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Developmental toxicity / teratogenicity

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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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
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 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 ahomogenous solution was prepared, separate formulations were prepared for each concentration, homogeneity was maintainted by magnetic stirrer

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly

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:
daily
Duration of test:
21 days post coitum
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22
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/day, resulting in no clinical findings or adverse effects on dams or embryo-fetal development up to and including 1000 mg/kg body weight/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 AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- All dams survived until the scheduled necropsy. No clinical symptoms related to the treatment with the test item were noted during the study at any dose level (see table1). 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.

- 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).

-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.

- 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. 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.
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.

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

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- 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 and 2 fetuses at the dose of 300 mg/kg. Because of lack of the dose-correlation, this finding was considered to be incidental.

- 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.

- 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. 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.

- 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
31% examined fetuses (in 86% litters) at the dose level of 300 mg/kg
35% examined fetuses (in 100% litters) at the dose level of 1000 mg/kg
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.

- 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
18% examined fetuses (in 68% litters) at the dose level of 300 mg/kg
20% examined fetuses (in 62% litters) at the dose level of 1000 mg/kg
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.

- 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
17% examined fetuses (in 64% litters) at the dose level of 300 mg/kg
19% examined fetuses (in 57% litters) at the dose level of 1000 mg/kg
No test item-related effects on the ossification stage and supernumerary ribs were noted at the dose level of 100 mg/kg. 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. 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 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. When calculated on a litter basis, statistically significant increase was noted only at the dose level of 300 mg/kg. The increased numbers of supernumerary ribs exceeded the historical control data and were considered to be test itemrelated. An increase of rudimentary supernumerary ribs indicate only minor and not adverse developmental disturbance as the rudimentary thoracolumbar supernumerary ribs are known to be 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.

- 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
2% examined fetuses (in 14% litters) at the dose level of 300 mg/kg
4% examined fetuses (in 19% litters) at the dose level of 1000 mg/kg
No test item-related effects on the cartilage development were noted at the dose level of 100 mg/kg. At the dose level of 1000 mg/kg, 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 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. When calculated on a litter basis, statistically significant increase was noted only at the dose level of 300 mg/kg. 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.

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: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

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:
Under the conditions described for this study, NOEL (No Observed Effect Level) was considered to be 1000 mg/kg body weight/day for pregnant rat. No adverse effects on fetal development were observed at any dose levels, therefore triethoxy(2,4,4-trimethylpentyl)silane was considered not to reveal teratogenic potential up to and including a dose of 1000 mg/kg which is the NOAEL (No Observed Adverse Effect Level).