Official pyrogen tests MCQs With Answer

Official pyrogen tests MCQs With Answer is a focused collection of multiple-choice questions designed for M.Pharm students studying Biological Evaluation of Drug Therapy. This set emphasizes official methods used to detect pyrogens — primarily the rabbit pyrogen test (RPT) and bacterial endotoxin tests (BET) such as LAL and newer alternatives like recombinant factor C and the Monocyte Activation Test. Questions cover principles, formats (gel-clot, chromogenic, turbidimetric), regulatory acceptance criteria, calculation of limits and MVD, sample preparation, inhibition/enhancement controls, depyrogenation, and validation requirements. This concise quiz will help reinforce conceptual understanding and practical application required for exam and industry practice.

Q1. Which two tests are recognized as the principal official pyrogen tests in major pharmacopoeias?

• Rabbit pyrogen test and bacterial endotoxin test (LAL)
• Sterility test and oxidase test
• Monocyte Activation Test and Western blot
• Chromatography and gel electrophoresis

Correct Answer: Rabbit pyrogen test and bacterial endotoxin test (LAL)

Q2. Which LAL methodology is based on clot formation of horseshoe crab amebocyte lysate in presence of endotoxin?

• Gel-clot LAL assay
• Chromogenic LAL assay
• Turbidimetric LAL assay
• Recombinant factor C fluorescence assay

Correct Answer: Gel-clot LAL assay

Q3. How is the endotoxin unit (EU) typically defined for routine testing purposes?

• An EU is a biological unit referenced to a pharmacopeial standard; approximately 1 EU ≈ 0.1 ng of E. coli O55:B5
• An EU equals exactly 1 microgram of any lipopolysaccharide
• An EU is based on optical density change per minute in turbidimetric assays
• An EU equals one colony forming unit (CFU) of Gram-negative bacteria

Correct Answer: An EU is a biological unit referenced to a pharmacopeial standard; approximately 1 EU ≈ 0.1 ng of E. coli O55:B5

Q4. Which formula is used to calculate the endotoxin limit (EU/mL) for a specific parenteral product?

• Endotoxin limit (EU/mL) = K / M where K is the threshold pyrogenic dose (EU/kg) and M is the maximum human dose in kg
• Endotoxin limit (EU/mL) = (LAL sensitivity × sample volume) / K
• Endotoxin limit (EU/mL) = M × K where M is molecular weight and K is a constant
• Endotoxin limit (EU/mL) = 1 / (dilution factor × recovery)

Correct Answer: Endotoxin limit (EU/mL) = K / M where K is the threshold pyrogenic dose (EU/kg) and M is the maximum human dose in kg

Q5. Which of the following is NOT an official LAL/BET assay format recognized in pharmacopeias?

• ELISA-based endotoxin assay
• Gel-clot assay
• Chromogenic assay
• Turbidimetric assay

Correct Answer: ELISA-based endotoxin assay

Q6. What is the primary biological principle of the Monocyte Activation Test (MAT)?

• Measurement of cytokine release (e.g., IL-6) from human monocytes in response to pyrogens
• Clotting of horseshoe crab blood components in presence of endotoxin
• Color change due to enzymatic cleavage by bacterial proteases
• Optical density increase caused by particulate aggregation

Correct Answer: Measurement of cytokine release (e.g., IL-6) from human monocytes in response to pyrogens

Q7. Which depyrogenation method is most effective for glassware and equipment intended to be pyrogen-free?

• Dry heat depyrogenation at around 250°C for specified time
• Autoclaving at 121°C for 15 minutes
• Rinsing with sterile water for injection only
• Filtration through 0.22 µm membrane filter

Correct Answer: Dry heat depyrogenation at around 250°C for specified time

Q8. In LAL testing, what is the purpose of the positive product control (PPC)?

• To determine whether the product matrix inhibits or enhances the LAL reaction
• To sterilize the sample prior to testing
• To calibrate the photometer used in turbidimetric assays
• To neutralize endotoxin present in the reagent

Correct Answer: To determine whether the product matrix inhibits or enhances the LAL reaction

Q9. Which compound is commonly used to bind and neutralize endotoxin during sample preparation or treatment?

