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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24.01.2008 to 02.03.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OPPTS 870.3650
Qualifier:
according to
Guideline:
other: OECD 422
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc, Portage.
- Age at study initiation: 9 weeks minimum
- Weight at study initiation: Males: 210.1 to 254.6 g ; Females: 159.6 to 201.7 g
- Fasting period before study: No
- Housing: Individually in suspended wire-mesh cages (except during cohabitation when female was put into male home cage, and pregnant animals were moved into shoebox cages).
- Diet: Ad libitum except during FOB and motor activity assessment.
- Water: Ad libitum.
- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 21.3
- Humidity (%): 39 - 60 %
- Air changes (per hr): 13.5
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28.01.2008 To: 07.06.2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test substance and vehicle were administered to each dose group at an equal dose volume (2ml/kg bw) based on the most recent body weight. Dosing solutions were individually prepared. Based on stability data, dosing solutions were prepared twice during the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the physical and chemical properties of the test substance, dried and deacidified peanut oil was considered to be the most appropriate vehicle for oral administration.
- Concentration in vehicle: Dependent on dose
- Lot/batch no. (if required): 080408 and 090408
- Purity: No data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing solution analyses were performed by a GC/FID method to verify concentration, stability, and homogeneity of the test substance in the vehicle. Dosing solutions were analysed prior to study inititation to ensure stability and homogeneity. Stability was determined on study days 0, 4, 15, 21 and 32. Dosing solutions were also analysed for concentration verification following each dose solution preparation.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Until evidence of mating
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually in a shoe box cage
Duration of treatment / exposure:
Males: 28 days
Toxicity phase females: 29 days
Reproductive phase females: two weeks prior to mating, then through pregnancy, and to post-partum day 3.
Frequency of treatment:
Daily, seven days per week
Duration of test:
14 April 2008 to 07 June 2008
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:
Ten
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on range-finding study

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed at least twice daily for mortality, morbidity and moribundity. General clinical observations were made at least once per day. Clinical observations were not performed on the day of the detailed clinical observations.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals received a detailed physical examination once before the first dose and weekly thereafter. Examinations included but were not limited to 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, stereotypies, difficult or prolonged parturition or bizarre behaviour were recorded.

From day 20 after evidence of mating, pregnant animals were checked at least three times daily for evidence of parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were determined beginning with randomisation into test groups, on the first day of dosing, at least weekly thereafter and the day of necropsy. During gestation the pregnant animals were weighed on gestation days 0, 7, 14 and 20, within 24 hours after parturition and on day 4 post-partum or upon terminal euthanasia of the dam. Litter weights were measured within 24 hours of parturition and on day 4 post-partum or upon terminal euthanasia of the dams.

FOOD CONSUMPTION:
Individual animal food consumption was recorded at least weekly on an individual animal basis for the periods listed below.
Male rats: Two week pre-mating only. Feeder weights were taken on days 1, 8 and 15.
Female toxicity phase: Day 1 of dosing to necropsy. Feeder weights were taken on days 1, 8, 15, 22 and the day prior to necropsy.
Reproductive phase females: Two week pre-mating period, gestation and post-partum. Feeder weights were taken on days 1, 8, 15 and on gestation days 0, 7, 14, 20 and on day 0 and 4 post-partum.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No
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
The uteri of females with positive evidence of mating that failed to produce a litter were stained to enable counting of possible reabsorbed implant sites.
Fetal examinations:
Pups were examined daily for survival, external abnormalities and clinical signs during the lactation period. Each litter was examined as soon as possible after delivery (within 24 hours) to determine the number and sex of the pups, the number of pups alive, number of pups dead, runts and the presence of any gross abnormalities. All pups were counted, weighed, sexed and the sex ratio calculated. Litter weights were taken within 24 hours of completion of parturition and on post-partum day 4 or earlier if the dam was euthanised prior to post-partum day 4. Developmental parameters evaluated included mean litter size, offspring survival, sex ratio and offspring body weight gain.
Statistics:
Mean values of the following were evaluated appropriately: body weights and body weight changes, food consumption, haematology and serum chemistry, terminal organ weights and body weight ratios, gestation length, litter size, live litter size, litter weight, ratio of live births/litter size, litter sex ratio, implantation sites, corpora lutea, mating and fertility indices and neurotoxicological parameters.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All reproductive group females in Group 1-3 survived until scheduled necropsy. One nongravid female with no evidence of mating was euthanised 25 days after the last day of mating. There was a dose-related increase in soiling around the nose and chin and generalised soiling around the muzzle in Group 4 reproductive toxicity females. These clinical findings were statistically significant (p<0.01) in reproductive toxicity group females. Three females with evidence of mating and positive evidence of pregnancy failed to deliver and were euthanised on gestation day 26, 25 and 25. Due to the severity of the clinical signs for Group 4 reproductivity phase females were euthanised between study days 24 and 43.

