Registration Dossier

Administrative data

Description of key information

Oral (OECD 408), rat: NOAEL = 300 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Jul 2019 - 04 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The Sprague Dawley rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 7 weeks old
- Weight at study initiation: 166 and 281 g
- Fasting period before study: no; however, Animals did not have access to diet during the conduct of the functional observational battery (FOB) and motor activity (MA) assessments
- Housing: Group housed (2–3 animals of the same sex and same dosing group together) in polycarbonate, solid-bottom cages containing appropriate bedding material
- Diet: PMI Nutrition International , LLC Certified Rodent LabDiet 5002 meal provided ad libitum, except during designated periods of fasting
- Water: Municipal tap water, treated by reverse osmosis and ultraviolet irradiation provided ad libitum
- Acclimation period: at least 7 days

DETAILS OF FOOD AND WATER QUALITY: Results of analysis for nutritional components and environmental contaminants were provided by the supplier and are on file at the Testing Facility. It was considered that there are no known contaminants in the feed at levels that would interfere with the objectives of the study.

Periodic analysis of the water as performed, and results of these analyses are on file at the Testing Facility. It was considered that there are no known contaminants in the water at levels that could interfere with the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 26
- Humidity (%): 30 to 70
- Air changes (per hr): Ten or more air changes per hour, 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing formulations of test material were prepared every 4 days or less. Dosing formulations were prepared at appropriate concentrations to meet dose level requirements.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was chosen as the appropriate vehicle based on the test substance’s characteristics and the subsequent relevant OECD testing guidelines.
- Concentration in vehicle: undiluted
- Amount of vehicle: The control group was administered 4 mL/kg
- Lot/batch no.: 2IC0148
- Purity: not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyzed dosing formulations contained 101% to 109% of the test substance which was within the protocol-specified range of target concentrations for suspensions (85% to 115%) and were homogeneous.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15 males and 15 females in the control and high-dose groups; 10 males and 10 females in the mid- and low-dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The target exposure concentrations were selected by the Sponsor Representative in consultation with the Study Director based on a previous repeated-dose study. In that study, the test substance was administered to up to 5 males and females via oral gavage for up to 14 consecutive days at dosage levels of 0, 90, 180, 300, and 500 mg/kg bw/day. Based on decreased body weights, dosage levels of 30, 100, and 300 mg/kg bw/day were selected for this study.
- Rationale for animal assignment: Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately.
- Fasting period before blood sampling for clinical biochemistry: yes; at least 8 hours (no more than 24 hours)
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality was observed at least twice daily (morning and afternoon), beginning upon arrival through termination/release. Cageside observations were made at least once daily on non-dosing/recovery days.
- Cage side observations checked in tables 1 though 5

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on Day 1 (prior to dosing) and then weekly (± 2 days) during the study period, and on the day of scheduled necropsy (animals selected for necropsy).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing), weekly (± 2 days) during the study period, on the day prior to the first day of scheduled necropsies, and on the day of the scheduled necropsy.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Near the end of the dosing period (Day 85)
- Dose groups that were examined: All Main and Recovery Study animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy (animals scheduled for necropsy)
- Anaesthetic used for blood collection: Yes; euthanized via carbon dioxide inhalation
- Animals fasted: Yes; at least 8 hours (no more than 24 hours)
- How many animals: Groups 1–4 (Main and Recovery Study animals)
- Parameters checked in table 6 and 7 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day of scheduled necropsy (animals scheduled for necropsy)
- Animals fasted: Yes; at least 8 hours (no more than 24 hours)
- How many animals: Groups 1–4 (Main and Recovery Study animals)
- Parameters checked in table 8 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: On the day of scheduled necropsy (animals scheduled for necropsy)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes; at least 8 hours (no more than 24 hours)
- Parameters checked in table 9 were examined.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: Yes; Functional Observational Battery (FOB); T3, T4, TSH hormones were analyzed, as well as estrous cycle
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see tables 10 and 11); Animals euthanized for humane reasons or at study termination were euthanized by carbon dioxide inhalation, followed by exsanguination. Main and Recovery Study animals were subjected to a complete necropsy examination, which included evaluation of the carcass; all external surfaces and orifices; the cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

