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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-01-30 to 2013-06-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
yes
Remarks:
The deviation according to the latest amendmend of the guideline is that the maternal animals were sacrificed on day 4 post partum.
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Economic Trade and Industry (METI), Ministry of Health, Labour and Welfa re (MHLW) and Ministry of Environment (MOE) Guidelines of 31 March 2011
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(trimethoxysilyl)propyl (2E,4E)-hexa-2,4-dienoate
EC Number:
642-902-2
Cas Number:
163802-53-7
Molecular formula:
C12H22O5Si
IUPAC Name:
3-(trimethoxysilyl)propyl (2E,4E)-hexa-2,4-dienoate
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 10 weeks
- Weight at study initiation: 305-353 g (males) and 185-247 g (females)
- Fasting period before study:
- Housing: individually in Makrolon type-3 cages with wire mesh tops and standard softwood bedding
- Diet: Pelleted standard rodent maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 8 days after health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 51
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

62 males and 102 females were obtained from the breeder. The surplus (reserve) animals were sacrificed after the commencement of treatment. This was documented in the raw data.
Group 1: 20 males and 30 females
Group 2: 10 males and 20 females
Group 3: 10 males and 20 females
Group 4: 20 males and 30 females

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Dried and deacidified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose concentrations:
Group 1: 0 mg/mL
Group 2: 6 mg/mL
Group 3: 60 mg/mL
Group 4: 200 mg/mL

Dose volume: 5 mL/kg body weight

VEHICLE
- Justification for use and choice of vehicle: The test substance was stable and soluble in dried and deacidified corn oil
- Lot/batch no. (if required): 492194511
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The dose formulations were analyzed using a GC/FID method provided by the Sponsor. Concentration, homogeneity and stability (after 8 days) of dose formulations were determined in samples (ca.1g sample volume) taken after experimental start. Concentration of the dose formulations were also determined in samples of the formulations prepared during the end of the treatment period.
The following acceptance criteria were applied to the analytical results:
1. Concentration: The achieved content should be +/-10% of the nominal content
2. Homogeneity: The individual recoveries should not deviate more than +/-15% from the corresponding
mean
3. Stability: The results obtained from storage stability samples should not deviate more than 10% from
time-zero reference (content or mean of homogeneity samples)
Details on mating procedure:
- Impregnation procedure:cohoused
- If cohoused: females were housed with sexually mature males until evidence of copulation was observed
- M/F ratio per cage: 1:1
- Length of cohabitation: Until evidence of copulation was confirmed.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
Duration of treatment / exposure:
28 days (Allocation A males)
29 days (Allocation B and allocation C animals)
ca. 6 weeks (Allocation A females)
Frequency of treatment:
Once, daily
Duration of test:
Until 4 days post partum
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
Group 1: 20 males and 30 females
Group 2: 10 males and 20 females
Group 3: 10 males and 20 females
Group 4: 20 males and 30 females

GROUP 1 (control)
Males
A: 1 - 10
C: 11 - 20
Females
A: 61 - 70
B: 71 - 80
C: 81 - 90
GROUP 2 (30 mg/kg bw/day)
Males
A: 21 - 30
Females
A: 91 - 100
B: 101 - 110
GROUP 3 (300 mg/kg bw/day)
Males
A: 31 - 40
Females
A: 111 - 120
B: 121 - 130
GROUP 4 (1000 mg/kg bw/day)
A: 41 - 50
C: 51 - 60
Females
A: 131 - 140
B: 141 - 150
C: 151 - 160
A= Animals that were paired for mating; the males were used for both reproduction and toxicity testing (treatment for 28 days); the females were used for reproduction testing
B= Satellite females used for toxicity testing (no mating; treatment for 29 days)
C= Recovery animals (no mating; treatment for 29 days followed by a 14 days recover)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on a previous non-GLP 14-day dose range-finding toxicity study in Sprague Dawley rats, using dose levels of 150, 400 and 1000 mg/kg bw/day. No adverse test item-related effects were observed.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily during the acclimatization period, treatment (within 3 hours post-dose) and recovery periods
- Cage side observations checked included: viability, mortality and behavioral abnormalities changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity, changes in gait, posture and response to handing as well as the presence of clonic and tonic movements, stereotypes or bizarre behavior.

