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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 March 2020 to 14 July 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimethoxyoctylsilane
EC Number:
221-338-7
EC Name:
Trimethoxyoctylsilane
Cas Number:
3069-40-7
Molecular formula:
C11H26O3Si
IUPAC Name:
trimethoxy(octyl)silane
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han) (Full Barrier)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approx. 7-8 weeks old
- Weight at study initiation: males: 165 – 195 g (mean: 181.2 g, ± 20 % = 145.0 – 217.5 g); females: 140 – 162 g (mean: 152.1 g, ± 20 % = 121.7 – 182.5 g)
- Fasting period before study:
- Housing: The animals were kept in groups of 5 animals / sex / group / cage in IVC cages (type IV, polysulphone cages) on Altromin saw fibre bedding
- Diet (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice, ad libitum
- Water (e.g. ad libitum): Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins BioPharma Product Testing Munich GmbH

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was dissolved in dried and de-acidified corn oil. The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item (w/w). The formulation was alternately vortexed and/or stirred until visual homogeneity was achieved. After homogenization the formulation was overlaid with a protective gas to prevent instability caused by repeated contact of the test item formulation with air. Formulates were kept under magnetic stirring during the daily administration. Based on the results of stability testing (Eurofins Munich Study No. 193078), the test item formulations were prepared freshly at least every 10 days as given by Eurofins Munich Study No. 193078. The prepared formulation was stored protected from light and at room temperature.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle dried and de-acidified corn oil was selected in consultation with the sponsor based on the test item’s characteristics.
- Concentration in vehicle: 0, 12.5, 25, 44 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw
- Lot/batch no. (if required): MKCK6411
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 193078). Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of HD and LD groups. As the test item was shown to be homogenous according to Eurofins Study No. 193078 (after 30 min without stirring), samples were not collected during the study for the investigation of homogeneity and only samples were taken for substance concentration in study week 1, 5, 9 and in the last week of treatment (16 samples in total). Duplicate samples were retained during the study for concentration analysis (Eurofins Munich Study Phase No. 93079). Dose formulation analytics showed that nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.
Duration of treatment / exposure:
90 days
Frequency of treatment:
7 days a week, daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control group
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Low dose group (LD)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Middle dose group (MD)
Dose / conc.:
175 mg/kg bw/day (actual dose received)
Remarks:
High dose group (HD)
No. of animals per sex per dose:
-10 male and 10 female animals per group, 80 animals in total
- 5 male and 5 female animals per recovery group, 20 in total
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses for the main 90-day oral repeated dose toxicity study were based on the results of a previous Combined Repeated Dose Oral Toxicity Study with the Reproduction / Developmental Toxicity Screening Test in Wistar Rats (OECD Test Guideline 422) study (BSL Munich Study No. 178263) and in consultation with the sponsor. The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
In the Combined Repeated Dose Oral Toxicity Study with the Reproduction / Developmental Toxicity Screening Test in Wistar Rats (OECD Test Guideline 422), at the high dose group of 600 mg/kg bw/day 1 male and 6 females had to be sacrificed in a moribund condition. After a dose reduction to 400 mg/kg bw/day additional 4 males and 10 females were sacrificed in a moribund condition. At the middle dose group of 300 mg/kg bw/day 4 females had to be euthanised prematurely and even after a dose reduction to 200 mg/kg bw/day additional 4 females were euthanised. Due to the longer treatment period of 90 days in this study a dose slightly below 200 mg/kg bw/day was chosen as high dose.

- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: yes, overnight fasting
- Rationale for selecting satellite groups: 10 animals per gender and group were subjected to necropsy one day after the last administration (end of treatment period). 5 animals per gender of the C and of the HD group were subjected to necropsy 28 days after the last administration (end of recovery period); these animals were used for assessment of possible delayed toxic effects .
- Post-exposure recovery period in satellite groups: 28 days
- Section schedule rationale (if not random): random
Positive control:
Not used

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed for clinical signs during the entire treatment period of 90 days. The recovery animals were observed for an additional period of 28 days following the last administration. General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
- Cage side observations checked included: the health condition of the animals was recorded, morbidity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
- Detailed clinical observations included: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment and recovery period.

