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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30.01.2013 - 26.06.2013
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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Economic Trade and Industry (METI), Ministry of Health, Labour and Welfare (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
Constituent 2
Reference substance name:
3-(trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate
IUPAC Name:
3-(trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate
Constituent 3
Reference substance name:
2,4-Hexadienoic acid, 3-(trimethoxysilyl)propyl ester, (2E,4E)-
IUPAC Name:
2,4-Hexadienoic acid, 3-(trimethoxysilyl)propyl ester, (2E,4E)-
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: (P) 10 weeks
- Weight at study initiation: (P) Males: 305 - 353 g; Females: 185 - 247 g
- Fasting period before study:
- Housing: individually in Makrolon type-3 cages with wire mesh tops and standard softwood bedding
- Diet: ad libitum
- 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 in dried and deacidified corn oil
- Lot/batch no. (if required): 492194511
Details on mating procedure:
- 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
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, homogneity 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)
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.
Details on study schedule:
FEMALES:

Treatment start: Day 1 of pre-pairing = day 1 of treatment
Pre-pairing: 14 days
Pairing: 1-4 days
Gestation: ca. 21 days
Treatment ends: On day 4 post partum; females not giving birth on day 24 post coitum
Blood and urine sampling: None
Necropsy: Dams on day 5 post partum, pups on day 4 post partum; Females not giving birth on day 25 post coitum


MALES:

Treatment start: Day 1 of pre-pairing (day 1 of treatment)
Pre-pairing: 14 days
Pairing: 1-4 days
Treatment ends: On the day before sacrifice
Blood and urine sampling: On the day of necropsy
Necropsy: After treatment for 28 days
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
30 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
300 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
10/Sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
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 recovery period)

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily throughout acclimatization, treatment and recovery period. Viability and mortality were checked twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity, changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes or bizarre behaviour were checked weekly in all animals. In Allocation A females the parameters were checked once prior to treatment start, weekly during pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period and on day 3 post partum.

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

OTHER:
FOB testing was conducted during acclimatization (Allocation A males, B females and C males and females), last treatment week (allocation A males and B females) and recovery week 2 (allocation C males and females).This included cage side observations, hand held observations, open field observations, reflexes, and measurements of counts (hind-limb/fore-limb grip strength, body temperature and landing footsplay and locomotor activity.

FOOD CONSUMPTION:
- Food consumption was recorded weekly during treatment and recovery periods for A males, B females and C males and females. No food consumption was recorded for A males during the pairing period. Food consumption for allocation A females was recorded weekly during pre-pairing, gestation days 0-7, 7-10, 10-14, 14-18, 18-21, and days 1-4 post partum. No food consumption was recorded during pairing period.

Blood and urine samples for clinical laboratory investigations were collected after treatment for 28 days for allocation A males, treatment for 29 days for allocation B females, treatment for 29 days followed by 2 weeks of recovery period for allocation C males and females. Blood samples were collected following fasting period of 18 hours and under light isoflurane anesthesia. Urine was collected during the 18-hour fasting period into a specimen vial, using a metabolism cage.
Haematology: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, total leukocyte count, differential leukocyte count (neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells), platelet count, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, reticulocyte count, reticulocyte maturity index (low, medium, high fluorescence). Methemoglobin, Heinz bodies, coagulation parameters.

Clinical biochemistry parameters: see parameters examined in table 1
Urinanalysis: see parameters examined in table 1
Oestrous cyclicity (parental animals):
Estrous cycle was determined by histopathological examination.
Sperm parameters (parental animals):
Qualitative assessment of the male reproductive organs was performed. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Postmortem examinations (parental animals):
SACRIFICE
Allocation A animals (Tox/repro males, repro females): Males were sacrificed after treatment for 28 days. The dams were sacrificed on day 5 post partum. Females not giving birth were sacrificed on day 25 post coitum.

Allocation B animals (Tox females): All allocation B animals (satellite females for toxicity testing) were sacrificed after treatment for 29 days.

Allocation C (Recovery) animals: Males and females were sacrificed 2 weeks after the last day of test item administration.

GROSS NECROPSY
All animals were examined macroscopically for any structural changes. Blood samples were taken from all adult animals except reproductive females (Group A), for possible thyroid hormone analysis. Special attention was directed at the organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively. Relative organ weights were calculated on the basis of body weight and brain weight. All organ and tissue samples which were collected (Table 1) were processed, embedded, cut and stained with haematoxylin and eosin. Additionally, section of testes and epididymides were also stained in PAS-haematoxylin.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on Day 4 post partum

GROSS NECROPSY
All animals were examined macroscopically for any structural changes.
Statistics:
Mean and median values and SD of various data included in the report, The Dunnett test, The Steel test, Fisher's exact test.
Reproductive indices:
Fertility indices, mean precoital time, post-implantation losses, mean litter size
Offspring viability indices:
Pup sex ratios, viability indices.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation and bedding in mouth of group 4 males after treatment. Considered to be test item-related but not toxicologically significant. One female from group 4 and another one from group 3 showed decreased activity, prostate position, ruffled fur, scabs at both eyes and tachypnea. These effects were considered related to gavage errors and were not considered to have toxicological significance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Three unscheduled deaths occurred, one control animal and two test-substance treated animals. The mortalities observed in animals dosed with 300 and 1000 mg/kg bw/day were considered related to gavage errors and were not considered to have toxicological significance.
Body weight and weight changes:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The observed effects were not toxicologically significant.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no test material related mortalities. No test item related clinical signs of toxicological significance were observed at the cage side or detailed examinations.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weights and body weight gain were considered to be unaffected by treatment with the test item. There were no test item related effects on food consumption.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No test item related effects were observed in estrous cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test item related effects were observed in mating performance, fertility, corpora lutea count and implantation rate, post-implantation loss and duration of gestation. The gestation indices (number of females with living pups as a percentage of females pregnant) were 90%, 87.5%, 88.9%, 87.5%, respectively at 0, 30, 300 and 1000 mg/kg/day). Litter sizes (at first litter check) and post-natal loss (days 0-4 post partum) were similar in control and test item - treated animals

ORGAN WEIGHTS (PARENTAL ANIMALS)
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 PATHOLOGY (PARENTAL ANIMALS)
There were no test item related macroscopic or microscopic findings.

OTHER FINDINGS (PARENTAL ANIMALS)
No test item related abnormalities were observed during treatment week 4 and during recovery. In addition, there were no test item-related effects on fore- and hindlimb grip strength, body temperature, landing foot splay or locomotor activity.

There was no test item related effect on food consumption.

There were no test item related changes in haematology and urine parameters. The following, test item related changes in clinical chemistry parameters were reported: decreased creatinine levels in males and females at all dose levels, increased cholesterol and phospholipid levels in females with 1000 mg/kg/day, increased plasma sodium, potassium, chloride, calcium and phosphorus in males treated with 300 and/or 1000 mg/kg/day and increased calcium levels in females treated with 30, 300 and 1000 mg/kg/day. Increased total protein and albumin concentrations in males treated with 300 or 1000 mg/kg/day. The sodium level was still increased in males after recovery. All test item related changes in clinical chemistry were considered minor and non-adverse.

MATING PERFORMANCE AND FERTILITY
No test item-related effects on mating performance and fertility were observed. All females were mated during the first pairing period. All females in the control group were pregnant. No test item-related effects on the number of corpora lutea were observed. The mean duration of gestation was unaffected by exposure to the test item. There were no test item related effects on implantation rate or post implantation loss. There were no test item related effects on litter size. There was no test-item related effect on postnatal loss.
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 reproduction/developmental toxicity).

Effect levels (P0)

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 were attributed to test material administration.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
No test-item related findings were noted for pups during first litter check and lactation. Sex ratios of pups were unaffected by exposure to the test item.

CLINICAL SIGNS (OFFSPRING)
No test-item related findings were noted for pups during first litter checks and lactation.

BODY WEIGHT (OFFSPRING)
There were no test item related effects on pup body weights and body weight gain.

GROSS PATHOLOGY (OFFSPRING)
No test item-related abnormalities were observed at necropsy of pups.


OTHER FINDINGS (OFFSPRING)
Sex ratio of pups was unaffected by exposure to the test item.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test material related effects were observed.

Overall reproductive toxicity

Reproductive 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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