DETAILED CLINICAL OBSERVATIONS: Yes; changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity, changes in gait, posture and response to handing as well as the presence of clonic and tonic movements, stereotypes or bizarre behavior.
- Time schedule: weekly starting from the treatment period on all allocation A males, allocation B females and recovery (allocation C) animals. Detailed clinical observations were also performed on allocation A females once prior to treatment start, weekly during the pre-paiting and pairing periods, on days 0, 6, 13 and 20 of the gestation period, and on day 3 post partum.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded during acclimatization, on day 1 of the ttreatment phase and then daily until the end of treatment. During recovery body weights were recorded on day 1, weekly thereafter and at termination

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 5 post partum; females not giving birth were sacrificed on day 25 post coitum
- Organs examined: see table 1

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: No data
- Number of late resorptions: No data
Fetal examinations:
- External examinations: Yes, all pups
- Gross anomalies: Yes, all pups
- The sex ratio of the pups was recorded
- Pups were weighed individually on days 0, 1 and 4 post partum
Statistics:
Mean and median values and SD of various data included in the report, The Dunnett test, The Steel test, Fisher's exact test.
Indices:
Fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios, viability indices

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical signs.
Decreased activity, prostrate position, ruffled fur, scabs at both eyes and tachypnea were observed prior to death in two reproduction test females that were sacrificed prior to scheduled death. These clinical signs observed were considered to be related to a gavage error. No other clinical signs were noted in any of the test animals.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two reproduction testing females from allocation group A and one control female died during the study, prior to scheduled death. Two of the animals allocation group A animalsl) exhibited moribund condition prior to death, which was concluded to be related to the gavage treatment. The death of the control animal could not be established.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changed in haematology parameters.
Methemoglobin levels were increase in males and females treated with 30 or 300 mg/kg bw/day and in females treated with 1000 mg/kg bw/day after 4 weeks of treatment. After recovery, methemoblobin levels of females treated with 1000 mg/kg bw/day were slightly lower than in the control group. Slight decrease in mean relative number of large unstained cells in females of 1000 mg/kg bw/day group after recovery. These differences were considered to be incidential since they were slight and contradictory in males and females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Changes in creatinine, cholesterol, phospholipids and calcium levels were observed in group 4 (1000 mg/kg bw/day) females after 4 weeks of treatment. All test item-related changes were considered minor. The values were within the range of the historical control data and/or they differed slightly from values obtained from control animals after the treatment or recovery period.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
In group 2 females (30 mg/kg bw/day) the protein concentration was significally lower to the control group values after the treatment. This effect was considered to be incidential as there was no dose-response relationship.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Increased mean absolute and relative kidney weights were observed in males and females treated at all doses, and decreased absolute uterine weights were observed at 1000 mg/kg/day. The changes were not considered adverse, since there were no corresponding microscopic findings and they were reversible.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item related macroscopic or microscopic findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related findings at histopathology.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
not examined
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
effects observed, treatment-related
Description (incidence and severity):
One female treated with 1000 mg/kg bw/day and one female treated with 30 mg/kg bw/day delivered only dead pups.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined

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

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
Dead pups were observed at first litter check: 1/16 pups of a control female, 3/15 pups of a 300 mg/kg bw/day female and 10/10 pups of a 1000 mg/kg bw/day female. This effect was considered to be incidential and not treatment-related.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights of pups in groups 3 and 4 (300 and 1000 mg/kg bw/day) were slightly lower when compared to the control group. However, the differences were not statistically significant and were considered to be incidential and not related to the treatment.
Changes in postnatal survival:
effects observed, non-treatment-related
Description (incidence and severity):
4 pups from control group, 1 pup from group 2 (30 mg/kg bw/day), 10 pups from group 3 (300 mg/kg bw/day) and 3 pups in group 4 (1000 mg/kg bw/day) died during days 0-4 post partum. These effects were considered to be incidential since there was no dose-response relationship.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Lack of milk in the stomach was observed for pups from groups 1, 3 and 4. No test-item related findings were noted.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not examined

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 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

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, 1000 mg/kg/day (the highest dose level tested) is the NOAEL (no-observed-adverse-effect-level) for general toxicity and the NOEL (no-observed-effect level) for reproductio or developmental toxicity).

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