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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:
08-10-2019 to XXX
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
25 June 2018
Deviations:
no
Remarks:
Minor deviations from the study plan occurred but were not considered to have compromised the validity and integrity of the study
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Animal model : Albino Rats (Outbred) VAF/Plus
CD (Sprague-Dawley derived)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Raleigh, North Carolina 27610
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 9 weeks old
- Weight at study initiation: 253 g to 362 g (males); 205 g to 293 g (females)
- Fasting period before study: No
- Housing: Male animals were pair-housed or triple-housed, by group, at start of study. During the study, single housing was required in Groups 1 and 5 because of an odd number of animals and because the animals would become too large to be triple-housed. Nominally 2 or 3 female animals per cage. Polycarbonate cages with a stainless steel mesh lid containing Teklad 7070C Certified Diamond Dry Cellulose Bedding
- Diet : Teklad Global 16% Protein Rodent Diet (Certified), ad libitum
- Water: Potable water from public supply via an automated watering system, ad libitum
- Acclimation period: At least 5 days

DETAILS OF FOOD AND WATER QUALITY:
Results of feed lot analysis used during this study as well as periodic analysis of the water were provided to the Testing Facility and are maintained on file at the Testing Facility. There were no known contaminants in the feed or in the water that were expected to interfere with the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26°C
- Humidity (%): 30 to 70%
- Air changes (per hr): Not specified
- Photoperiod : 12 hours light/12 hours dark

IN-LIFE DATES: From: October 2019 To: 27 January 2020

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Since this test item is not volatile and thus unlikely to result in significant inhalation exposure, daily administration via the oral route (as per Reach/ECHA guidance) for 13 weeks was used in this study.
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Appropriate amounts of the test item were mixed with the vehicle. The test item was used as supplied when calculating quantities to be used during dose preparation. Fresh formulations were prepared once weekly and were stored at room temperature under nitrogen when not in use. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure and was capped except for dispensing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on the test substance’s characteristics and the subsequent relevant OECD testing guidelines.
- Concentration in vehicle: 0, 6.25, 25, 62.5, 100 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg body weight
- Lot/batch no. (if required): 2IC0148, 2IH0387
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were collected for concentration analysis at the first and last sample preparation. Samples for homogeneity were collected at the same intervals, from the low and top doses only.
Analyses were performed by the Department of Formulation and Inhalation Analysis at the Testing Facility.
Duration of treatment / exposure:
90-day exposure
Frequency of treatment:
Once daily, 7 days per week, for 13 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Negative control group; Group 1
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Group 4
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
Group 5
No. of animals per sex per dose:
10 animals per sex per dose. To determine reversibility of potential effects of the test item, additional recovery animals (5/sex in Group 5 i.e. at dose level 400 mg/kg bw/day) were included in the study. Additional recovery animals (5/sex in Group 1 i.e. at dose level 0 mg/kg bw/day) were required for comparison purposes.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses for this study were selected based on the results of a 4-week preliminary study (Covance SX91YQ) in rats orally dosed daily at 400, 600 or 800 mg/kg bw/day for up to 4 weeks. Beginning on Day 15, females dosed at ≥600 mg/kg bw//day exhibited neurological clinical signs of abnormal gait, hindlimb splayed, impaired locomotion, flattened posture and/or decreased activity. Six females, one at 600 mg/kg/day and three at 800 mg/kg/day, were euthanized for welfare reasons between Days 15 and 21 and the remaining two 800 mg/kg bw/day females were electively euthanized on Day 22. In the brain of female rats, white matter vacuolation/degeneration was present in white matter tracts in the cerebellum and medulla at ≥600 mg/kg bw/day. White matter vacuolation/degeneration was also present in all 3 levels of the spinal cord of female rats at ≥600 mg/kg bw/day. The incidence of this finding increased distally with the lumbar spinal cord more commonly affected that the cervical spinal cord. Nerve roots attached to the lumbar spinal cord also had vacuolation. Degeneration of the tibial and sciatic nerves was present in females at ≥600 mg/kg bw/day.