There were statistically significant changes in the incidence of neurological clinical signs only in Group 4 reproductive group females compared to controls. Hind limb dragging (5/10) and uncoordinated gait (5/10) occurred in Group 4 reproductive group females. None of these neurological signs were noted until study day 32, with the exception of one animal that exhibited uncoordinated gait on day 21 and dragging hind limbs by day 25. These effects were also associated with a generalised decrease in overall activity (p<0.01) in 5/9 affected females. Rapid respiration occurred in 3/10 animals (p<0.05) beginning on the day prior to and/or on the day these animals were euthanised in extremis.
Mortality:
no mortality observed
Description (incidence):
One nongravid female demonstrating no evidence of copulation was euthanised 25 days after the last day of mating procedure. Three females with evidence of copulation and positive evidence of pregnancy failed to deliver and were euthanised on gestation day 26, 25, and 25, respectively. Due to the severity of the clinical signs, three females in the following Group 4 reproductive group females were euthanized prior to scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights and body weight gains across all reproductive group female dose levels were comparable to control throughout the premating period. A 19.7% decrease in gestation week 2 mean body weight gains occurred in Group 3 reproductive group females compared to the controls (p<0.05) but were similar to control values throughout the remainder of the study. Mean body weights of Group 4 reproductive group females were statistically decreased compared to controls throughout gestation (p<0.01). Associated body weight gains in this group were also decreased by 45.9% (p<0.01) by the end of gestation week 1 and 29.1% (p<0.01) by the end of the gestation period. There was also a significant decrease in Group 4 reproductive female mean body weights throughout the post-partum period compared to controls (p<0.01). Although Group 4 reproductive group females exhibited a 73.9% decrease in mean body weight gains between post-partum day 0 to 4, this did not attain significance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease in average daily food consumption of the Group 4 reproductive group females occurred throughout gestation (17.8%) and the post-partum period until terminal sacrifice (48.9%) (p<0.01) compared to controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Organ weight findings including organ / body weight ratios:
not examined
Description (incidence and severity):
Organs were not weighed in maternal animals.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The Group 4 reproductive phase females exhibited various gross findings that included a generalised thinness of the body in 8/10 animals, decreased thymus size in 6/10 animals and spleen size in 7/10 animals. Five of these animals also had notable decreases in hind leg skeletal muscle mass. These macroscopic findings are consistent with the clinical observations of uncoordinated gait and/or hindlimb dragging and microscopic neurological findings that occurred in all of these animals. In addition to the other gross findings, two animals had decreased ingesta in all or parts of the intestinal tract. No gross findings occurred at the lower dose levels.

The uteri of reproductive group females with positive evidence of mating that failed to produce a litter were stained to enable counting of possible reabsorbed implant sites. No implantation sites were found in these animals.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Histopathological findings in the liver were associated with an increase in mean absolute and relative liver weights in the Group 4 female toxicity group and are consistent with common adaptive changes that occur in the liver.

Epithelial hyperplasia of the urinary bladder was observed in 7/10 Group 4 reproductive group females and not in the controls (p<0.01). Although two reproductive group females in Group 2 had this finding in the urinary bladder, there was no dose-related effect as this finding did not occur in Group 3.

A statistically significant increase in the incidence of mild to moderate atrophy of the spleen (p<0.02) and thymus (p<0.02) was identified in 5/10 Group 4 reproductive group females and not in controls. These findings are consistent with the gross findings of decreased spleen and small thymus in several of the reproductive group females.

The main finding in the CNS was white matter degeneration of the brain and spinal cord in Group 4 reproductive group females. The cerebellum and medulla appeared to be the most prominently affected areas of the brain. In Group 4 8/10 reproductive toxicity females there was an increase in the incidence of minimal white matter degeneration (vacuolation) of the brain, which attained statistical significance (p<0.01). Minimal to marked demyelination of the spinal cord also occurred in 9/10 Group 4 reproductive group females and no controls (p<0.01).

In the peripheral nerves examined, the sciatic and tibial nerves, there was a statistically significant increase in the incidence of minimal to severe demyelination/degeneration in 8/10 (sciatic) (p<0.01) and 9/9 (tibial) (p<0.01) Group 4 reproductive group animals and not in controls. Affected animals tended to show the change in multiple sites unless only the spinal cord was involved.

