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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
28 March 2019 to 30 October 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
This study serves as a dose-range finding study (DRF study) for a subsequent repeated dose toxicity study. This study followed the procedures indicated by internal BSL Munich SOPs. Procedures and facilities comply with the requirements of Directive 2010/63/EU [and the national legislation defined in the animal protection law concerning the protection of animals used for experimental and other scientific procedures].
GLP compliance:
no
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Dichloro(diphenyl)silane
EC Number:
201-251-0
EC Name:
Dichloro(diphenyl)silane
Cas Number:
80-10-4
Molecular formula:
C12H10Cl2Si
IUPAC Name:
dichloro(diphenyl)silane
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females, nulliparous and non-pregnant: yes
- Age at study initiation: 12-13 weeks old
- Weight at study initiation: males: 332 – 363 g; females: 206 – 229 g
- Fasting period before study: no; overnight fasting for hematology and clinical chemistry parameters
- Housing: housed 2 animals / sex / group / cage in IVC cages (type III, polysulphone cages) on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet was available ad libitum
- Water: tap water available ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: For tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals). Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item formulation and vehicle were administered as a single daily dose to the animals by oral gavage. The application volume for all groups was 4 mL/kg body weight.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared plastic vial and the vehicle was added to give the appropriate final concentration of the test item. The formulation was vortexed and/or stirred until visual homogeneity was achieved. Formulates were constantly stirred until daily administration. The test item formulations were prepared freshly on each administration day before the administration procedure and administered directly after its preparation. The vehicle was used as control item.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected in consultation with the sponsor based on the test item’s characteristics.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
14 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
375 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
2 animals/sex/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses for the first dose group and the control group were selected in consultation with the sponsor prior to the start of the study. The remaining doses were selected sequentially in consultation with the sponsor during the course of the treatment period of this study. The treatment started with 25 and 50 mg/kg/day. In case of no effects at a particular dose level, the dose levels for the remaining groups increased stepwise one dose level at a time until effects are seen. Before starting dosing with a new higher dose level at least 3 treatments per animal and dose group were made.
- Rationale for animal assignment: Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups while achieving a mostly homogenous variation in body weight throughout the groups of males and females, respectively (randomisation performed with IDBS Workbook 10.1.2 software).
- Fasting period before blood sampling for clinical biochemistry: overnight
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day, approximately at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the start of the treatment period a detailed clinical observation outside the home cage was made.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before assignment to the experimental groups and on study days 1, 3, 6, 8, 11 and 14 during the treatment periods as well as on the day of necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; food consumption was measured on study days 1, 8 and 14 for each dose group.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After overnight fasting, blood from the abdominal aorta of the animals was collected as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Yes; ketamine, xylazin
- Animals fasted: Yes
- How many animals:
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After overnight fasting, blood from the abdominal aorta of the animals was collected as part of the sacrifice of the animals
- Animals fasted: Yes
- How many animals:
- Parameters checked in table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. On study day 15, all surviving animals of the study were sacrificed using anesthesia (ketamine, xylazin) followed by exsanguination and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. All animals found dead and/or intercurrently euthanised for animal welfare reasons were subjected to a gross necropsy and the organs preserved for a histopathological examination. All macroscopic findings were recorded and organs showing gross abnormalities and all organs listed in Table 3 were preserved in 4 % neutral-buffered formaldehyde except testes and epididymides, which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol. Organs that were weighed are presented in Table 4.

HISTOPATHOLOGY: Yes; organs presented in Table 3 were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining for all animals sacrificed at the end of the treatment period and any animal found dead or euthanised before the planned day of sacrifice.
