Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Aug 2019 to 30 Jan 2020
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 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 11 - 12 weeks old
- Weight at study initiation: between 200 and 250 g
- Fasting period before study: not reported
- Housing: individually housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal) was provided ad libitum
- Water: Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system.
- Acclimation period: prior to the initiation of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12-hr light/12-hr dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing formulations were prepared every 4 days and an adequate amount of each formulation was dispensed into daily aliquots, which were stored at room temperature, protected from light, under nitrogen, until use.

VEHICLE
- Lot/batch no. (if required): 2IC0148
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyzed dosing formulations contained 107% to 114% of the test substance which was within the protocol-specified range of target concentrations for suspensions (85% to 115%) and were homogeneous.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
single daily oral gavage dose during Gestation Days 6 through 20
Frequency of treatment:
single daily dose
Duration of test:
from Gestation Days 6 through 20
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 dams/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were based on a dose range-finding study, in which 5 males and 5 females (non-pregnant) Sprague Dawley rats per dose group via oral gavage for up to 14 consecutive days at dosage levels of 0, 90, 180, 300, and 500 mg/kg bw/day. At 500 mg/kg/day, the test substance caused moribundity, leading to unscheduled euthanasia, and thus exceeds a maximum tolerated dose. Therefore, the dose levels selected for this study were 0, 50, 150, and 400 mg/kg bw/day.
- Rationale for animal assignment: Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Animals in poor health or at extremes of body weight range were not assigned to groups.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once in the morning and once in the afternoon.
- Cage side observations included, but were not limited to: labored breathing, fur staining, thinning fur, abnormal gait, decreased activity, and increased salivation.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily, beginning on the day of receipt and lasting through euthanasia

BODY WEIGHT: Yes
- Time schedule for examinations: individually on Gestation Days 0 (by supplier) and 5 - 21 (daily)

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: liver and kidneys

OTHER: Blood samples for thyroid hormone analyses were collected (prior to noon in order to avoid diurnal fluctuations in thyroid hormone levels) via a jugular vein into tubes without anticoagulant
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Placentae were also examined
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and are reported at the 1% and 5% levels.

Body weight; body weight gain; food consumption; gravid uterine weight and corrected maternal body weights; litter means; organ weights; and anogenital distance: Levene's test was 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.

Ovarian and uterine content; litter % of fetuses with external/visceral/skeletal abnormalities: The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparison was conducted using Dunn's test.

Parental indices and mortality; macroscopic findings; microscopic findings: A Fisher’s Exact Test was used to conduct pairwise group comparisons of interest.

Thyroid hormone analysis: The groups were compared using an overall one-way ANOVA F-test. If the overall F-test is found to be significant, then pairwise comparisons will be conducted using Dunnett's test.
Indices:
Postimplantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early or Late) per Group) / (No. Gravid Females per Group) X 100
Summation Per Group (%) = (Sum of Postimplantation Loss per Litter) / (No. Litters per Group) X 100
Postimplantation Loss/Litter (%) = (No. Dead Fetuses, Resorptions (Early or Late) per Group) / (No. Implantation Sites per Litter) X 100
Historical control data:
Charles River Ashland has historical data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Labored breathing and/or abnormal breathing sounds were noted for 17 of 25 females in the 400 mg/kg bw/day group at the daily examinations and/or 1 - 2 hours following dose administration generally throughout the study (Gestation Days 8 - 21). Abnormal breathing sounds were also noted for 8 of 25 females in the 150 mg/kg bw/day group during Gestation Days 7 - 19, albeit to a lesser extent than noted in the 400 mg/kg bw/day group.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female in the 150 mg/kg bw/day group was found dead on gestation day 18. No clinical observations or noteworthy changes in body weight were noted for this female prior to death, and no macroscopic findings were observed at necropsy. In the absence of unscheduled deaths in the high-dose group, this mortality was not considered test substance-related.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean maternal body weights, body weight gains, corrected body weights, corrected body weight gains, and gravid uterine weights in the 50, 150, and 400 mg/kg bw/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exceptions. A statistically significantly higher mean body weight gain during Gestation Days 6 - 7 and lower mean body weight gain during Gestation Days 20 - 21 were noted in the 400 mg/kg bw/day group compared to the control group. Because these body weight changes failed to produce a noteworthy effect on the overall body weight gain and absolute mean body weights in this group, they were considered transient and unrelated to test substance administration.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 150, and 400 mg/kg bw/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effects on mean TSH, T3, or T4 concentrations were noted at any dose level. Mean TSH concentration in the 400 mg/kg bw/day group females was 21.8% lower than the control group. This decrease was not considered test substance-related because the difference from the control group was not statistically significant, and there were no effects on thyroid gland weight or histopathology in the thyroid at this dose level.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not specified
Details on maternal toxic effects:
Intrauterine growth and survival were unaffected by test substance administration at dose levels of 50, 150, and 400 mg/kg bw/day. Parameters evaluated included mean litter proportions of postimplantation loss, mean number of viable/live fetuses, mean fetal body weights, and fetal sex ratios. Differences from the control group were slight, not statistically significant, and/or did not occur in a dose-related manner.

Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed

Maternal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related skeletal malformations were observed in fetuses in this study. Two fetuses in the 150 mg/kg bw/day group each had an absent lumbar vertebra. This finding was not considered test substance-related because no skeletal malformations were noted in the high-dose group. No other skeletal malformations were noted in this study.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related visceral malformations were observed in fetuses in this study. One fetus in the 50 mg/kg bw/day group had a retroesophageal right subclavian artery; however, this finding was not observed in a dose-related manner, and therefore was not considered test substance-related. No other visceral malformations were noted in this study.
Other effects:
no effects observed
Description (incidence and severity):
Mean anogenital distance (absolute and relative to cube root of body weight) was unaffected by test substance administration at all dose levels. Differences from the control group were slight and not statistically significant.
Details on embryotoxic / teratogenic effects:
Mean mean number of viable/live fetuses, mean fetal body weights, and fetal sex ratios were unaffected by treatment. Mean anogenital distance (absolute and relative to cube root of body weight) was unaffected by test substance administration at all dose levels. No test substance-related skeletal or visceral malformations were observed in fetuses in this study.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In an oral gavage developmental study conducted in accordance to OECD 414 and to GLP, the NOAEL for N-[3-(dimethoxymethylsilyl)-2-methylpropyl]ethylenediamine for maternal toxicity and embryo/fetal development was 400 mg/kg bw/day, as no significant adverse effects were observed up to the highest dose of 400 mg/kg bw/day tested in rats.