Pyrogen Test in Pharmaceutical Microbiology: Comprehensive Guide to Rabbit Test (Sham Test) and Its Significance
1️⃣ Introduction
The Pyrogen Test, also known as the Rabbit Test or Sham Test, is a classical method used in pharmaceutical microbiology to detect pyrogens — fever-causing substances that may be present in parenteral products (such as injectables, vaccines, and medical devices). Even trace amounts of pyrogens can cause severe fever, shock, or death when injected into the bloodstream. Therefore, pyrogen testing is a critical quality control step to ensure the safety of sterile pharmaceutical preparations.
2️⃣ What Are Pyrogens?
Pyrogens are substances that trigger fever when introduced into the body.
🔹 Types of Pyrogens
- Endotoxins: Derived from Gram-negative bacteria, heat-stable lipopolysaccharides.
- Exotoxins: Produced by both Gram-positive and Gram-negative bacteria.
- Non-bacterial Pyrogens: Originating from viruses, fungi, or chemical contamination.
3️⃣ Purpose of the Rabbit Pyrogen Test
- To detect pyrogenic substances in pharmaceutical preparations.
- To ensure safety of parenteral products.
- To validate depyrogenation procedures.
- To comply with pharmacopoeial standards (IP, BP, USP, EP).
4️⃣ Principle of the Rabbit Test (Sham Test)
The test is based on measuring the rise in body temperature of healthy rabbits after intravenous injection of the test solution. If pyrogens are present, they stimulate the thermoregulatory center in the hypothalamus, resulting in a fever response. The temperature rise of the test solution is compared with the control (pyrogen-free saline).
5️⃣ Materials and Equipment Required
| Item | Purpose |
|---|---|
| Healthy rabbits (1.5–3.0 kg) | Test animals |
| Test solution | Product to be tested |
| Pyrogen-free saline | Control |
| Rectal thermometer/probe | Temperature measurement |
| Syringes & needles | Injection |
| Animal restrainer | To hold rabbit during testing |
| Water bath (37°C ± 2°C) | To warm test solution |
6️⃣ Step-by-Step Procedure
Step 1: Preparation of Animals
Select healthy, infection-free rabbits. House them individually under controlled conditions. Fast for 18 hours before the test (water allowed).
Step 2: Pre-Test Temperature Measurement
Record rectal temperatures at 30-minute intervals for 90 minutes. Variation should not exceed 0.2°C.
Step 3: Injection of Test Solution
Warm the test solution to 37°C ± 2°C and inject slowly into the marginal ear vein within 10 minutes.
Step 4: Post-Injection Temperature Recording
Record rectal temperatures every 30 minutes for 3 hours after injection.
Temperature Rise (°C) = Max Temperature after Injection − Initial Temperature
Step 5: Interpretation of Results
| Rabbit No. | Initial Temp (°C) | Max Temp (°C) | Rise (°C) |
|---|---|---|---|
| 1 | 38.6 | 39.1 | 0.5 |
| 2 | 38.8 | 39.0 | 0.2 |
| 3 | 38.7 | 39.1 | 0.4 |
Total Temperature Rise = 1.1°C → Pass
Since the total temperature rise is less than 1.4°C, the sample is non-pyrogenic.
7️⃣ Acceptance Criteria (As per Pharmacopoeia)
| Parameter | Criteria |
|---|---|
| Individual Rabbit | Rise ≤ 0.6°C |
| Combined (3 Rabbits) | Total rise ≤ 1.4°C |
| If 1 Rabbit > 0.6°C but < 1.0°C | Test 3 more rabbits |
| Final Decision | Total rise (6 rabbits) ≤ 3.7°C → Pass |
8️⃣ Example Calculation
ΔT = T₂ − T₁ If ΣΔT ≤ 1.4°C → PASS If ΣΔT > 1.4°C → FAIL
9️⃣ Advantages of Rabbit Pyrogen Test
- Detects both endotoxin and non-endotoxin pyrogens.
- Useful for biologicals and vaccines.
- Pharmacopoeia-approved method.
- No complex equipment required.
🔟 Limitations
- Ethical concerns due to live animals.
- Time-consuming and labor-intensive.
- Variable animal sensitivity.
- Low throughput compared to LAL test.
11️⃣ Alternatives to Rabbit Test
- LAL Test (Limulus Amebocyte Lysate): Detects endotoxins.
- rFC Test (Recombinant Factor C): Animal-free version of LAL.
- MAT (Monocyte Activation Test): Detects both endotoxin and non-endotoxin pyrogens.
12️⃣ Conclusion
The Rabbit Pyrogen Test (Sham Test) remains a trusted and regulatory-accepted method for detecting pyrogens in pharmaceutical preparations. Although modern alternatives like LAL and MAT are more ethical and efficient, the Rabbit Test is still relevant where non-endotoxin pyrogens are a concern. Ensuring pyrogen-free products is a vital part of pharmaceutical quality assurance and patient safety.
💬 About the Author
Siva Sankar is a Pharmaceutical Microbiology Consultant and Auditor with extensive experience in sterility testing, validation, and GMP compliance. He provides consultancy, training, and documentation services for pharmaceutical microbiology and cleanroom practices.
📧 Contact: siva17092@gmail.com
📱 Mobile: 09505626106