Other examinations:
None
Statistics:
Toxicology and pathology data were captured either on paper according to appropriate SOPs or using the validated computerised system Ascentos® System (version 1.3.4, Pathology Data Systems Ltd.).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Two high-dose males that were euthanized for animal welfare reasons showed the clinical signs of moving the bedding, increased salivation, apathy, hypotonia (muscle), reduced spontaneous activity (slight/severe), slow movements, piloerection (moderate) and half eyelid close (both) on treatment day 6. One high-dose female, which was euthanized for animal welfare reasons on day 3, showed the clinical signs of moderate piloerection, apathy, moderate reduced spontaneous activity, half eyelid close (both) and hypothermia on treatment day 3. Another high-dose female, which was also euthanized for animal welfare reasons on day 6, showed the clinical signs of moving the bedding, lacrimation (left), hypothermia, reduced spontaneous activity (slight) and piloerection (slight to moderate) on treatment day 6. One male in the 375-mg/kg bw/day dose group showed moving the bedding on days 2-14 and another showed similar findings and also moderate salivation on day 14. One female in the 375-mg/kg bw/day showed moving the bedding on days 4-14, slight edema (skin/fur) on days 7-10 and scratch/cut at snout on days 11-14. Another female was showed moving the bedding on day 4 and was found dead on day 5. Moving the bedding was also observed at 50 and 100 mg/kg bw/day on different days of treatment period. There were no clinical signs at 25 mg/kg bw/day (group 2) during the entire treatment period in both males and females. Moving the bedding was transiently observed in 1/2 males of group 3 (50 mg/kg bw/day) and in both males of group 4 (100 mg/kg bw/day) and in all female animals of groups 3 and 4 (50 /100 mg/kg bw/day). This is assumed to be due to discomfort caused by a local reaction to the test item.
Mortality:
mortality observed, treatment-related
Description (incidence):
During the treatment period, 2/2 males and 2/2 females of group 5 (500 mg/kg bw/day) were euthanized on day 6 in a moribund condition due to animal welfare reasons. One rat at 375 mg/kg bw/day was found dead on treatment day 5. Inflammatory and degenerative findings in the stomach were considered to be the cause of mortality. All remaining animals survived until the scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
Males of the control, group 2 (25 mg/kg bw/day), group 3 (50 mg/kg bw/day) and group 4 (100 mg/kg bw/day) tended to gain body weight throughout the study period and comparable to control. Males of group 5 (500 mg/kg bw/day) and group 6 (375 mg/kg bw/day) showed a tendency towards a loss of body weight. Individual mean body weight change from treatment day 1 to 14 was 37 g (animal no. 1) and 48 g (animal no. 2) in group 1, 26 g (animal no. 4) and 16 g (animal no. 4) in group 2, 49 g (animal no. 5) and 42 g (animal no. 6) in group 3 and 27 g (animal no. 7) and 21 g (animals no. 8) in group 4, respectively. Two male were euthanized on treatment day 6 (animals no. 9 and 10) in group 5 and body weight change was found to be reduced on days 3-6. One male in group 6 (animal no. 11) tended to show reduced body weight gain on days 3 and 6 and later slightly gained weight. Animal no. 12 showed increased body weight until day 8 and later reduced weight on days 11 and 14.

Females:
Body weight change of test item-treated females showed only a slight trend towards body weight gain and mean body weight gain of test item-treated groups was comparable to control females up to 100 mg/kg bw/day. Females of group 5 (500 mg/kg bw/day) showed a tendency towards a loss of body weight prior to their euthanization on treatment days 3 (no. 21) and 6 (no. 22); body weight change was found to be reduced on days 1-3 and 3-6, respectively. One female in group 6 (animal no. 24) was found dead on day 5, and it had shown a trend towards decreased body weight loss on day 3. Overall, the treated group mean body weights were comparable to the respective controls and there were no adverse effects on body weight development in both genders up to 100 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
Mean WBC levels were moderately higher in group 3 (50 mg/kg bw/day, deviation from control: 53%), in group 4 (100 mg/kg bw/day, deviation from control: 40%) and in group 6 (375 mg/kg bw/day, deviation from control: 48%). Slightly higher mean PLT in group 6 (375 mg/kg bw/day, deviation from control: 24%) was observed when compared to the corresponding control group.

