4	0-1	0.8±0.8	0-2	0.0±0.0	0-0	0.4±0.9	0-2	0.0±0.0	0-0	0.8±0.4	0-1	0.0±0.0	0-0	0.2±0.4	0-1
25	0.2±0.4	0-1	0.8±0.8	0-2	0.2±0.4	0-1	0.4±0.9	0-2	0.0±0.0	0-0	0.8±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0
26	0.4±0.9	0-2	0.6±0.5	0-1	0.4±0.9	0-2	0.4±0.9	0-2	0.0±0.0	0-0	0.8±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0
27	0.4±0.9	0-2	0.6±0.5	0-1	0.4±0.9	0-2	0.4±0.9	0-2	0.0±0.0	0-0	0.8±0.4	0-1	0.0±0.0	0-0	0.0±0.0	0-0
28	0.4±0.9	0-2	0.6±0.5	0-1	0.4±0.9	0-2	0.6±0.9	0-2	0.0±0.0	0-0	0.8±0.4	0-1	0.0±0.0	0-0	0.6±0.5	0-1
29	0.4±0.9	0-2	0.6±0.5	0-1	0.4±0.9	0-2	0.6±0.9	0-2	0.0±0.0	0-0	0.8±0.4	0-1	0.0±0.0	0-0	1.2±1.1	0-3

Conclusions:

A dermal repeated dose toxicity conducted according to the OECD TG 410 and in compliance with GLP with bis(trimethoxysilylpropyl)amine (CAS 82985-35-1) is available, although the results were disregarded as there were positive skin reactions observed with the vehicle control, light paraffin oil. The mild to moderate microscopic skin lesions found for animals treated with the vehicle alone suggests that the vehicle and/or test procedure contributed to the skin irritation observed in the Silane-treated animals. Bis(trimethoxysilylpropyl)amine (CAS 82985-35-1) was tested on both male and female New Zealand White rabbits to determine specific target organ toxicity after repeated skin contact. Solutions of differing concentrations of the test item (0.5, 1, and 5%, referring to 8.4, 16.8, and 84.0 mg/kg bw) in a light paraffin oil were applied to the clipped dorsal trunk skin under an impervious covering for 6 hours/day, 5 days/week for 4 weeks. No mortality occurred throughout the study period. Decreased body weight gain and decreased food consumption were observed in both treated males and females as compared to the controls, but the effects were not statistically significant. A treatment related decrease in absolute and relative liver weights was observed in mid and high dose males as compared to the controls. However, the effects were not statistically significant. Mild to moderate skin irritation was seen at the lowest dosage level, 8.4 mg/kg/day, whereas mild to marked irritation was noted at dosage levels of 16.8 mg/kg/day and above. Treatment-related gross and microscopic lesions were observed only in the treated skin of both males and females of all treatment and control groups, with higher severity and/or incidence of severity tending to the treatment groups. The predominant finding was hyperkeratosis. However, degenerative effects to the skin were not observed. Since some findings were also observed with the solvent control animals, the irritation seen in the test groups may in part have been caused by the solvent. Among this, light paraffin oil is known to be irritating to the skin (Nessel et al. (1999) Toxicological Sciences 49:48-55). The study provided evidence for cumulative irritation from repeated skin contact with the test item, but there was no evidence of systemic toxicity. The other effects observed in this study (decreases in food consumption, body weight gain, and liver weight for males in the mid and high dose groups) were not indicative of a consistent systemic effect of the test material and may have been partially related to debility resulting from skin irritation. Therefore, no clear result on both the local and the systemic repeated dose toxicity hazard could be obtained from this study..

Additional information

There are only reliable data available on toxicity after repeated oral exposure (subacute) to the registered substance, but no adequate information about specific organ toxicity after subchronic repeated oral exposure is available for bis(trimethoxysilylpropyl)amine (CAS 82985-35-1) or any structurally related silane containing secondary amine groups. Therefore, a testing proposal is included into this dossier. However, to conduct a quantitative risk assessment, reliable data from a subacute repeated dose toxicity study performed via the oral route with the registered substance is available.