The organs identified in Table 11 were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

HISTOPATHOLOGY: Yes (see tables 10 and 12); Tissues identified in Table 12 (except animal identification and bone marrow smears) from animals found dead, euthanized in extremis, and in the control and high-dose groups at the terminal necropsy were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. In addition, gross lesions and the testes were prepared from all animals in the low- and mid-dose groups at the terminal necropsy and all animals at the recovery necropsy
Other examinations:
For the functional observational battery tests, animals (all main Ssudy and recovery animals) were evaluted during the final week of test substance administration and near the end of the recovery period. Animals were evaluted for sensory activity, neuromuscular observations, and motor activity.

For the thyroid hormone analysis, sample were collected on on the days of scheduled necropsy. For total T3 and T4 analysis, hormone samples were analyzed using a validated UHPLC/MS/MS assay. For TSH analysis, hormone samples were analyzed using a qualified radioimmunoassay procedure.

Estrous cycle was evaluted on the day of scheduled necropsy for all main and recovery study animals. To determine estrous cycle, vaginal lavages were performed to prepare vaginal smears; from the smears, Giemsa stained slides were prepared from all females and read.
Statistics:
Body weight, body weight gains, food consumption, hematology variables, coagulation variables, clinical chemistyr variables, urinalysis variables, organ weights, and organ weights relative to body and brain weight were analysed through a parametric/non-parametric analysis. For the parametric/non-parametric analysis, Levene’s test as used to assess the homogeneity of group variances.

The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse test substance-related clinical observations.
However, several males and females in the 300 mg/kg bw/day group and a single male and female in the 100 mg/kg bw/day group were noted with abnormal breathing sounds over the course of the study. Due to the properties of the test substance/formulation (likely irritant, viscous nature), these findings were considered to be related to administration, rather than a systemic effect of the test substance. Two males in the 300 mg/kg bw/day group were still noted with these findings throughout the recovery period, although it was not observed during the functional observational battery evaluations during Week 16.

Additionally, several males and females in the 100 and 300 mg/kg/day group were noted with wet fur around the mouth. This also was considered to be a response to being dosed with a viscous formulation, rather than a systemic effect of the test substance.

All other clinical observations in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no clear test substance-related deaths.

Six unscheduled deaths occurred in the 300 mg/kg bw/day group males and females during the dosing period. Five of the unscheduled deaths (Animal Nos. 4001, 4002, 4008, 4512, and 4513) were attributed to laryngeal and/or tracheal neutrophilic inflammation and were consistent with gavage-associated reflux. The cause of the unscheduled death of Animal No. 4509 was undetermined and a relationship to the test substance was uncertain. However, given limited sampling of the larynx/trachea, clinical observations of gasping, and gross observations of gas filled tissues (suggestive of respiratory distress), gavage-associated reflux could not be excluded for this animal also.In addition, clinical observations noted in these animals prior to death included abnormal breathing sounds, suspected dehydration, labored breathing, abdominal distention, thin, ungroomed fur, wet fur around mouth, muzzle, and/or urogenital area, ventral cervical area, red staining of fur on muzzle and/or periorbital area, and/or yellow fur stating on thoracic and/or urogenital area; body weight losses were noted in a single male (No. 4002; 5.5%) and all females (7.9% to 22.0%) for the interval prior to death.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related lower body weights were noted in the 300 mg/kg bw/day group males.