Based on the severity of the clinical signs of neurotoxicity, the microscopic findings in the central and peripheral nervous systems, and the need to euthanize animals at dose levels ≥600 mg/kg bw/day following less than 28 days of exposure, the high dose level selected for this 90-day study was 400 mg/kg bw/day. The mid-high, mid-low and low dose levels of 250, 100 and 25 mg/kg bw/day were selected to provide dose-response data and to establish a NOAEL.

- Rationale for animal assignment (if not random): One animal at a time was allocated in each cage on arrival, until an appropriate number of animals per cage was achieved (normally 2 or 3 animals per cage). Each sex was allocated separately. Prior to dosing, animals were re-distributed in an attempt to equalize group mean body weights and replace animals with clinical signs or ophthalmology findings.

- Fasting period before blood sampling for clinical biochemistry: yes, animals were fasted overnight prior to each blood collection interval.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals are observed twice daily for mortality and general condition. On treatment days, animals are observed for signs of toxic or pharmacologic effects once prior to and 1 to 2 hours after dose administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made for each animal outside its cage at least twice pre-test and weekly (once every 7 days) during the treatment and recovery periods. The observations focused on general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as an evaluation of respiration and palpation for tissue masses.

BODY WEIGHT: Yes
- Time schedule for examinations: At least twice during pre-test and weekly during the rest of the treatment and recovery periods for non-fasted body weights. Fasted body weights are obtained on the day of the scheduled necropsy intervals.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumption was measured (weighed) weekly, beginning one week prior to treatment.


FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Once during the pre-test, at end of dosing (week 13) and at end of recovery (Week 4). Lids, lacrimal apparatus and conjunctiva were examined visually. The cornea, anterior chamber, lens, iris, vitreous humor, retina and optic disc were examined by indirect ophthalmoscopy. The pupils of each animal were dilated prior to examination using tropicamide ophthalmic solution.
- Dose groups that were examined: all (surviving) animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of dosing (week 14) and at the end of recovery (week 5)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: up to 10 animals/sex/group at termination of dosing, and 5 at the end of recovery
- Parameters checked in table were examined. : yes, see Table 1

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of dosing (week 14) and at the end of recovery (week 5)
- Animals fasted: Yes
- How many animals: up to 10 animals/sex/group at termination of dosing, and 5 at the end of recovery
- Parameters checked in table were examined: yes, see Table 1

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12 (prior to dosing) and Recovery Week 4, time of testing counter-balanced across treatment groups
- Dose groups that were examined: all main study and recovery animals; blind evaluations
- Battery of functions tested: Functional observational battery (FOB) assessment was performed and the following parameters were observed: home cage evaluations (posture, palpebral closure, vocalizations, motor movements); handling evaluations (ease of removal, reactivity to handling, chromodacryorrhea, lacrimation, salivation, coat); open field evaluations (gait and posture; locomotion; arousal, piloerection, exophthalmia, urine, faecal pellets); motor movements (fasciculations, tremors, convulsions); reflex assessments (visual approach, audition assessment, pinna reflex, proprioception, pain perception, pupil response, air righting); grip strength; landing foot splay; body temperature

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 2)

HISTOPATHOLOGY: Yes (see Table 2)
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit. The parameters to analyse were identified as either continuous (greater than 6 distinct values), discrete (between 3 and 6 distinct values) or binary (2 distinct values).

