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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 09, 2009 to April 06, 2009
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
This species and strain of animal is recognized as appropriate for short-term toxicity studies. The Sprague Dawley rat was selected because it is a widely used strain for which significant control data are available. The animals were approximately 7 weeks old at the initiation of dose administration.
Sex:
male/female
Details on test animals and environmental conditions:
ANIMAL RECEIPT AND ACCLIMATION/PRETEST PERIOD
37 male and 37 female Crl:CD(SD) rats were received in good health on 24 February 2009, from Charles River Laboratories, Inc., Raleigh, NC. The animals were approximately 38 days old at receipt. Each animal was examined by a qualified technician on the day of receipt and weighed 3 days later. Each animal was uniquely identified with a subcutaneous microchip (BMDS system) in the scapular area. All animals were housed for a 13-day acclimation/pretest period. During this period, each animal was observed twice daily for mortality and changes in general appearance or behavior. Individual body weights and food consumption were recorded and detailed physical examinations were performed periodically during the pretest period.

ANIMAL HOUSING
Upon arrival, all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. Racks were rotated within the animal room at least once every 2 weeks during the study to change the physical location from one area of the room to another to ensure similarly varied environmental exposure for all animals. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Nylabones® were provided to all animals throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

DIET, DRINKING WATER AND MAINTENANCE
The basal diet fed ad libitum during the acclimation period and to the control group throughout the study was PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal). The certified feed was analyzed by the manufacturer and results were provided to WIL Research Laboratories, LLC. Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, the test diet, and the basal diet were provided ad libitum throughout the study, except during designated periods of fasting when food, but not water, was withheld. Municipal water supplying the facility was analyzed for contaminants according to the standard operating procedures. The results of the diet and water analyses are maintained at the test facility.

ENVIRONMENTAL CONDITIONS
All animals 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 environmental conditions of 71 ± 5 °F (22 ± 3 °C) and 50 ± 20% relative humidity. Room temperature and relative humidity were controlled and monitored using the Metasys®® DDC Electronic Environmental control system. Actual mean daily temperature ranged from 69.7 °F to 72.2°F (20.9 °C to 22.3 °C) and mean daily relative humidity ranged from 42.5% to 56.1% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/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 a minimum of 10 fresh air changes per hour.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
Four days prior to the initiation of dose administration, all available animals were weighed and examined in detail for physical abnormalities. Based on the review of all appropriate pretest data, animals judged suitable for assignment to the study were selected for use in a computerized randomization procedure. A printout containing the animal numbers, corresponding body weights and individual group assignments was generated based on body weight stratification in a block design. The animals were then arranged into groups. Individual body weights at randomization were within ±20% of the mean for each sex. Groups 1-6 each consisted of 5 males and 5 females. Individual body weights ranged from 179 g to 212 g for males and from 150 g to 178 g for females at randomization.

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The basal (control) diet or test diets were fed ad libitum for 28 consecutive days, through the day prior to the scheduled necropsy. Initial concentrations were based on average food consumption and body weights determined during pretest period. Concentration was adjusted weekly, based on the expected average weight and food consumption.
Vehicle:
other: meal
Details on oral exposure:
PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal) was used to prepare the control and test diets. Each lot used was documented.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The control and test diets were prepared approximately weekly, placed in labeled storage bags and stored at room temperature. Dietary concentrations for the test diets were adjusted weekly based on the expected average weight and food consumption.
Samples for homogeneity determination were collected from the top, middle, and bottom strata of the 50 and 15,000 ppm pretest formulations.
Samples for stability determinations were collected from the middle strata of these same pretest formulations and from the basal diet. Samples for stability determination were analyzed following room temperature storage for 0, 5, 8, and 10 days. Samples for concentration analysis were collected from the first and last formulation preparation from the middle stratum of each dietary formulation (including the control group). All samples were stored frozen (approximately -65° to -85°C) until shipment for analysis.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Basal (control) diet
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Dosage level changed from 10 mg/kg/day to 800 mg/kg/day on study day 18
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
The test substance, was offered on a continuous basis in basal diet for 28 consecutive days to five groups (Groups 2-6) of Sprague Dawley (Crl:CD®[SD]) rats at dosage levels of 10, 50, 100, 300, and 1000 mg/kg/day, respectively. A concurrent control group (Group 1) received the basal diet on a comparable regimen. On study day 18, the dosage level for Group 2 was changed from 10 mg/kg/day to 800 mg/kg/day for the remainder of the study. All groups (Groups 1-6) consisted of five animals/sex. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded approximately weekly. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals at the scheduled necropsy (study week 4). Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Tissues were retained for possible future microscopic examination.