Test substance-related lower mean body weight gains were noted in the 300 mg/kg bw/day group males throughout the dosing period when compared to the control group, which resulted in 7.1% lower mean body weight at the end of the dosing period. Higher mean body weight gains were noted in the 300 mg/kg/day group males throughout the recovery period (statistically significant for the first 2 weeks of the recovery period) when compared to the control group, which resulted in 1.0% lower mean body weight at the end of the recovery period. These higher body weight gains reduced the difference in mean body weights between the end of the dosing and recovery periods in the 300 mg/kg bw/day group males from 7.1% to 1.0%, respectively, when compared to the control group.

There were no other test substance-related effects on body weight in males and no test substance related effects in females. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. These were not considered test substance-related and were instead attributed to normal variation.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was no test substance-effects on food consumption during the dosing phase of the study. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. These were not considered test substance-related and were instead attributed to normal variation.

During the recovery period, the 300 mg/kg/day group males had higher mean food consumption throughout the recovery period when compared to the control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
NEED TO ENTER AFTER REPORT IS FINALIZED; EDITS REQUESTED IN DRAFT UNDER COAGULATION

Hematology parameters were unaffected by test substance administration. However, some statistically significant differences were observed when the control and test substance-treated groups were compared; however, differences were not considered test substance-related and were instead attributed to normal biological variation.

Test substance-related changes in coagulation parameters on Days 91/92 were limited to lower fibrinogen in the 300 mg/kg bw/day group females. Additionally, test substance-related changes in clinical chemistry parameters on Day 91/92 included higher alanine aminotransferase in the 300 mg/kg bw/day group males and females and lower cholesterol, high density lipoprotein, and low density lipoprotein in the 300 mg/kg bw/day group females. There were no other test substance-related changes in clinical pathology parameters at the terminal necropsy or at the recovery necropsy.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related changes in clinical chemistry parameters were noted in the 300 mg/kg bw/day group males and females.

Test substance-related minimally increased mean alanine aminotransferase (ALT) was noted in the 300 mg/kg bw/day group males and females on Day 91/92 when compared to control group. It was noted that in females, the mean for the concurrent control was artificially elevated by an outlier (Female No. 1507). When this outlier value was removed, the mean ALT was changed from 104.7 U/L to 57 U/L. Compared to the adjusted mean control group female value, the 300 mg/kg bw/day group females were considered to be elevated. Additionally, test substance-related lower mean cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were noted in the 300 mg/kg bw/day group females when compared to the control group. There were no correlating test substance-related microscopic changes in the liver at the primary necropsy. There were no test substance-related changes in ALT, cholesterol, HDL, or LDL noted at the recovery necropsy. Accordingly, the noted clinical chemistry effects were not considered adverse. Remaining differences in clinical chemistry parameters, regardless of statistical significance, were not considered not test substance-related based on their absence of a dose response, general overlap of individual values with the range of control values, and/or were of a magnitude of change commonly observed in rats under similar study conditions.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant differences when the test substance treated males and females were compared to the control group at the Week 16 evaluations were limited to higher mean urination in the 300 mg/kg bw/day group males and lower mean rearing in the 300 mg/kg bw/day group females at Week 16; however, these differences were not considered test substance-related because they were consistent with Charles River Ashland historical data.

No other urinalysis findings were noted in the study report.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Home cage observations, open field observations, sensory and neuromuscular observations were unaffected by test substance administration.
Test substance-related lower total and ambulatory motor activity counts were noted in the 300 mg/kg bw/day group females. In females, statistically significant lower mean total and ambulatory activity counts were noted at the Week 13 evaluation in the 300 mg/kg bw/day group. The largest difference between the control and 300 mg/kg bw/day groups was noted during the first 10 minutes. The activity curves in the 300 mg/kg bw/day group females were lower throughout the testing period and were essentially parallel to the concurrent control group. Because statistical significance was only noted in the 300 mg/kg bw/day group females for both total and ambulatory activity, this was considered to be test substance-related effect. Mean counts for both total and ambulatory activity were within the range of means in the Charles River Ashland historical control data. Based on the lack of further observations in the functional observational battery and/or during routine examinations, lower mean total and ambulatory activity counts were not considered adverse. There were no effects on total and ambulatory activity at the Week 16 recovery assessment on females compared to the control group. Motor activity patterns (total and ambulatory activity counts) in males were unaffected by test substance administration.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related organ weight changes were noted at the terminal euthanasia. There were isolated organ weight values that were statistically different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to administration of the test substance.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
T3, T4, and TSH were unaffected by test substance administration. Any differences, including those that reached statistical significance, were not considered test substance-related based on their absence of a dose response, and/or general overlap of individual values with the range of control values.