Based on clinical signs in the Group 4 reproductive group females, two skeletal muscle sections of the hind limb were evaluated (the adductor muscles of the inner thigh and the gastrocnemius (calf) muscle). There was a statistically significant increase in Group 4 reproductive group female adductor muscle degeneration (10/10) compared to controls (4/10) (p<0.01). There was a trend toward significance in the incidence of degeneration of the gastrocnemius muscle in Group 4 reproductive group females (9/10) compared to controls (5/10), and statistically significant increase in the severity of degeneration compared with controls. Animals affected with neurological findings also showed gross atrophy, diffuse decreased muscle fibre size, fibre fragmentation, increased density of myobibre nuclei and focal inflammation around necrotic fibres. The animals with these skeletal muscle findings also had notable decreases in hind leg skeletal muscle mass. These findings are consistent with denervation atrophy and considered secondary to the nerve changes. Although the Group 4 reproductive group animals that demonstrated clinical signs also exhibited microscopic evidence of neuronal degeneration, the severity of the neurological lesions in the nerves did not necessarily correlate with the severity of neurological clinical signs observed in these animals.

These data indicate that the severity of the clinical and microscopic tissue neuromuscular effects is a likely reflection of duration of exposure rather than an effect specific to the reproductive status of the animal, as the toxicity group females were only dosed for 29 days compared to up to 45 days for the reproductive group females. There were no neurological findings in any of the lower dose groups.

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Mean percent pre-implantation and post-natal loss were unaffected by treatment. Mean percent post-implantation and post-natal loss were unaffected by treatment.
Mean percent post-implantation loss, however, was statistically increased in Group 4 (39.9%) compared to controls (5.7%) (p<0.01).
Post-implantation loss values include loss of fetuses and/or pups that occurred between implantation and first litter check.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Abnormalities:
effects observed, treatment-related
Localisation:
other: urinary bladder

Results (fetuses)

Fetal body weight changes:
not specified
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The number of live male and female pups/dam at first litter check (PND 0) in Group 4 were statistically significantly decreased compared to controls (p<0.01), resulting in a 39.3% decrease (p<0.01) in percent viability pups/dam at first litter check compared to controls. Pup viability was unaffected at all lower doses. On PND 4, several dams in Group 4 had been euthanised due to severity of various clinical signs and/or difficulty during labour. Only four dams continued through PND 4. Of these litters, the total viable pups on PND 4 was decreased compared to controls, resulting in a 25.2% decrease in percent viability of pups/dam on PND 4 compared to controls (p<0.01). This significant decrease was due to a single dam that had a 14.3% post-natal loss of offspring. The remaining dams had no post-natal loss of pups between PND 0-4.
Changes in sex ratio:
effects observed, non-treatment-related
Description (incidence and severity):
There was a statistically significant increase in the mean ratio of male to female pups/litter on post natal day (PND) 0 in Group 2 and in the number of live male pups/litter on PND 4 after adjusting for the mean litter size. These effects did not occur at any other dose levels and are likely not attributable to the test substance.
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Total litter size was unaffected. On PND 0, mean litter weights and average pup body weights were similar to controls. By PND 4, weights in Group 4 were decreased compared to control weights (p<0.02). Average pup weights on PND 4 of the surviving pups were decreased by 25.2% and average pup weight gains of the surviving pups were decreased by 60% compared to controls (p<0.01). The loss of pup body weights in the Group 4 surviving pups resulted in a 50.1% decrease in mean live litter weights compared to controls.
All of the dams with complications during parturition and/or changes in litter parameters exhibited various degrees of hind limb weakness that likely contributed to the difficulty in delivering the pups and consequent decreases in average pup weights and pup body weight gains and pup viability on PND 0 through to PND 4.
External malformations:
not specified
Skeletal malformations:
not specified
Visceral malformations:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Litter observations indicated that there was one runt in Group 3. Gross observations in Group 4 litters included milk not present in the stomach of several pups in 6/7 litters of dams and two dams that had several pups that were very thin compared to controls. There were no gross findings at any lower dose levels.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
Remarks on result:
other: The developmental effects only occurred at the 1000 mg/kg bw/day dose level in association with marked maternal toxicity. As such it is not possible to determine with confidence if the 1000 mg/kg bw/day dose level represents the NOAEL.
Remarks:
Therefore, the developmental toxicity NOAEL is considered to be 300 mg/kg bw/day.

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
not specified

Any other information on results incl. tables

See Section 7.8.1, Table 3, for full reproductive and development results.

Applicant's summary and conclusion

Conclusions:
In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test conducted according to OECD 422 and in compliance with GLP (reliability score 1) on triethoxy(octyl)silane, the developmental effects (reduced postnatal pup viability and body weights) only occurred at the 1000 mg/kg bw/day dose level in association with marked maternal toxicity. As such it is not possible to determine with confidence if the 1000 mg/kg bw/day dose level represents the NOAEL. Therefore, the developmental toxicity NOAEL is considered to be 300 mg/kg bw/day. The NOAELs for maternal toxicity and teratogenicity were 300 mg/kg bw/day and ≥1000 mg/kg bw/day, respectively.