FOOD CONSUMPTION:
- Time schedule for examinations: Food consumption was measured weekly during the treatment and recovery period. Inadvertently, in the second treatment week food was refilled without weight recording for the cage of LD males 16 to 20 in between the scheduled time points for food weight determination.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the first administration and in the last week of the treatment period as well as at the end of the recovery period in the recovery animals.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery period as part of the sacrifice of the animals
- Anaesthetic used for blood collection: No
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters checked in table [No.1 & 2] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment and recovery period as part of the sacrifice of the animals
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters checked in table [No.3] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: prior to or as part of the sacrifice of the animals. Additionally, urine colour / appearance were recorded.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes (overnight)
- Parameters checked in table [No.4] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once before the first exposure and towards the end of the exposure period but not earlier than in week 11 as well as in the last week of the recovery period.
- Dose groups that were examined: all animals
- Battery of functions tested: functional observational battery of tests

OTHER: Thyroid hormone analysis
- Time schedule for examinations: at the end of treatment and recovery period
- How many animals: all animals
- Parameters checked in table [No.5] were examined.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see tables Nos. 6 and 7)

HISTOPATHOLOGY: Yes (see tables Nos. 6 and 7)
Statistics:
A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Furthermore, statistical comparisons of data acquired during the recovery period may be performed with a Student’s t-Test or Mann-Whitney U-Test when appropriate. These statistics were performed with Ascentos 1.3.4 software or GraphPad Prism V.6.01 software (p<0.05 is considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs such as scratches and hairless areas were considered to be incidental and not test item related or toxicologically relevant.
Slightly increased salivation for 6 days in 1 out of 15 control female was considered as local reaction provoked by the oral application route. Slight to moderately increased salivation was observed in 3 out of 10 MD males, while in 2 out of 15 HD males slight to severely increased salivation was recorded. Abnormal breathing occurred in 1 out of 10 MD females and in 3 out of 15 HD males. No clinical signs were observed during the recovery period of the study. Findings during the treatment phase might be a reaction to the irritating nature of the test item, but were not considered as toxicologically relevant as only few animals exhibited these clinical signs and they ceased during the recovery period.
No clinical signs were observed during the recovery period of the study.
No test item-related findings were recorded during detailed clinical observations or ophthalmologic examinations during the treatment or recovery period of the study.
Mortality:
no mortality observed
Description (incidence):
No test item-related mortality occurred during the treatment or recovery phase of the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related effects on body weight or body weight change were observed during the treatment or recovery phase of the study.
A statistically significantly increased body weight gain in main LD females (27.00 g) compared to controls (11.40 g) in the first treatment week (study day 1 to 8) was considered as incidental as in the following weeks no such effect was observable.
Slight, but statistically significant increases in body weights on study day 112 of HD recovery females (263 g) compared to controls (246 g) were considered as incidental and not toxicologically relevant as differences were marginal.
HD males exhibited a statistically significantly reduced body weight gain between the recovery days 105 and 112 (0.80 g) compared to controls (7.40 g). This reduction can be explained by a noticeably high body weight gain in control recovery male no. 41 (16 g) and was thus considered as incidental.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related effects on food consumption were recorded during the treatment or recovery period of the study.
A quite low food consumption in LD males (7.99 g) compared to the other groups (C: 21.21 g, MD: 21.39 g and HD: 20.74 g) between study day 8 and 15 can be explained by a negative food consumption of -5 g/day/animal in cage 8 (LD males no. 16 - 20). This was caused by an inadvertent refilling of food without weight recording in between the scheduled time points for food weight determination. Hence, this finding has no toxicological relevance.
A relatively high food consumption in HD females (29.00 g) compared to the other groups (C: 13.30 g, LD: 14.26 g and MD: 14.22 g) between study day 85 and 90 can be explained by a noticeably high food consumption of 46 g/day/animal in cage 18 (HD males no. 81 - 85). This finding was considered as incidental.
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant effects were observed.
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related effects on haematology or coagulation were determined at the end of the treatment or recovery period of the study.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant effects on parameters of clinical biochemistry were determined at the end of the treatment or recovery period of the study.
In general, statistical significant reductions regarding parameters of clinical biochemistry were not considered as toxicologically relevant and were thus not discussed in detail.