Females:
Mean WBC levels were slightly higher in group 4 (100 mg/kg bw/day, deviation from control: 26 %) and slightly lower in group 3 (100 mg/kg bw/day, deviation from control: 18%). The values in group 6 (375 mg/kg bw/day) have to be considered carefully as only 1/2 females survived until the scheduled sacrifice. However, observed differences followed no dose-dependency. Treatment with the test item had no dose-dependent effect on haematology parameters of males and females in any of the test item-treated groups. Differences between test item-treated males or females and their respective controls showed no dose dependency or consistency and thus were not considered toxicologically relevant.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males:
Mean ALAT level was slightly lower in group 6 (375 mg/kg bw/day, deviation from control: 28%). Mean creatinine level was moderately lower in groups 2 and 3 (25 and 50 mg/kg bw/day, deviation from control: 38 % and 48 %, respectively) and higher in groups 4 and 6 (100 and 375 mg/kg bw/day, deviation from control: 45 % in each). Mean ASAT level was moderately lower in group 4 (100 mg/kg bw/day, deviation from control: 51%). Mean AP level was moderately lower in group 6 (375 mg/kg bw/day, deviation from control: 42%) and slightly higher in group 3 (50 mg/kg bw/day, deviation from control: 24%). Mean K level was moderately lower in groups 2-4 and 6 (25-100 and 375 mg/kg bw/day, deviation from control: 25-35%). Mean total protein was slightly lower in groups 4 and 6 (100 and 375 mg/kg bw/day, deviation from control: 18% and 26%, respectively). Mean cholesterol level was moderately higher in group 6 (375 mg/kg bw/day, deviation from control: 45%). Mean urea level was slightly lower in group 4 (100 mg/kg bw/day, deviation from control: 20%).

Females:
Mean ALAT level was moderately higher in groups 3 and 4 (50 and 100 mg/kg bw/day, deviation from control: 48% and 39%, respectively). Mean creatinine level was moderately higher in groups 2 and 4 (25 and 100 mg/kg bw/day, deviation from control: 38% and 81% respectively). Mean ASAT level was slightly lower in group 4 (100 mg/kg bw/day, deviation from control: 26%). Mean AP level was moderately lower in groups 2 and 3 (25 and 50 mg/kg bw/day, deviation from control: 44% and 45%, respectively). Mean K level was moderately higher in group 2 (25 mg/kg bw/day, deviation from control: 43%). Mean total protein was slightly lower in group 4 (100 mg/kg bw/day, deviation from control: 25%). Mean cholesterol level was moderately higher in groups 2 and 4 (25 and 100 mg/kg bw/day, deviation from control: 60% and 89%,respectively). Mean urea level was slightly higher in group 2 (25 mg/kg bw/day, deviation from control: 23 %). The values of female group 6 (375 mg/kg bw/day) have to be considered carefully as only one animal survived until the scheduled necropsy. Slight differences between test item-treated males or females and corresponding controls followed no dose-dependency or consistency between the genders and thus were not considered toxicologically relevant. Treatment with the test item had no effects on parameters of clinical biochemistry of males and females in any of the test item treated groups which were considered toxicologically relevant.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related gross lesions were noted in the stomach, small and large intestine at ≥375 mg/kg bw/day in both genders. Gross lesions in the stomach consisted of abnormal content (mucoid, white) and green hard abnormal surface (crateriform, white fundic area). In the duodenum/ileum abnormal green content was observed. Dark spotted thymus was found in male animal no. 7 at 375 mg/kg bw/day, a dark thymus was found in a female at 375 mg/kg bw/day and small thymus at 500 mg/kg bw/day. At 100 mg/kg bw/day, there was a single incidence of dark spotted thymus in one male. Single incidence of abnormal color (dark red) mesenterium at 375 mg/kg bw/day was observed in one male. The adrenal gland was enlarged at both sides in one female at 375 mg/kg bw/day and was small in one female at 500 mg/kg bw/day. Brain, lungs, liver were autolytic in one female at 375 mg/kg bw/day which was found dead on treatment day 5.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Inflammatory and degenerative lesions were noted in animals at 375 and 500 mg/kg bw/day. The findings consisted of forestomach inflammation in all animals at both doses. In most animals, there were either erosion and/or ulceration in the forestomach and/or glandular stomach. In addition, in one female at 375 mg/kg bw/day, there was glandular stomach inflammation, and, in one animal per sex at this dose, there was serosal inflammation (due to perforating ulceration). Other findings consisted of a single case of forestomach squamous epithelial degeneration in one female at 500 mg/kg/ bw/day, as well as of hyperkeratosis in one female at 500 mg/kg bw/day, and in three animals at 375 mg/kg bw/day. The inflammatory lesions were furthermore associated with squamous hyperplasia in both males at 500 mg/kg bw/day, and in three animals at 375 mg/kg bw/day. There were findings secondary to the stomach lesions, i.e., there were stress-related thymic and splenic atrophy in one female at 500 mg/kg bw/day, and diffuse cortical hypertrophy in the adrenal gland in the decedent female at 375 mg/kg bw/day. No histopathological gastro-intestinal lesions were noted up to 100 mg/kg bw/day.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
375 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
intestine
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion