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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:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out in accordance with an appropriate OECD test guideline and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
ANIMAL RECEIPT AND ACCLIMATION
One hundred female and fifty male, sexually mature Crl:CD®(SD)IGS BR rats were received from Charles River Laboratories, Inc., Raleigh, North Carolina on April 27, 2004. The animals were 57 days old upon receipt. Each animal was examined by a qualified technician on the day of receipt, sexed and weighed 1 day after receipt. Each rat was uniquely identified by a Monel® metal eartag displaying the animal number and housed for 15 days for acclimation purposes. During the acclimation period, the rats were observed twice daily for general changes in appearance or behavior. The animals were allowed a pretreatment week during which they did not receive the test article but their clinical conditions and body weight and food consumption data were recorded during that time.
ANIMAL HOUSING
Upon arrival and until pairing, all animals were individually housed in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed three times per week. The reproductive phase females were paired for mating in the home cage of the toxicity phase male of the same treatment group. Following positive evidence of mating, the reproductive phase females were individually housed in plastic maternity cages with nesting material, ground corncob bedding (Bed-O'Cobs®; The Andersons, Industrial Products Division, Maumee, Ohio). The nesting material is periodically analyzed by the manufacturer for contaminants. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at WIL Research Laboratories, LLC. The females were housed in these cages through lactation day 4, the scheduled day of necropsy. Females for which there was no evidence of mating were placed in plastic maternity cages with nesting material upon completion of a 14-day mating period. Females that did not deliver were necropsied on post-mating day 25. Animals were
housed in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The animal facilities at WIL Research Laboratories, LLC are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).
DIET, DRINKING WATER AND MAINTENANCE
The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002, is a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research Laboratories, LLC. Feeders were changed and sanitized once per week. Municipal water supplying the facility is sampled for contaminants according to standard operating procedures. The results of the diet and water analyses are maintained at WIL Research Laboratories, LLC. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. The basal diet and reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system were provided ad libitum throughout the acclimation period and during the study.
ENVIRONMENTAL CONDITIONS
All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain daily averages of 71 ± 5°F (22 ± 3°C) and 50 ± 20% relative humidity. Room temperature and relative humidity were monitored using the Metasys DDC Electronic Environmental control system and were recorded approximately hourly. These data are summarized in Appendix B. Actual mean daily temperature ranged from 69.9°F to 75.2°F (21.1°C to 24.0°C) and mean daily relative humidity ranged from 39.0% to 48.9% during the study. Light timers were calibrated to provide a 12-hour light (6 a.m. to 6 p.m.)/12-hour dark photoperiod. The 12-hour light/12-hour dark photoperiod was interrupted as necessary to allow for the performance of protocol-specified activities. Air handling units were set to provide approximately 10 fresh air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The vehicle and test article formulations were administered orally by gavage, via 15-gauge, polypropylene-shafted silicone-bulb-shaped-tipped dosing cannulae (Instech Solomon, Plymouth Meeting, Pennsylvania) once daily.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the initiation of dosing (May 5, 2004), representative control and test article formulations were prepared. Duplicate samples (1 mL each) for homogeneity determination were collected from the top, middle and bottom strata of the 25, 75 and 250 mg/kg/day dosing formulations and from the middle stratum of the control group preparation. The aliquots dispensed for homogeneity were representative of the size of aliquots dispensed for daily dosing procedures and included resuspension analysis over the longest period of aliquot storage. Samples from the control formulation and samples from the top and bottom strata were withdrawn from the aliquots after 6 and 12 days of refrigerated storage to confirm resuspension homogeneity and stability of the test article in the formulations. Fourteen-day refrigerated stability was assessed by comparison of the test article concentrations from samples collected from the middle stratum of each formulation on the day of preparation and following 14 days of storage. Duplicate samples (1 mL each) were collected from the middle stratum of each formulation, including the control group, for study weeks 0, 1, 2, 4 and 6 for confirmation of concentration. Characterization of the test article structure was performed by gas chromatography (GC) with mass selective detection (MSD). The determination of vinyl-tris(2-methoxyethoxy)silane in dried/deacidified corn oil formulations was performed by gas chromatography (GC) with mass selective detection (MSD).
Details on mating procedure:
Following a minimum of 14 days of treatment, one female assigned to the reproductive toxicity phase was cohabited with one male rat of the same treatment group. The selected males and females were approximately 12 weeks old when paired for mating. Animals were selected for pairing based on ear tag number; the male with the lowest animal number was paired with the female in the same dose group with the lowest animal number, continuing in increasing numerical eartag order until all animals were paired. A breeding record containing the male and female identification numbers and start of cohabitation was prepared. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Each mating pair was examined daily. The day on which evidence of mating was identified was termed gestation day 0, and the animals were separated. A maximum of 14 days was allowed for mating. Females that had not shown evidence of mating in 14 days were placed in plastic maternity cages containing nesting material.
Duration of treatment / exposure:
Exposure period: 28 days
Four groups of male and female Crl:CD(SD)IGS BR rats (10/sex/group) were administered the test article, vinyl-tris(2-methoxyethoxy)silane, in the vehicle, dehydrated, deacidified corn oil, daily by oral gavage for 28 days; the males were treated 14 days prior to mating and continuing throughout mating. Additionally, four groups of female rats (10 per group) were mated with the treated males and were also administered the test article by oral gavage daily for a minimum of 14 days prior to mating, throughout mating and gestation and continuing through lactation day 3.
Frequency of treatment:
daily
Duration of test:
Premating exposure period (males): 14 days
Premating exposure period (females): 14 days
Duration of test: 14 days prior to mating, throughout mating and gestation and continuing through lactation day 3
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 75 and 250 mg/kg/day
Basis:

No. of animals per sex per dose:
10/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Four groups of male and female Crl:CD (SD)IGS BR rats (10/sex/group) were administered the test article, vinyl-tris(2-methoxyethoxy)silane, in the vehicle, dehydrated, deacidified corn oil, daily by oral gavage for 28 days; the males were treated 14 days prior to mating and continuing throughout mating. Additionally, four groups of female rats (10 per group) were mated with the treated males and were also administered the test article by oral gavage daily for a minimum of 14 days prior to mating, throughout mating and gestation and continuing through lactation day 3. Dose levels were 0, 25, 75 and 250 mg/kg/day for the mated males, unmated females (toxicity phase) and mated females (reproductive phase).

