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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
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 oral 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.Using gas chromatography with flame ionization detection (GC/FID), the test article was analyzed for purity prior to the start of dosing. 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).
Duration of treatment / exposure:
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
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 75 and 250 mg/kg/day
Basis:
other: nominal
No. of animals per sex per dose:
10/sex/group
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Observations and examinations performed and frequency:
All animals were observed twice daily for appearance and behavior. Clinical observations, body weights and food consumption were recorded at appropriate intervals. In addition, detailed clinical observations (functional observational battery [FOB] conducted out of the home cage) and locomotor activity were evaluated for all adult male and toxicity phase females once prior to the start of test article administration (baseline evaluations) and again during the last week of the test article administration.
Sacrifice and pathology:
Clinical pathology assessments (hematology and serum chemistry) and macroscopic and microscopic examinations (including organ weights) were also performed on the appropriate groups of adult males and toxicity phase females. 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.
Statistics:
All statistical tests were performed using appropriate computing devices or programs. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test article-treated group to the control group by sex. Mean body weights and body weight changes, maternal food consumption, gestation lengths, implantation sites, unaccounted for sites, pre-coital intervals, numbers of pups born, live litter sizes, ambulatory counts measured in locomotor activity assessment, functional observational battery, clinical pathology values (excluding differential white cell counts other than lymphocytes and neutrophils) and organ weight data (absolute and relative) subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences (Snedecor and Cochran, 1980). If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test article-treated groups to the control group. 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. Functional observations battery data which yielded scalar and descriptive data and qualitative histopathological findings of the control group were compared to each treated group by the Fisher’s Exact Test (Steel and Torrie, 1980).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
All males and all toxicity phase females survived to the scheduled necropsies.  There were no test article-related clinical findings in the males or toxicity phase females.  There were no test article-related effects observed at the FOB or locomotor activity evaluations in the males or toxicity phase females at any dose level. Food consumption was reduced in the 250 mg/kg/day group males during study days 0-7; mean body weight gains were reduced in these males during study days 14-21 and 21-28.  Mean body weight in the 250 mg/kg/day group males was 6.7% lower than the control group value on study day 28. There were no test article-related effects on mean body weights, body weight gains or food consumption in the toxicity phase females. Decreases in mean red blood cell counts, hemoglobin levels, hematocrit, mean corpuscular hemoglobin concentration, reticulocyte counts, platelet counts, basophil counts and eosinophil counts were observed in both sexes in the 250 mg/kg/day toxicity phase group.  Examination of the bone marrow smears revealed an overall decrease in the myeloid:erythrocyte (M/E) ratio.  When evaluated in combination with the blood count data and bone marrow histopathology, the decreased M:E ratio does not indicate erythroid hyperplasia, but rather a disproportionate mild supression of both myeloid and erythroid elements.  However, ineffective erythropoiesis or an early regenerative response cannot be excluded.  Increases in monocyte counts were also observed in these animals.  These findings correlated with the microscopic finding of hypocellularity in the sternal bone marrow, in which aggregates of mature granulocytes were absent.  Mean albumin, total protein and globulin levels were also reduced in the 250 mg/kg/day toxicity phase male and female groups, resulting in increased mean albumin/globulin ratios.   Test article-related macroscopic changes, microscopic changes and/or reductions in organ weights were observed in the 75 mg/kg/day group males and the 250 mg/kg/day group males and females.  Small and/or soft testes and/or epididymides were observed in the 250 mg/kg/day group males.  Mean absolute and relative (to final body and brain weights) testes and epididymides weights were also reduced in this group.  Microscopically, small/soft testes correlated with seminiferous tubule degeneration observed in all males in the 250 mg/kg/day group.  Secondary to the loss of spermatogenesis in the testes was hypospermia and luminal cellular debris in the epididymides, corresponding to macroscopic findings.  Mean absolute and relative prostate weights in the 75 and 250 mg/kg/day group males were reduced; the reduction correlated microscopically with decreased secretion and/or atrophy.  Mean absolute and relative seminal vesicle weights were also reduced in the 250 mg/kg/day group males; there were no correlating microscopic findings.  Adhesions and/or white areas on the spleen were observed in the 75 mg/kg/day group males and the 250 mg/kg/day group males, toxicity phase females and a reproductive phase female.  These findings corresponded with capsular fibrosis microscopically.  Small thymus was observed in the 250 mg/kg/day group males and toxicity phase females.  Mean absolute and relative (to final body and brain weights) thymus weights were also reduced in males and females at 250 mg/kg/day and correlated to the microscopic finding of lymphoid depletion.  Lymphoid depletion was also observed in the mesenteric and/or mandibular lymph nodes in the 250 mg/kg/day group males and toxicity phase females.  Mean absolute and relative adrenal gland weights were reduced in the 250 mg/kg/day group males and toxicity phase females; there was no microscopic correlate to this decrease.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: Based on effects on hematology and serum chemistry parameters and effects on lymphoid tissues for females at 250 mg/kg/day, which were similar to the effects noted for males, the NOAEL for female systemic toxicity was 75 mg/kg/day.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on decreased body weights, body weight gains and food consumption, effects on hematology parameters (decreased
blood cell counts and changes in the granulocyte and erythroid series) and serum chemistry parameters (decreased albumin, protein and globulin) at 250 mg/kg/day and effects on lymphoid tissues (macroscopic and microscopic findings and/or decreased organ weights) at 75 and/or 250 mg/kg/day, the no observed adverse-effect level (NOAEL) for male systemic toxicity was 25 mg/kg/day. Based on effects on hematology and serum chemistry parameters and effects on lymphoid tissues for females at 250 mg/kg/day, which were similar to the effects noted for males, the NOAEL for female systemic toxicity was 75 mg/kg/day.