The number of females in estrus, diestrus, or proestrus were comparable between the control and test substance-treated groups at the terminal and recovery euthanasia.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no definitive test substance-related unscheduled deaths or findings that were considered adverse
Key result
Critical effects observed:
no
Conclusions:
In an oral gavage study conducted in accordance with OECD 408 and GLP the NOAEL for N [3 (dimethoxymethylsilyl)-2-methylpropyl]ethylenediamine (CAS 23410-40-4) was considered to be 300 mg/kg bw/day based on the lack of definitive test substance-related unscheduled deaths or findings that were considered adverse.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study was conducted according to an appropriate OECD test guideline, and 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

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In the available key study (CRL, 2020) the registration substance was investigated for repeated dose toxicity via oral route in a subchronic toxicity test in rats, conducted according to OECD 408 and in compliance with GLP. There were no test substance-related unscheduled deaths. Two males were found dead and 3 females and 1 male were euthanized in extremis, all in the 300 mg/kg bw/day group. However, the unscheduled deaths in all of the males and in 2 of the 3 females were consistent with gavage-associated reflux. The cause of moribundity in the remaining female was undetermined. The relationship to the test substance was uncertain, but given the limited sampling of the larynx/trachea, clinical observations of gasping, and gross observations of gas-filled tissues (suggestive of respiratory distress), gavage-associated reflux could not be excluded for this animal. All other animals survived to the scheduled necropsies. There were no test substance-related clinical observations or effects on food consumption, hematology, urinalysis, estrous cycle, hormone analyses (T3, T4, and TSH), or organ weights. There were no test substance-related ophthalmic, macroscopic, microscopic findings. Test substance-related lower body weights were limited to the 300 mg/kg bw/day group males throughout the dosing period. At the end of the dosing period, the mean body weight in the 300 mg/kg bw/day group males was 7.1% lower than the control group. During the recovery period, body weight gains for the remaining males in this group were higher than or similar to the control group. Test substance-related effects on FOB parameters were noted at Week 13 in the 300 mg/kg bw/day group males and females and included rales (abnormal sound accompanying breathing) and lower body weight in males. These findings were consistent with observations/data collected during the dosing period. There were no test substance-related effects on FOB during the recovery period. Test substance-related effects on motor activity were noted in the 300 mg/kg bw/day group females at Week 13 and included lower total and ambulatory activity counts. There were no other test substance-related effects on motor activity at Week 13 or during the recovery period. Test substance-related changes in coagulation parameters on Days 91/92 were limited to lower fibrinogen in the 300 mg/kg bw/day group females. Additionally, test substance-related changes in clinical chemistry parameters on Day 91/92 included higher alanine aminotransferase in the 300 mg/kg bw/day group males and females and lower cholesterol, high density lipoprotein, and low density lipoprotein in the 300 mg/kg bw/day group females. There were no other test substance-related changes in clinical pathology parameters at the terminal necropsy or at the recovery necropsy.
In conclusion, administration of N-[3-(dimethoxymethylsilyl)-2-methylpropyl]ethylenediamine by once daily oral gavage to Crl:CD(SD) rats at dose levels of 30, 100, and 300 mg/kg bw/day for a minimum of 90 days resulted in no definitive test substance-related unscheduled deaths or findings that were considered adverse. Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg bw/day.


Justification for classification or non-classification

The available data on repeated dose toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and is therefore conclusive but not sufficient for classification of the registered substance.