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical observations were noted at any dose level during the dosing or recovery phases.
Circling was noted on two occasions (Days 57 and 76) for one female administered 25 mg/kg bw/day. Given isolated occurrence and lack of dose response, this observation was considered incidental.
Other clinical observations including salivation, lost appendages, kinked tail, hair loss, encrusted or dry skin, abnormal colour of the skin or chromodacryorrhea, and broken teeth were observed across all dose groups, including controls. These appeared rather infrequently, were transient, occurred during pre-dose period, or were with comparable incidences as controls; therefore, they were considered not test item related.
Mortality:
no mortality observed
Description (incidence):
One female administered 400 mg/kg bw/day was euthanized for welfare reasons on Day 31 of the dosing phase with clinical observations that included irregular/laboured breathing. Relevant macroscopic findings included a mass in the thymus, enlarged liver and spleen, and pale femoral bone marrow. The major factor contributing to death was systemic lymphoma. Given the isolated occurrence, and no test item-related histologic findings, this early death was considered incidental.
All other animals survived to their scheduled sacrifice during dosing or recovery phase.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related alterations in body weight or body weight change were noted at any dose level during dosing or recovery phase.
A few statistically significant differences, compared with control, were noted for males administered 250 mg/kg bw/day (Days 12 to 19 of the dosing phase) or 400 mg/kg bw/day (Days 12 to19 and Days 40 or 47 of the dosing phase) and for females administered 25, 100, 250, or 400 mg/kg bw/day (Days 5 to 12 of the dosing phase) or females administered 400 mg/kg bw/day (Days 5 to 12 of the recovery phase). These differences were considered incidental as they lacked any consistent pattern or direction, and overall mean body change was comparable across all dose groups.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects on food consumption were noted at any dose level during dosing or recovery phase.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cataract or opacity of the lens in the posterior subcapsular and capsular region was noted in four females administered 400 mg/kg bw/day at the terminal interval and in two females administered 400 mg/kg bw/day at the recovery interval.
Although these findings were noted at the highest dose, these findings were likely incidental and not test item-related per the veterinary ophthalmologist.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At the termination of dosing, there were no test item-related haematology changes in animals administered 25 mg/kg bw/day. Test item-related haematology changes at ≥ 100 mg/kg bw/day in males only included decreases in red cell distribution width (0.90 to 0.93X control) that corresponded to decreases in reticulocytes (0.71X control) in males administered 400 mg/kg bw/day only. These changes were considered non-adverse due to their relatively small magnitude. All other differences in mean values between control and test item-treated animals, including those that were statistically significant, were considered not test item-related as they lacked a dose relationship and/or there was general overlap between individual control and test item-treated values. There were no test-item related coagulation changes at any dose.

At the end of 4-week recovery, there were no test-item related haematology changes in animals previously administered 400 mg/kg bw/day indicating complete recovery of those changes identified at the termination of dosing. All differences in mean values between control and test item-treated animals, including those that were statistically significant, were considered not test item-related as there was general overlap between individual control and test item-treated values and/or similar changes were not identified at the termination of dosing. There were no test-item related coagulation changes at any dose.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At termination of dosing, there were no test-item related clinical chemistry changes at any dose. All differences in mean values between control and test item-treated animals, including those that were statistically significant, were considered not test item-related as they lacked a dose relationship and/or there was general overlap between individual control and test item-treated values.
At the end of 4-week recovery, there were no test-item related clinical chemistry changes in animals previously administered 400 mg/kg bw/day. One animal had minimal to mild increases in aspartate and alanine aminotransferase
activities. As these changes were not identified at the termination of dosing they were not considered test item-related. All differences in mean values between control and test item-treated animals, including those
that were statistically significant, were considered not test item-related as there was general overlap between individual control and test item-treated values.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related effects were noted on functional observational battery tests during dosing or recovery phase.
Abnormal observations included completely closed eyelids that were not dose-dependent and was noted for controls and thus was not treatment-related.

