13-Week Toxicity Study
In addition to obtaining toxicological data, the purpose of this study is to determine the treatments for each strain and species to be used in the 2-year toxicology/carcinogenesis study.
After a 10- to 14-day quarantine period, animals are assigned at random to treatment groups. The study includes five treatment groups each administered a different concentration of the test article plus a control group. Each group contains 10 animals per sex per species. The animals receive the subject chemical by a designated route of exposure. Controls receive untreated water or feed or vehicle alone in gavage and dermal studies. For dosed-feed and dosed-water studies, animals are exposed for 90 days after which they are sacrificed with no recovery period. For inhalation, gavage and dermal studies animals are exposed five times per week, weekdays only until the day prior to necropsy. Male mice are housed individually and rats and female mice are housed in groups of five animals per cage except for inhalation and dermal exposure studies in which all rats and mice are housed individually.
Animals Sexes Species Test
Total Treatment 10 x 2 x 2 x 5 = 200 Controls 10 x 2 x 2 x 1 = 40 Special "rats" for clinical lab studies 10 x 2 x 1 x 5 = 100 Special controls for clinical lab studies 10 x 2 x 1 x 1 = 20 ___ Total 360
Animals are weighed individually on day one on test, after seven days, and at weekly periods thereafter. Animals are observed twice daily, at least six hours apart (before 10:00 AM and after 2:00 PM), including holidays and weekends, for moribundity and death. Animals found moribund or showing clinical signs of pain or distress are humanely euthanized. Formal clinical observations are performed and recorded weekly. For dosed-feed or dosed-water studies, food consumption/water consumption is measured and recorded weekly.
Necropsy and Histopathologic Evaluation:
Liver, thymus, right kidney, right testis, heart, and lung weights are recorded from all animals surviving until the end of the study.
A complete necropsy is performed on all treated and control animals that either die or are sacrificed. All tissues required for complete histopathology are trimmed, embedded, sectioned and stained with hematoxylin and eosin for histopathologic evaluation. (See necropsy examination)
A complete histopathologic evaluation inclusive of treatment-related gross lesions shall be done on all early death animals regardless of dose group, all control animals, all animals, and all animals in the highest treatment group with at least 60% survivors at the time of sacrifice plus all animals in higher treatment groups. Treatment-related lesions (target organs) shall be identified and these organs plus gross lesions shall be examined to a no-effect level. (list of tissues)
Specific Toxicologic Parameters Evaluated in the 13-Week Study
Clinical Laboratory Studies
Blood is collected from both sexes of "special study" rats, at days 4 ± 1 and 21 ± 2 and from the core study rats at the end of the study. These are processed for hematology and clinical chemistry determinations. Blood is collected from core study mice at the end of the study for hematology determinations. (clinical measurements)
Blood for Micronuclei
Blood samples are taken from mice and rats at study termination for micronuclei determinations.
Sperm Morphology and Vaginal Cytology Evaluations (SMVCE)
SMVCE are conducted on core study rats and mice. Mortality, body weight changes and clinical signs of toxicity are used to determine the 3 treatment groups used for the SMVC evaluations.