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The “28-Day Repeated Dose Study” refers to a type of preclinical toxicity study conducted in the field of pharmacology and toxicology. This study is designed to assess the effects of a substance (such as a drug or chemical compound) when administered repeatedly over a period of 28 days, typically to laboratory animals. The aim is to evaluate the substance’s potential toxicity and its impact on various organs and systems within the body.

During the study, animals are usually exposed to the test substance daily for 28 consecutive days, and observations are made regarding any adverse effects, changes in behavior, clinical signs, and physiological parameters. At the end of the study, necropsies (post-mortem examinations) may be performed to examine internal organs and tissues for any abnormalities or lesions.

The 28-Day Repeated Dose Study is a crucial step in the safety assessment of substances intended for human use, providing valuable information for researchers and regulatory authorities to determine the substance’s safety profile and potential risks associated with prolonged exposure. These studies are often conducted in accordance with regulatory guidelines to support the development and approval of pharmaceuticals, chemicals, and other products.


Sample 28-Day Repeated Dose Study Protocol


In this example of the 28-Day Repeated Dose Study the primary objective of this study is to assess the repeated dose toxicity of ANLC-TX024 over a 28-day period in C57BL/6 mice. The study aims to identify any potential adverse effects, changes in behavior, and physiological alterations associated with the drug. This investigation is crucial for evaluating the safety profile of ANLC-TX024 in the context of its intended use for the treatment of a rare hypothalamic tumor.

Study Design:


  • C57BL/6 mice will be procured from a reputable supplier.
  • Mice will be acclimated to laboratory conditions for a minimum of 7 days before the start of dosing.

Dosing Groups:

  • Mice will be randomly assigned to different dosing groups (including control group) based on body weight, ensuring a homogeneous distribution.
  • Dosing groups will receive ANLC-TX024 at escalating doses (to be determined based on prior toxicity studies and regulatory guidelines).

Dosing Route:

  • ANLC-TX024 will be administered daily via the chosen route (e.g., oral gavage) for 28 consecutive days.

Control Group:

Parameters to be Assessed

Clinical Observations:

  • Daily monitoring for changes in behavior, activity, and general appearance. Recording of clinical signs and symptoms:
    • General Appearance:

      • Fur condition (e.g., grooming behavior)
      • Posture and body position
      • Activity level and mobility
    • Behavioral Changes:

      • Locomotor activity (ambulation, exploration)
      • Aggressiveness or social interactions
      • Nesting behavior (if applicable)
    • Neurological Function:

      • Coordination and balance
      • Tremors or convulsions
      • Response to external stimuli (e.g., touch, sound)
    • Respiratory Function:

      • Respiratory rate and pattern
      • Labored breathing or dyspnea
    • Ocular Observations:

      • Eye appearance (clearness, discharge)
      • Pupillary reflex
    • Oral Cavity:

      • Salivation
      • Presence of oral lesions or abnormalities
    • Gastrointestinal Function:

      • Presence of diarrhea or changes in fecal consistency
      • Abdominal distension or discomfort
    • Urinary Function:

      • Frequency of urination
      • Changes in urine color or volume
    • Food and Water Intake:

      • Monitoring of daily food and water consumption
    • Body Weight:

      • Regular weighing to track changes over the study period
    • Skin and Fur:

      • Presence of lesions, erythema, or edema
      • Scratching or grooming behavior
    • Temperature Regulation:

      • Monitoring for signs of hypothermia or hyperthermia
    • Reproductive Function (if applicable):

    • Clinical Signs of Distress:

      • Hunched posture
      • Ruffled fur
      • Inactivity or lethargy

Body Weight:

  • Weighing of mice at regular intervals throughout the study.

Food and Water Consumption:

  • Measurement of daily food and water intake.

Hematology and Clinical Chemistry:

  • Blood samples collection for hematological and biochemical analysis at the end of the dosing period.
  • Hematology:
    • Complete Blood Count (CBC):

      • White Blood Cell Count (WBC)
      • Red Blood Cell Count (RBC)
      • Hemoglobin concentration (Hb)
      • Hematocrit (Hct)
      • Platelet Count (Plt)
      • Differential White Blood Cell Count (Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils)
    • Coagulation Parameters:

      • Prothrombin Time (PT)
      • Activated Partial Thromboplastin Time (APTT)
  • Clinical Chemistry:
    • Liver Function:
      • Alanine Aminotransferase (ALT)
      • Aspartate Aminotransferase (AST)
      • Alkaline Phosphatase (ALP)
      • Total Bilirubin
      • Albumin
    • Kidney Function:
      • Blood Urea Nitrogen (BUN)
      • Creatinine
      • Electrolytes (Sodium, Potassium, Chloride)
    • Metabolic Markers:
      • Glucose
    • Urinary Function:
      • Urinalysis (including examination for protein, glucose, and blood)

Organ Weight and Macroscopic Examination:

  • Necropsy at the end of the study to assess organ weights and perform a macroscopic examination of internal organs.


  • Tissue samples from major organs (including liver, kidney, spleen, and tumor site) will be collected for histopathological examination.

Data Analysis:

  • Data collected will be analyzed using appropriate statistical methods.
  • Any statistically significant findings will be further investigated.
  • Histopathology:
    • Liver:

      • Hepatocellular hypertrophy
      • Hepatocellular necrosis
      • Inflammatory cell infiltration
      • Steatosis (fatty liver)
      • Kupffer cell hyperplasia
    • Kidneys:

      • Tubular degeneration or necrosis
      • Glomerular changes
      • Interstitial inflammation
      • Hyaline droplet formation (indicative of specific protein accumulation)
    • Spleen:

      • Changes in white pulp (lymphoid tissue)
      • Presence of extramedullary hematopoiesis
    • Hypothalamic Tumor Site:

    • Other Organs:

      • Examination of major organs such as the heart, lungs, stomach, and intestines for any pathological changes.
    • Lymphoid Organs (e.g., Thymus and Lymph Nodes):

      • Evaluation of lymphoid organ weight and architecture
      • Assessment of lymphoid depletion or hyperplasia
    • Endocrine Organs:

      • Assessment of adrenal glands for changes in the cortex and medulla
      • Evaluation of thyroid and parathyroid glands
    • Reproductive Organs (if applicable):

      • Examination of testes and ovaries for changes in germinal epithelium, interstitial tissue, and follicular development
    • Nervous System:

    • Muscles:

      • Assessment of skeletal muscles for any pathological changes

Ethical Considerations:

  • The study will be conducted in accordance with ethical standards and animal welfare regulations.
  • Approval from the Institutional Animal Care and Use Committee (IACUC) will be obtained before the initiation of the study.


  • A comprehensive report will be prepared, summarizing study design, dosing, observations, and data analysis.
  • Results will be interpreted in the context of safety for ANLC-TX024 use in the treatment of a rare hypothalamic tumor.

This protocol is a guideline, and specific details may need to be adjusted based on the characteristics of the drug, regulatory requirements, and emerging scientific considerations.