Morpholine, 4-[(triethoxysilyl)methyl]-

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance with an appropriate OECD test guideline and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines (Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000)

Principles of method if other than guideline:

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and fetus consequent to exposure of the female to the test item from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section).

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

480-370-1

EC Name:

-

Cas Number:

21743-27-1

Molecular formula:

Hill formula: C11H25NO4Si CAS formula: C11H25NO4Si

IUPAC Name:

4-[(triethoxysilyl)methyl]morpholine

Test material form:

solid - liquid: suspension

Test animals

Species:

rat

Strain:

other: HanRcc: WIST(SPF)

Details on test animals or test system and environmental conditions:

Animals: Rat, HanRcc: WIST(SPF)
Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
Number of Animals: 88 mated females (In order to complete mating within a reasonable time period, 97 female rats were obtained from the breeder. The surplus females were sacrificed after commencement of treatment for the last mated females); 22 mated females per group
Age (Day 0 Post Coitum): 11 weeks
Body Weight Range (Day 0 Post Coitum): 183 to 234 g
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Computer-generated random algorithm.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Husbandry:
Room Number, Füllinsdorf: 15B
Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was 12-hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland).
Diet: Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst/Switzerland) was available ad libitum (batch no. 60/09).
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Four groups of females were treated by oral gavage with (N-Morpholinomethyl)triethoxysilan once daily at dose levels of
0 (control), 100, 300 or 1000 mg/kg body weight/day on days 6-20 post coitum. The dose levels were selected based on a previous
dose range finding toxicity study in Han Wistar rats, Harlan Laboratories Study C66154, using dose levels of 0, 100, 300 and 1000 mg/kg/day. A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Formulations:
The dose formulations were prepared weekly using the test item as supplied by the Sponsor. (N-Morpholinomethyl)triethoxysilan was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added and the mixture was mixed for at least 15 minutes.
Separate formulations were prepared for each concentration. All dose formulations were divided into daily aliquots.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Storage of Dose Formulations:
Dose formulations were divided into daily aliquots and stored in glass beakers at room temperature (20 ± 5 °C) under nitrogene, protected from moisture and light. Based upon the results of stability analyses performed within the non-GLP Harlan Laboratories study no. C66154 (Dose Range-Finding Prenatal Developmental Toxicity Study in the Han Wistar Rat), dose formulations were stable for at least one week.

Analysis of Dose Formulations:
On the first treatment day sample from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (7 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations
were frozen (-20 ± 5 °C) and delivered on dry ice to Dr. K. Morgenthal (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis. The samples were analyzed by atomic absorption spectroscopy. A certified reference solution was used as analytical standard.
Analyzed samples were not discarded without written consent from the study director.

The test item concentrations of the samples were determined by analysis of the silicon concentration using an atomic absorption spectrometry (AAS) method.
The test item content in five out of a total of nine samples was found to be in the range of 80.2% to 83.9% and, thus, for these samples the required content limit of ±20% with reference to the nominal concentration was met.
Dose formulations for treatment in the low dose group (target dose 100 mg/kg bw/day) prepared on the first treatment day (top, middle and bottom) as well as during the last week of treatment (middle) contained test item in concentration of 69.6% to 77.0% of the nominal concentration.
These values were slightly below the required content limit. The difference between the actual and nominal concentration was considered possibly to be a result of adsorption of the test item on a container wall.
During the course of this study no effects which were considered to be test item-related were observed up to and including dose level of 1000 mg/kg bw/day. Therefore the lower amount of the test item in the low dose formulation had been considered not to have any impact on the reliability or validity of the results.
The homogenous distribution of the test item in the preparations was approved because single results found deviate not more than 2.8% (accepted limit: ≤15%) from the corresponding mean. In addition, the test item was found to be stable in application formulations when kept seven days at room temperature due to recoveries which met the variation limit of 10% from the timezero (homogeneity) mean.

Details on mating procedure:

After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day of mating was designated day 0 post coitum. Male rats of the same source and strain were used only for mating. These male rats are in the possession of Harlan Laboratories and were not considered part of the test system. The fertility of these males had been proven and was continuously monitored.

