Trimethoxy(2,4,4-trimethylpentyl)silane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Feb - 27 Jul 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines: MAFF, Test Data for Registration of Agricultural Chemicals, 12 Noshan No. 8147, Teratology (2-1-18), 2000

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Triethoxy(2,4,4-trimethylpentyl)silane

EC Number:

252-558-1

EC Name:

Triethoxy(2,4,4-trimethylpentyl)silane

Cas Number:

35435-21-3

Molecular formula:

C14H32O3Si

IUPAC Name:

triethoxy(2,4,4-trimethylpentyl)silane

Test material form:

solid - liquid: suspension

Details on test material:

- Name of test material (as cited in study report): SILRES® BS 1701
- Physical state: colorless liquid
- Lot/batch No.: KH07241
- Expiration date of the lot/batch: 12 July 2009
- Stability under test conditions: 10 days at room temperature (20±5°C)
- Storage condition of test material: room temperature (20±5°C), protected against moisture (kept in container tightly closed)

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Ltd., Fuellingsdorf, Switzerland
- Age at study initiation: 11 weeks
- Weight at study initiation: 181 to 223 g
- Housing: individually in makrolon type-3 cages with wire mesh top and sterilized standard softwood bedding
- Diet: pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba, Switzerland), ad libitum
- Water: community tap-water from Fuellingsdorf, ad libitum
- Acclimation period: minimum 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Test substance was weighed into a glass beaker and vehicle was added (w/w), using a homogenizer a homogenous solution was prepared, separate formulations were prepared for each concentration, homogeneity was maintainted by magnetic stirrer

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no.: 37899577

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples 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. 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. D. Flade (Harlan Laboratories Ltd., Itingen /Switzerland) and stored there at -20±5 °C until analysis.
The samples were analyzed by GC coupled to a flame ionisation detector following an analytical procedure provided by the sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analysed samples were not discarded without written consent from the study director.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: females were housed with sexually mature males in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period
- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation was observed
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal smear was sperm positive/copulation plug observed, defined as day 0 post coitum

Duration of treatment / exposure:

from day 6 post coitum until day 21 post coitum (1 day prior to caesarean section)

Frequency of treatment:

once daily, 7 days/week

Duration of test:

21 days post coitum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

22 mated females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Han Wistar rats, Harlan Laboratories Study C16981, using dose levels of 0, 100, 300 and 1000 mg/kg bw/day, resulting in no clinical findings or adverse effects on dams or embryo-fetal development up to and including 1000 mg/kg bw/day.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for viability/mortality and for clinical signs once daily during acclimatisation and up to day of necropsy

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: recorded daily from day 0 to day 21 post coitum

FOOD CONSUMPTION: Yes
- Time schedule for examinations: recorded at 3-day intervals: days 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, position of fetuses in the uterus and the number of corpora lutea, uteri of all females with live fetuses were weighed during necropsy on day 2 to enable calculation of corrected body weight gain

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: at least one half per litter
- Skeletal examinations: Yes: the remaining per litter
- Head examinations: Yes: at least one half per litter

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• Means and standard deviations of various data were calculated.
• The Dunnett-test [see References (C.W. Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Statist. Assoc. 50, pp. 1096-1121 (1955))] (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 [see References (R.G. Miller: Simultaneous Statistical Inference, Springer Verlag, New York (1981))] (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 [see References (R.A. Fisher: Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950))] was applied if the variables could be dichotomized without loss of information.

Historical control data:

historical control data was provided

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical symptoms related to the treatment with the test item were noted during the study at any dose level (see table 1). In the control group, 1 female had a hairless region on the left flank (observed from day 18 to 21 post coitum). This finding was incidental.

Mortality:

no mortality observed

Description (incidence):

All dams survived until the scheduled necropsy

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weight and mean body weight gain were not affected by treatment with the test item at any dose level (see table 3). The overall differences in mean body weight gain were by +49.5%, +45.7%, +47.1 and +46.4% in order of ascending dose levels (percentages refer to the alterations within the treatment period). Mean corrected body weight gain (corrected for the weight of the gravid uterus) was similar in all dose groups: 12.4%, 10.3%, 12.8% and 10.1% in order of ascending dose levels.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effects on mean food consumption were noted at any dose level (see table 2). The overall differences in food consumption during the treatment period were by +0.5%, +4.2% and -0.9% in order of ascending dose levels (percentages refer to the value of the control group).

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

Description (incidence and severity):

No findings were noted during macroscopical examination at any dose level.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No test item-related effects were observed at any dose level.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Incidental statistically significantly lower number of embryonic resorptions was observed at the dose level of 100 mg/kg. This effect was considered to be a result of biological variability.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Mean number of live fetuses was similar in all groups and was 13.1, 12.2, 12.1 and 12.4 in order of ascending dose levels.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

No test item-related effects on the relevant reproduction data (post implantation loss and number of live fetuses at termination) were observed at any dose level.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed in maternal animals.