Examinations

Observations and examinations performed and frequency:
SURVIVAL
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

CLINICAL OBSERVATIONS
Clinical examinations were performed once daily for all animals. The absence or presence of findings was recorded for individual animals at the scheduled intervals. Detailed physical examinations were conducted on all animals beginning during the acclimation period, upon individual housing of the animals, on the day of randomization, and approximately weekly thereafter.

BODY WEIGHTS
Individual body weights were recorded approximately weekly, beginning at least one week prior to test substance administration and on the day prior to the scheduled necropsy. Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded for all animals on the day of the scheduled necropsy.

FOOD AND TEST SUBSTANCE CONSUMPTION
Individual food consumption was recorded at least weekly during the pretest period and throughout the study. Food intake was calculated as g/animal/day and g/kg/day for the corresponding body weight intervals.
The mean amounts of test substance consumed (mg/kg/day) by each sex per dose group were calculated from the mean food consumed (g/kg/day) and the appropriate target concentration of test substance in the food (mg/kg). Food efficiency (body weight as a percent of feed consumed) was also calculated for all animals.
Sacrifice and pathology:
CLINICAL PATHOLOGY
Blood and urine samples for clinical pathology evaluations (hematology, coagulation, serum chemistry, and urinalysis) were collected from all animals prior to the scheduled necropsy (study day 28). The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection. Blood was collected for hematology and serum chemistry evaluation via the retro-orbital sinus of animals anesthetized by inhalation of isoflurane. Blood was collected for coagulation parameters at the time of euthanasia via the vena cava of animals euthanized by inhalation of carbon dioxide. Blood was collected into tubes containing potassium EDTA (hematology), sodium citrate (coagulation) or no anticoagulant (serum chemistry).

HEMATOLOGY AND COAGULATION
All red blood cells, white blood cells, plate indices and coagulation related parameters were sampled for analysis. Additionally serum chemistry and urinalysis were conducted.

ORGAN WEIGHTS
The following organs were weighed from all animals at the scheduled necropsy: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Testes, Lungs, Ovaries with oviducts, Pituitary, Prostate, Spleen, Thymus, Thyroid with parathyroids.

MACROSCOPIC EXAMINATION
A complete necropsy was conducted for all animals. Animals were euthanized by carbon dioxide inhalation followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.
Statistics:
All statistical tests were performed using the Toxicology Data Management System (WTDMS™). Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Body weight, body weight change, food consumption, clinical pathology (except gamma glutamyltransferase), and organ weight data were subjected to a parametric one-way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Gamma glutamyltransferase values under range were assigned a value of 0.1 (half the lower limit of quantitation) for statistical analysis and reporting. Gamma glutamyltransferase data were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significance (p<0.05), Mann-Whitney U-test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight loss and/or lower body weight gains were noted for the 300, 800, and 1000 mg/kg/day group males and females throughout the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Lower food consumption were noted for the 300, 800, and 1000 mg/kg/day group males and females throughout the study. Clinical observations of decreased defecation in the 800 mg/kg/day group females correlated to lower food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The higher red blood cell counts, hemoglobin, hematocrit, and MCV levels and the lower reticulocyte counts in the 800 mg/kg/day group animals may be related to dehydration or malnutrition, correlating to the lower food consumption noted in this group. The opposite effect on reticulocyte counts and red cell mass in the 1000 mg/kg/day group animals may be due to an over compensation for an initial reduction in red blood cell counts.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Lower glucose levels in the 800 mg/kg/day group animals and 1000 mg/kg/day group males in the serum may have resulted from inadequate food consumption and/or depletion of glycogen stores (Levin, 1993).
Lower values for liver enzymes alkaline phosphatase (800 mg/kg/day and 1000 mg/kg/day group females) and AST (all test substance-treated group females), in the absence of correlating gross organ observations, were likely due to reduced metabolic liver function corresponding to decreased food consumption.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Higher urea nitrogen in the 800 and 1000 mg/kg/day group animals and increased albumin and total protein in the 800 and 1000 mg/kg/day group males were most likely due to dehydration and malnutrition from lower food consumption.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related differences in organ weights consisted of lower liver, spleen, and thymus weights (absolute and relative to body and/or brain) in the 800 mg/kg/day group females. Liver enzymes were also altered and lower spleen and thymus weights may be due to stress. Direct test-substance effects cannot be confirmed without histopathologic examination.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the study conditions, systemic toxicity of the test substance offered in the diet to rats for 28 d was observed at 800 (beginning on Day 18) and 1000 mg/kg bw/day, as evidenced by lower body weight gains, lower food consumption, as well as effects on hematology, serum chemistry parameters and organ weights. Therefore, the 28 d NOAEL, with no microscopic examination of tissues, was 300 mg/kg bw/day for males and females.
Executive summary:

A study was conducted to determine the potential toxicity of the test substance when administered to rats via the diet for 28 consecutive days and to assist in dose selection for the 90 d dietary definitive reproductive toxicity study according to OECD Guideline 407, in compliance with GLP. The test substance was offered on a continuous basis in basal diet for 28 consecutive days to 5 groups (Groups 2-6) of Sprague Dawley (Crl:CD (SD) rats at dosage levels of 10, 50, 100, 300 and 1000 mg/kg bw/day, respectively. A concurrent control group (Group 1) received the basal diet on a comparable regimen. On Day 18, the dosage level for Group 2 was changed from 10 to 800 mg/kg bw/day for the remainder of the study. All groups (Groups 1-6) consisted of 5 animals/sex. All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly. Individual body weights and food consumption were recorded approximately weekly. Clinical pathology parameters (hematology, coagulation, serum chemistry and urinalysis) were analyzed for all animals at the scheduled necropsy (Week 4). Complete necropsies were conducted on all animals and selected organs were weighed at the scheduled necropsy. Tissues were retained for possible future microscopic examination. There were no test substance-related effects on survival, urinalysis parameters or macroscopic findings. However, lower food consumption was noted for the 800 and 1000 mg/kg bw/day males and females as compared to controls. The following effects were considered secondary to test substance-related effects of dehydration and lower food consumption: decreased defecation at 800 mg/kg/day; lower body weights and lower body weight gains at 300 (females only), 800 and 1000 mg/kg/day; higher red blood cell counts, hemoglobin, hematocrit and lower reticulocyte counts at 800 mg/kg bw/day and MCV levels in the 800 mg/kg bw/day group females; lower red blood cell counts, hemoglobin, hematocrit in the 1000 mg/kg bw/day females, and higher reticulocyte count levels in the 1000 mg/kg bw/day group animals; lower eosinophils at 800 and 1000 mg/kg bw/day; higher albumin and total protein at 800 and 1000 mg/kg bw/day, and increased urea nitrogen for 800 and 1000 mg/kg bw/day group animals. Additional possible test substance effects included lower glucose at 800 mg/kg (males and females) and 1000 (males) mg/kg bw/day, slightly lower values for liver enzymes at 800 and 1000 mg/kg bw/day; and lower liver, spleen and thymus weights (absolute and relative to body and/or brain) were noted in the 800 mg/kg/day group females. Under the study conditions, systemic toxicity of the test substance offered in the diet to rats for 28 d was observed at 800 (beginning on Day 18) and 1000 mg/kg bw/day, as evidenced by lower body weight gains, lower food consumption, as well as effects on hematology, serum chemistry parameters and organ weights. Therefore, the 28 d NOAEL, with no microscopic examination of tissues, was 300 mg/kg bw/day for males and females (Matthew, 2010).