Duration of treatment / exposure:

On days 6-20 post coitum

Frequency of treatment:

daily

Duration of test:

Up to day 21 post coitum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

22 mated females/dose group

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

The following observations were recorded as follows:
Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy).
Food Consumption: Recorded at 3-day intervals: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.
Body Weights: Recorded daily from day 0 until day 21 post coitum.

At the scheduled necropsy on day 21 post coitum, females were sacrificed by CO2 asphyxiation and the fetuses removed by Caesarean section. Post mortem examination included gross macroscopic examination of all internal organs.

Ovaries and uterine content:

Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count and position of fetuses in the uterus was performed and the data recorded. The uteri (and contents) of all females with live fetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.

Fetal examinations:

Fetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
1. Microdissection technique (sectioning/dissection technique). At least one half of the fetuses from each litter was fixed in Bouin's fixative (one fetus per container). They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ethanol (one fetus per container). Descriptions of any abnormalities and variations were recorded.

2. The remaining fetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol, glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations were recorded. The specimens were preserved individually in plastic vials.

If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites.

Terminology Used in the Assessment of the Data:
Empty Implantation Site: Very early resorption or aborted implantation
Embryonic Resorption: Amorphous mass being resorbed
Fetal Resorption: Clearly defined fetal body being resorbed
Dead Fetus: Appearance of live fetus but without induced respiration or movement
Live Fetus: Breathing and/or moving fetus
Abnormality: A structural change in a fetus that would probably impair its health or development.
Variation: A fetal change that is unlikely to adversely affect survival or health. This includes a delay in growth or morphogenesis that has otherwise followed a normal pattern of development.

Statistics:

The following statistical methods were used to analyze food consumption, body weights, reproduction and skeletal examination data:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Indices:

From the on-line recorded reproduction data, the following parameters were calculated: pre- and post-implantation losses, embryonic and fetal deaths, live and dead fetuses, abnormal fetuses, fetal sex ratios and fetal body weights.

For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis. Mean fetal weights were calculated from the individual weights both on a per group and on a per litter basis.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
General Tolerability: All females survived until the scheduled necropsy. No clinical symptoms were noted during the study at any dose level.

Food Consumption and Body Weights: Food consumption, body weights and body weight gain were not affected by the treatment with the test item at any dose level.


Reproduction Data: The relevant reproduction data (post-implantation loss and the number of fetuses per dam) were not affected by treatment with the test item at any dose level.

Macroscopic Findings: No test item-related macroscopical findings were noted during necropsy of the dams.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
External Examination: No abnormal findings were noted during the external examination of the fetuses at any dose level.

Sex Ratios: No effects on sex ratio were observed at any dose level.

Body Weights: No test item-related effects on fetal body weight were observed at any dose level.

Visceral Examination: No test item-related abnormalities or variations were noted during the visceral examination of fetuses at any dose level.

Skeletal and Cartilage Examination: No abnormalities, which were considered to be test item-related, were noted during examination of fetal skeletons and cartilages at any dose level.

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the variations on development of axial skeleton

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on these observations, NOEL (No Observed Effect Level) for pregnant rat was considered to be 1000 mg/kg body weight/day.
NOEL for embryo and fetal development was considered to be 1000 mg/kg body weight/day.
Consequently, under the conditions described for this study, (N-Morpholinomethyl)triethoxysilan did not reveal teratogenic potential up to and including the dose level of 1000 mg/kg/day.

Executive summary:

In order to detect effects on the pregnant rat and development of the embryo and fetus, (N-Morpholinomethyl) triethoxysilan was administered orally by gavage once daily to pregnant females from day 6 through to day 20 post coitum at dose levels of 100, 300 and 1000 mg/kg/day.

Test item did not show any toxic potential on pregnant females up to and including the dose level of 1000 mg/kg bw/day.

(N-Morpholinomethyl)triethoxysilan had no influence on the relevant reproduction data (postimplantation loss and the mean number of fetuses per dam) up to and including the dose level of 1000 mg/kg bw/day.

Based on these observations, NOEL (No Observed Effect Level) for pregnant rat was considered to be 1000 mg/kg body weight/day.

NOEL for embryo and fetal development was considered to be 1000 mg/kg body weight/day.

Consequently, under the conditions described for this study, (N-Morpholinomethyl)triethoxysilan did not reveal any teratogenic potential up to and including the dose level of 1000 mg/kg/day.