Examinations

Maternal examinations:
All animals were observed twice daily for appearance and behavior. Clinical observations, body weights and food consumption were recorded at appropriate intervals. For females that delivered or had macroscopic evidence of implantation, the numbers of former implantation sites and corpora lutea were recorded. Recognizable fetuses for the females euthanized in extremis were examined externally and preserved in 10% neutral-buffered formalin. For females that failed to deliver, a pregnancy status was determined.Uteri with no macroscopic evidence of implantation were opened and subsequently placed in a 10% ammonium sulfide solution for detection of early implantation loss
Ovaries and uterine content:
Uteri with no macroscopic evidence of implantation were opened and subsequently placed in a 10% ammonium sulfide solution for detection of early implantation loss
Fetal examinations:
All reproductive phase females were allowed to deliver and rear their offspring to lactation day 4; surviving dams and pups were euthanized and examined on lactation day 4.On the day parturition was initiated (PND 0), the pups were sexed and examined for gross malformations, and the numbers of still born and live pups were recorded. Individual gestation length was calculated using the date delivery started. Abnormal behavior of the offspring was recorded. The dam and litter remained together until PND 4. Intact offspring dying from PND 0 to 4 were necropsied. Cannibalized pups were discarded without necropsy. Tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by gross findings. The carcass of each pup was then discarded.
Statistics:
Mating, fertility, copulation and conception indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Mean litter proportions (percent per litter) of pup viability and percent males at birth were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences (Kruskal and Wallis, 1952). If the ANOVA revealed statistically significant (p<0.05) intergroup variance, the Mann-Whitney U-test (Kruskal and Wallis, 1952) was used to compare the test article-treated groups to the control group.
Indices:
Male (Female) Mating Index (%)
Male Fertility Index (%)
Male Copulation Index (%)
Female Fertility Index (%)
Female Conception Index (%)
On the day parturition was initiated (PND 0), the pups were sexed and examined for gross malformations, and the numbers of still born and live pups were recorded.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
There were no test article-related effects on mean body weights, body weight gains or food consumption during the pre-mating period in the reproductive phase females. However, mean gestation body weight gains and food consumption were reduced in the 250 mg/kg/day group females throughout gestation; mean body weight on gestation day 20 was 22.1% lower than the control group value. Reductions in mean body weight and body weight gain late in gestation were attributed to the increased number (five of nine) of entirely resorbed litters in the 250 mg/kg/day group. Evaluation of lactation body weight and food consumption in the 250 mg/kg/day group was precluded by reduced fertility, embryonic death and total litter loss. No test article-related effects on mean gestation or lactation body weight gains or food consumption were observed in the 25 and 75 mg/kg/day group reproductive phase females.
In the reproductive phase, one female in the 75 mg/kg/day group was euthanized in extremis on gestation day 22. The cause of moribundity for this female was considered to be dystocia. In addition, one female in the 250 mg/kg/day group had total litter loss on lactation day 0 and one female in the 75 mg/kg/day group had total litter loss on lactation day 2. All other reproductive phase females survived to the scheduled necropsy. There were no test article-related clinical findings in the reproductive phase females.
Test article-related effects on reproductive performance (fertility indices) were observed in the 250 mg/kg/day group males and reproductive phase females. Male and female fertility indices were 60.0% in this group compared to 90.0% in the control group. The mean number of days between pairing and coitus was increased in the 250 mg/kg/day group. Mean gestation length was increased in the 75 mg/kg/day group and in the single female in the 250 mg/kg/day group that delivered.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Of the nine reproductive phase females in the 250 mg/kg/day group with evidence of mating, three were nongravid and five had entirely resorbed litters. Only one female in this group delivered, but all pups were found dead on postnatal day (PND) 0, precluding evaluation of pup body weights. In the 75 mg/kg/day group, the mean numbers of implantation sites, pups born and live litter size on PND 0 were reduced while the number of unaccounted for implantation sites was increased. These effects were considered test article-related; however, only the live litter size on PND 0 was statistically significantly different (p<0.01) from the control group. Postnatal survival in the 75 mg/kg/day group was reduced throughout the lactation period (days 1-4), primarily due to one female with total litter loss on lactation day 2; the differences from the control group were not statistically significant but were considered test article-related. A slight increase was observed in the number of pups found dead or missing and presumed cannibalized in the 75 mg/kg/day group. The general physical condition and mean body weights and body weight gains of pups in the 25 and 75 mg/kg/day groups were unaffected by maternal test article administration. No internal findings in the pups found dead or at the scheduled necropsy were attributed to maternal test article administration.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 75 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The NOAEL for fetotoxicity/developmental toxicity is 75 mg/kg/day. A role for developmental toxicity in the reduced postnatal survival at this dose cannot be excluded. The NOAEL for teratogenicity is >= 75 mg/kg/day.