 

Repeated dose toxicity: oral (subacute)

A reliable key repeated dose toxicity study (28-day) with bis(trimethoxysilylpropyl)amine (CAS 82985-35-1) is available and was performed according to OECD TG 407 (Chemicals Evaluation and Research Institute, 2014) and in compliance with GLP. Groups of 5 Crl:CD rats of each sex were administered the test material at doses of 100, 300 and 1000 mg/kg bw/day or vehicle alone (olive oil) via oral gavage for 28 days on 7 consecutive days per week. Five additional animals per sex per dose were included as recovery group for analysis of possible post-exposure reversibility of intoxication symptoms. Food efficiency, water consumption and ophthalmoscopy were not performed. Clinical observations, body weight changes, food consumption, haematology, clinical chemistry, urinalysis, neurobehaviour and organ weight measurements demonstrated no treatment-related adverse effects. Gross and histopathological examinations revealed edema (submucosal layer), slight hyperplasia of the surface epithelial cells (fundic mucosa) and slight necrosis (pyloric mucosa) in 1/5 males of the 1000 mg/kg bw/day group. In the recovery group, slight hyperplasia of the surface of epithelial cells in the pyloric mucosa and slight focal necrosis of the fundic mucosa related to the macroscopic findings in the glandular stomach were also observed in 1/5 male animals of the 1000 mg/kg bw/day dose group. Slight hyperplasia of the surface epithelial cells in the pyloric mucosa in the glandular stomach was observed in 1/5 females of the 1000 mg/kg bw/day dose group. Additionally, these effects were also noted in 1/5 females of the 1000 mg/kg bw/day dose group of the recovery group. Taken together, as no adverse effects were observed with respect to general/detailed clinical observations, haematological/clinical chemistry/neurobehaviour examination, body weights, organ measurements or urinalysis, the NOAEL (systemic) was considered to be 1000 mg/kg bw/day. Nevertheless, local adverse effects on gross pathology and histopathological examination following treatment with the test material at 1000 mg/kg bw/day were recorded in 1 male and female animal each (including 1 animal of each sex of the recovery group). Therefore, a NOAEL (local) of 300 mg/kg bw/day was derived.

 

Repeated dose toxicity: inhalation

No data available.

 

Repeated dose toxicity: dermal (subacute)

A dermal repeated dose toxicity study conducted according to the OECD TG 410 and in compliance with GLP with bis(trimethoxysilylpropyl)amine(CAS 82985-35-1) is available, although the study was disregarded for risk assessment as there were positive skin reactions observed with the vehicle control, light paraffin oil. The mild to moderate microscopic skin lesions found for animals treated with the vehicle alone suggests that the vehicle and/or test procedure contributed to the skin irritation observed in the Silane-treated animals. Bis(trimethoxysilylpropyl)amine (CAS 82985-35-1) was tested on both male and female New Zealand White rabbits to determine specific target organ toxicity after repeated skin contact. Solutions of differing concentrations of the test item (0.5, 1, and 5%, referring to 8.4, 16.8, and 84.0 mg/kg bw) in a light paraffin oil were applied to the clipped dorsal trunk skin under an impervious covering for 6 hours/day, 5 days/week for 4 weeks. No mortality occurred throughout the study period. Decreased body weight gain and decreased food consumption were observed in both treated males and females as compared to the controls, but the effects were not statistically significant. A treatment related decrease in absolute and relative liver weights was observed in mid and high dose males as compared to the controls. However, the effects were not statistically significant. Mild to moderate skin irritation was seen at the lowest dosage level, 8.4 mg/kg/day, whereas mild to marked irritation was noted at dosage levels of 16.8 mg/kg/day and above. Treatment-related gross and microscopic lesions were observed only in the treated skin of both males and females of all treatment and control groups, with higher severity and/or incidence of severity tending to the treatment groups. The predominant finding was hyperkeratosis. However, degenerative effects to the skin were not observed. Since some findings were also observed with the solvent control animals, the irritation seen in the test groups may in part have been caused by the solvent. Among this, light paraffin oil is known to be irritating to the skin (Nessel et al. (1999) Toxicological Sciences 49:48-55). The study provided evidence for cumulative irritation from repeated skin contact with the test item, but there was no evidence of systemic toxicity. The effects observed in this study (decreases in food consumption, body weight gain, and liver weight for males in the mid and high dose groups) were not indicative of a consistent systemic effect of the test material and may have been partially related to debility resulting from skin irritation. Therefore, no clear result on both the local and the systemic repeated dose toxicity hazard could be obtained from this study.

Justification for classification or non-classification

The available data are reliable and suitable for classification. Based on this data, classification for repeated dose toxicity according to 67/584/EEC and EC/1272/2008 is not warranted.