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

No test item-related effects on mean weights of live fetuses were observed at any dose level. Slightly but statistically significantly higher mean body weights of live fetuses were observed at the dose levels of 100 and 1000 mg/kg bw/day. Mean fetal body weights calculated on an individual basis were 4.8 g in both groups compared to 4.7 g in the control group. Both values were in the range of historical control data (mean fetal body weight in control groups comprised values from 4.7 to 4.9 g) therefore this effect was considered not to be test item-related but a result of biological variability.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No effects on sex ratio of the fetuses were observed at any dose level. Proportions of male fetuses were 50.0%, 46.1%, 45.9% and 46.5% in order of ascending dose levels.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No test item-related abnormal findings were noted during external examination of the fetuses at any dose level (see table 4). A malrotaded hind limb was found in 1 fetus at the dose of 100 mg/kg bw/day and 2 fetuses at the dose of 300 mg/kg bw/day. Because of lack of the dose-correlation, this finding was considered to be incidental.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Bone and Cartilage Abnormalities and Variations:
During skeletal examination of fetuses, findings were noted in:
17% examined fetuses (in 55% litters) in the control group
27% examined fetuses (in 73% litters) at the dose level of 100 mg/kg bw/day
18% examined fetuses (in 68% litters) at the dose level of 300 mg/kg bw/day
20% examined fetuses (in 62% litters) at the dose level of 1000 mg/kg bw/day
The type and frequencies of the noted skeletal variations were similar in the groups receiving the test item and the control group and did not indicate any dose-dependency, therefore they were considered not to be test item-related. No test item-related abnormalities were observed. A supernumerary greater horn of hyoid arch was noted in 1 fetus in the control group. A malpositioned and/or shortened and fused costal cartilage was found in 1 fetus each at the dose levels of 100 mg/kg and 1000 mg/kg. This finding was considered to be incidental.
Bone Examination of Fetuses (Ossification Stage and Supernumerary Ribs): During bone examination of fetuses, findings were noted in:
16% examined fetuses (in 50% litters) in the control group
22% examined fetuses (in 68% litters) at the dose level of 100 mg/kg bw/day
17% examined fetuses (in 64% litters) at the dose level of 300 mg/kg bw/day
19% examined fetuses (in 57% litters) at the dose level of 1000 mg/kg bw/day
No test item-related effects on the ossification stage and supernumerary ribs were noted at the dose level of 100 mg/kg bw/day.

When compared to the control values statistically significantly lower numbers of non-ossified cervical and caudal vertebrae and incompletely ossified sternal bodies were noted at the dose level of 1000 mg/kg bw/day. Statistically significantly lower number of non-ossified digits and toes was noted in all groups treated with the test item. Most of these values were in the range of the historical control data and therefore these findings were considered not to be test item-related but a result of biological variability. Compared to the control values, a statistically significantly increased incidence of supernumerary rudimentary ribs was noted at the dose levels of 300 and 1000 mg/kg bw/day when calculated on an individual basis. This effect did not correlate with the dose levels; it was most pronounced at the dose level of 300 mg/kg bw/day. When calculated on a litter basis, statistically significant increase was noted only at the dose level of 300 mg/kg bw/day. The increased numbers of supernumerary ribs exceeded the historical control data and were considered to be test item-related. An increase of rudimentary supernumerary ribs indicate only minor and not adverse developmental disturbance as the rudimentary thoracolumbar supernumerary ribs are known to be transien transient [see References (N. Chernoff, J. M. Rogers: Supernumerary Ribs in Developmental Toxicity Bioassays and in Human Populations: Incidence and Biological Significance, J. Toxicol. Environ. Health. Part B, 7, pp. 437- 449 (2004))], therefore this effect was considered not to be adverse.

Cartilage Examination of Fetuses (Additional Variations): During cartilage examination of fetuses, findings were noted in:
2% examined fetuses (in 14% litters) in the control group
10% examined fetuses (in 41% litters) at the dose level of 100 mg/kg bw/day
2% examined fetuses (in 14% litters) at the dose level of 300 mg/kg bw/day
4% examined fetuses (in 19% litters) at the dose level of 1000 mg/kg bw/day
No test item-related effects on the cartilage development were noted at the dose level of 100 mg/kg bw/day. At the dose level of 1000 mg/kg bw/day, statistically significantly lower number of skull cartilaginous structures with small hole was observed when compared to the control value. This value remained in the range of the historical control data and was therefore considered not to be test item-related but a result of biological variability. Compared to the control value, an increased incidence of long or interrupted costal cartilages was noted at the dose levels of 300 and 1000 mg/kg bw/day when calculated on an individual basis. This effect did not correlate with the dose levels; it was most pronounced at the dose level of 300 mg/kg bw/day. When calculated on a litter basis, statistically significant increase was noted only at the dose level of 300 mg/kg bw/day. The increased numbers of long or interrupted costal cartilages exceeded the historical control data and were considered to be test item-related. Although long or interrupted costal cartilages are considered as permanent structural changes, they are minor and most unlikely to adversely affect further development and postnatal live of the animal therefore this effect was considered not to be adverse.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