Marginal non-significant, but dose dependent increases in total bile acid (TBA) were observed at the end of the treatment period in test item treated males (LD: 16.749 µmol/L, MD: 17.174 µmol/L, HD: 20.813 µmol/L) compared to controls (14.482 µmol/L). At the end of the recovery period TBA levels of HD males were still clearly increased (27.866 µmol/L, deviation vs. control: 142.735 %) compared to controls (11.480 µmol/L). No such effects were observed in females. However, these findings were not considered as toxicologically relevant as no histopathological findings were recorded and differences at the end of the treatment period were very small.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No noticeable, test item-related changes were determined during urinalyses at the end of the treatment or recovery period of the study.
Some main HD males exhibited slightly increased erythrocyte counts at the end of the treatment period (no. 31: approx. 250 cells/µL, no. 32, 33 and 38: approx. 50 cells/µL) compared to the other dose groups (approx. 10 cells/µL), but this finding is considered as incidental as no other peculiarities were noticed in the urine of the animals.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant effects were observed during functional observation battery performed before first application and in the last treatment and recovery week of the study. Statistical significant differences before treatment cannot be test item-related.
In the last treatment week (week 13), statistically significantly less/more HD males were sleeping/ moving around in the cage compared to controls (sleep score: 0.60 vs. 0.93 in controls, moving score: 0.40 vs. 0.07 in controls). The bad experience (e.g. taste) of being dosed with the HD formulation might have induced an increased awareness in HD animals, but was not considered as toxicologically relevant.
LD females exhibited a statistically significantly increased finger approach response (score: 5.60) than controls (score: 4.13), but as no dose dependency was observed, this effect was considered as incidental and not as toxicologically relevant.
Inadvertently, for recovery animals these observations were not performed in the last recovery week.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes regarding organ weights were recorded at the end of the treatment or recovery period of the study.
Absolute pituitary gland weights as well as pituitary gland to brain weight ratios were statistically significantly reduced in LD and HD males compared to controls:
Mean pituitary weight: C: 0.0104 g, LD: 0.0088 g, HD: 0.0090 g
Mean pituitary/brain ratio: C: 0.5100 %, LD: 0.4291 %, HD: 0.4457%
As differences were marginal, no dose-dependency was observable and as no findings were recorded during histopathological examinations, these effects were not considered as toxicologically relevant.
Moreover, absolute liver weights as well as liver to brain weight ratios were statistically significantly increased in HD recovery females (liver: 7.082 g, liver/brain: 368.201 %) compared to recovery controls (liver: 6.018 g, liver/brain: 312.051 %).
As no effects were seen in main HD females at the end of the treatment period and as main LD liver weights were higher than weights of HD recovery females, these effects were not considered as toxicologically relevant.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings recorded were gross lesions within the range of normal background changes which may be observed in rats of this strain and age, accidental lesions related to the gavage procedure, incidental appearances without corresponding histomorphological changes, or macroscopic alterations representing normal physiology.
A cyst was found on the left kidney of control female no. 54, which was histopathologically identified as lymphangiectasis and the left part of the uterus was dilated in control female no. 56, which is related to the oestrous cycle. The thymus of LD male no. 19 had an abnormal red spotted colour due to haemorrhage. The right kidney of MD male no. 28 exhibited pelvic dilatation. The left mandibular lymph nodes of HD male no. 31 were enlarged. In HD male no. 36 the lung exhibited caudal adhesion and a beige colour at this location, which was histopathologically further specified as pleural fibrosis. Similarly also the lungs of MD female no. 79 exhibited caudal adhesion and a beige colour at this location. Moreover, the thoracic cavity was filled with a clotted beige content. These findings were histopathologically identified as granulomatous inflammation. Moreover, a single mass microscopically further specified as granuloma was detected on the lateral side of the oesophagus of HD female no. 79. The thoracic cavity of HD female no. 82 was filled with fluid.
During necropsy of the recovery animals, a bladder stone was found in the urinary bladder of control male no. 45.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No histomorphological changes that could be attributed to treatment with the test item were observed in the high dose (175 mg/kg bw/day) animals that were sacrificed at the end of the treatment period. All microscopic findings were within the range of normal background lesions which may be observed in rats of this strain and age, accidental lesions related to the gavage procedure, or microscopic alterations representing normal physiology, and therefore, were deemed not to be treatment-related.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No toxicologically relevant effects on hormones were determined at the end of the treatment or recovery period of the study.
A statistical significant reduction in T3 levels was determined in LD males at the end of the treatment period (4.52 ng/mL) compared to controls (5.35 ng/mL). As no dose-dependency or other toxicologically relevant findings could be observed, this finding was not considered as toxicologically relevant.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
175 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no treatment-related adverse effects observed during the treatment or recovery period.

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

See attachments for data tables.

Applicant's summary and conclusion

Conclusions:
In the 90-day oral repeated dose toxicity study for trimethoxy(octyl)silane, conducted according to OECD Test Guideline 408 and in compliance with GLP, the systemic NOAEL was concluded to be 175 mg/kg bw/day based on no observed test-substance related adverse systemic effects up to the highest dose tested.