No clearly treatment-related effects were noted on motor activity tests during dosing or recovery phase although a few statistically significant differences were noted in females:
Horizontal Assessments
Significantly decreased motor activity, compared with control, was noted for interval 4 (p≤0.046, Williams’ test) for females administered 100, 250, or 400 mg/kg bw/day and for interval 3 (p=0.034, Williams’ test) for females administered 400 mg/kg bw/day during Week 12 of the dosing phase. No significant changes were noted during recovery phase assessment for females administered 400 mg/kg bw/day.
No test item-related effects in motor activity were observed for males during Week 12 of dosing phase. However, significantly increased motor activity, compared with control, was noted for interval 12 (p=0.032, t-test) during recovery phase assessment for males administered 400 mg/kg bw/day.
Vertical Assessments
Significantly decreased motor activity, compared with control, was noted for interval 4 (p≤0.041, Williams’ test) during Week 12 of the dosing phase for females administered 100, 250 and 400 mg/kg bw/day. On the contrary, significantly increased motor activity was noted for interval 5 (p=0.036, t-test) during recovery phase assessment for females administered 400 mg/kg bw/day.
No test item-related effects in motor activity were observed for males during Week 12 of dosing phase or Week 4 of recovery phase.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At terminal necropsy, organ weight changes related to the test item were present in the liver at ≥250 mg/kg bw/day. Compared to controls, liver weights were higher in males and females which correlated microscopically with hepatocellular hypertrophy.
At recovery necropsy, test item-related organ weight changes were not present in recovery animals at 400 mg/kg bw/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At terminal necropsy, test item-related macroscopic findings were not present in main study animals. All macroscopic findings occurred sporadically or at similar incidence and severity in control and test item treated groups and were considered incidental and due to biological variability.
At recovery necropsy, test item-related macroscopic findings were not present in recovery animals.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At terminal necropsy, axonal degeneration (minimal), characterized by shrunken axons surrounded by clear halos, was present in the lumbar spinal nerve roots and sciatic nerve in one female at 400 mg/kg bw/day.
This finding was of minimal incidence and severity but was considered adverse as it was also present in one recovery female at 400 mg/kg bw/day. An additional finding of white matter vacuolation in the lumbar spine (minimal) was also observed in this recovery female at 400 mg/kg bw/day.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Centrilobular hepatocellular hypertrophy, characterized by enlargement of centrilobular hepatocytes, was present in all males (minimal to slight) at 250 mg/kg bw/day and all females (minimal) at 400 mg/kg bw/day. All other microscopic findings occurred sporadically or at similar incidence and severity in control and test item treated groups and were considered incidental and due to biological variability. As hepatocellular hypertrophy recovered completely, it was considered as non-adverse effects.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
neuropathology

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
400 mg/kg bw/day (actual dose received)
System:
peripheral nervous system
Organ:
spinal cord
other: sciatic nerve, lumbar spine
Treatment related:
yes
Dose response relationship:
no
Relevant for humans:
not specified

Any other information on results incl. tables

 Table 1. Test item-related haematology changes in rats dosed for 4 weeks

Sex

M

M

M

M

F

F

F

F

Dose (mg/kg/day)

25

100

250

400

25

100

 

250

 

400

Reticulocytes

-

-

-

0.71**

-

-

-

-

RDW

-

0.93**

0.93**

0.90**

-

-

-

-

RDW: Red Cell Distribution Width.

Changes are expressed as fold change versus concurrent control.

Statistical significance based on absolute values: ** = p < 0.01; * = p < 0.05

- : no test item-related change

Table‎ 2: Test item-related organ weight changes (% difference relative to controls) in rats dosed with triethoxy(octyl)silane for 13 weeks

Group/sex

2M

3M

4M

5M

2F

3F

4F

5F

Dose (mg/kg/day)

25

100

250

400

25

100

250

400

Liver

 

 

 

 

 

 

 

 

     Absolute weight (%)

8

-

3

7

-

3

8

12

     vs. body weight (%)

2

3

8a

10a

-

-

8a

17a

     vs. brain weight (%)

9

-

2

6

-

-

8

12

aStatistically significant difference between mean values for test item-treated and control groups.

-= not test item-related.

Table‎ 3: Test item-related findings in the liver in rats dosed with triethoxy(octyl)silane for 13‑weeks

Group/sex

1M

2M

3M

4M

5M

1F

2F

3F

4F

5F

Dose (mg/kg/day)

0

25

100

250

400

0

25

100

250

400

Centrilobular Hypertrophy

 

 

 

 

 

 

 

 

 

 

Minimal

0

0

0

2

8

0

0

0

0

10

Slight

0

0

0

0

1

0

0

0

0

0

Total

0

0

0

2

9

0

0

0

0

10

 

 

 

 

 

 

 

 

 

 

 

Number of tissues examined

10

10

10

10

10

10

10

10

10

10

Applicant's summary and conclusion

Conclusions:
In a 90-day oral repeated dose toxicity study with the registered substance triethoxy(octyl)silane, conducted according to OECD Test Guideline 408 and in compliance with GLP, the NOAEL for systemic effects was 250 mg/kg bw/day based on minimal adverse effects in peripheral nerves at 400 mg/kg bw/day.