During visceral examination of fetuses, findings were noted in:
34% examined fetuses (in 100% litters) in the control group
36% examined fetuses (in 91% litters) at the dose level of 100 mg/kg bw/day
31% examined fetuses (in 86% litters) at the dose level of 300 mg/kg bw/day
35% examined fetuses (in 100% litters) at the dose level of 1000 mg/kg bw/day
The type and frequencies of the noted variations were similar in the groups receiving the test item and the control group and did not indicate any dose-dependency, therefore these findings were considered not to be test item-related. All found abnormalities (situs invertus noted in 2 fetuses/2 litters, small pituitary noted in 1 fetus and interventricular septal defect of the heart noted in 1 fetus) were noted in the control group.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: No adverse developmental effects observed.

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Clinical Signs or Observations (Dams)

	0 mg/kg	100 mg/kg	300 mg/kg	1000 mg/kg
Number of females examined	22	22	22	22
Flank, left, hairless region	1	0	0	0
No clinical signs or observations	21	22	22	22

Table 2: Differences in Mean Food Consumption (g/animal/day) of dams

	Days post coitum
	0 -3	3 -6	6 -9	9 -12	12 -15	15 -18	18 -21	6 -21**
mg/kg	g (%)*	g (%)*	g (%)*	g (%)*	g (%)*	g (%)*	g (%)*	g (%)*
0	18.7	21.2	19.2	21.0	22.2	23.3	21.8	21.5
100	19.0 +1.6	21.2 +0.5	19.5 +1.6	21.7 +3.3	22.3 +0.5	23.3 +0.0	21.0 -3.4	21.6 +0.5
300	19.1 +2.1	21.2 +0.5	20.0 +4.2	22.2 +5.7	23.0 +3.6	24.1 +3.4	22.7 +4.1	22.4 +4.2
1000	18.5 -1.1	20.3 -3.8	19.0 -1.0	20.6 -1.9	21.1 -5.0	24.2 +4.3	21.4 -1.8	21.3 -0.9

*Percentages refer to the value of group I

**The calculation of food consumption during the treatment period started on day 6 post coitum (immediately prior to the first administration) and ended on day 21 post coitum (approximately 24 hours after the last administration).

Table 3: Differences in Mean Body Weight Gain of dams

	Days post coitum								corrected body
	0 -3	3 -6	6 -9	9 -12	12 -15	15 -18	18 -21	6 -21	weight gain (%)#
	g (%)*	g (%)*	g (%)*	g (%)*	g (%)*	g (%)*	g (%)*	g (%)*
0	12 +6.1	12 +5.7	11 +5.0	16 +6.9	17 +6.8	33 +12.4	33 +11.0	110 +49.5	12.5
100	12 +6.0	12 +5.7	11 +4.9	16 +6.8	16 +6.4	31 +11.7	28 +9.4	102 +45.7	10.3
300	12 +6.0	12 +5.6	11 +4.9	17 +7.2	17 +6.7	31 +11.5	30 +10.0	106 +47.1	12.8
1000	13 +6.5	12 +5.7	9 +4.0	18 +7.7	16 +6.4	31 +11.6	30 +10.1	104 +46.6	10.1

* Alteration within the respective period.

** The calculations of body weight gain during the treatment period started on day 6 post coitum (immediately prior to the first administration) and ended on day 21 post coitum (approximately 24 hours after the last administration).

# Body weight gain on day 21 post coitum corrected for uterus weight.

Table 4: External Examination Fetuses

mg/kg	No. of fetuses examined	Type of abnormal findings	Litter No.	Fetus No./sex
0	288	No findings
100	269	Hind limb right, malrotated	33	585/F
300	266	Hind limb right, malrotated	52 61	440/F 924/F
1000	260	No findings

 

Applicant's summary and conclusion

Conclusions:

In the prenatal developmental toxicity study in rats, conducted according to OECD TG 414 and in compliance with GLP, the NOAEL for maternal and developmental effects was concluded to be ≥1000 mg/kg bw/day based on no adverse systemic or developmental effects. An increased incidence of supernumerary rudimentary ribs and long or interrupted costal cartilages observed at the dose levels of 300 and 1000 mg/kg bw/day indicate a treatment-related minor disturbance in the development of the axial skeleton at these dose levels. Both types of variations belong to the most common seen in the control groups. They are of minor nature and not likely to affect further development and postnatal live of the animal. Therefore the increase of their incidence was considered not to be adverse.