• Polymyxin B
• Penicillin G
• Sodium chloride
• Ethanol

Correct Answer: Polymyxin B

Q10. Which statement about recombinant factor C (rFC) assays is correct?

• rFC assays use a recombinant form of the initial endotoxin-sensitive protein (factor C) and are accepted as an alternative in many pharmacopeias
• rFC assays measure fever response in rabbits and are animal-based
• rFC assays rely on beta-glucan activation of factor G to produce a clot
• rFC assays are qualitative only and cannot be used for quantitation

Correct Answer: rFC assays use a recombinant form of the initial endotoxin-sensitive protein (factor C) and are accepted as an alternative in many pharmacopeias

Q11. What is the correct formula for calculating the Maximum Valid Dilution (MVD) for an LAL test?

• MVD = (Endotoxin limit × Sample concentration) / LAL test sensitivity
• MVD = LAL sensitivity / (Endotoxin limit × Sample concentration)
• MVD = Endotoxin limit + LAL sensitivity
• MVD = 1 / (Endotoxin limit × recovery percentage)

Correct Answer: MVD = (Endotoxin limit × Sample concentration) / LAL test sensitivity

Q12. Which substance commonly causes false-positive LAL results by activating the factor G pathway?

• (1→3)-β-D-glucans
• Polysorbate 80
• Sodium chloride
• Glucose

Correct Answer: (1→3)-β-D-glucans

Q13. What is the pharmacopeial threshold pyrogenic dose (K) for intrathecal administration used in endotoxin limit calculations?

• 0.2 EU/kg body weight
• 5.0 EU/kg body weight
• 10 EU/kg body weight
• 1.0 EU/kg body weight

Correct Answer: 0.2 EU/kg body weight

Q14. Which official pyrogen test is animal-based and measures febrile response rather than direct endotoxin quantity?

• Rabbit pyrogen test (RPT)
• Gel-clot LAL assay
• Chromogenic LAL assay
• Recombinant factor C assay

Correct Answer: Rabbit pyrogen test (RPT)

Q15. What is the primary measured endpoint in the rabbit pyrogen test?

• Change in rectal (core) body temperature of test animals
• Optical density change of lysate reaction
• Level of IL-6 in blood plasma measured by ELISA
• Clotting time of amebocyte lysate

Correct Answer: Change in rectal (core) body temperature of test animals

Q16. Which statement best describes the gel-clot LAL assay?

• It is a qualitative/endpoint assay that indicates presence of endotoxin above a defined sensitivity by clot formation
• It provides continuous quantitative kinetic data on endotoxin concentration
• It measures cytokine release from monocytes
• It uses recombinant enzymes to generate fluorescent signal only

Correct Answer: It is a qualitative/endpoint assay that indicates presence of endotoxin above a defined sensitivity by clot formation

Q17. What is used as the reference material to calibrate endotoxin assays and demonstrate LAL reagent potency?

• Pharmacopeial Reference Standard Endotoxin (RSE)
• Polymyxin B
• Buffered saline blank
• Sterility test control

Correct Answer: Pharmacopeial Reference Standard Endotoxin (RSE)

Q18. For turbidimetric LAL assays, what preparation step helps prevent sample-induced turbidity interference?

• Clarify the sample by centrifugation or filtration and perform appropriate dilutions before assay
• Always run the assay at pH 2.0 to prevent turbidity
• Increase protein concentration to mask turbidity
• Boil the sample for 60 minutes to remove color

Correct Answer: Clarify the sample by centrifugation or filtration and perform appropriate dilutions before assay

Q19. Which description correctly defines bacterial endotoxin?

• Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria that can induce pyrogenic responses
• Protein exotoxins secreted by Gram-positive bacteria causing targeted enzymatic effects
• Viral capsid proteins that trigger innate immune activation
• Fungal metabolites that are non-immunogenic to mammals

Correct Answer: Lipopolysaccharide (LPS) from the outer membrane of Gram-negative bacteria that can induce pyrogenic responses

Q20. What is the typical acceptable recovery range for spike recovery (inhibition/enhancement) during validation of an LAL test?

• 50% to 200% recovery
• 10% to 30% recovery
• 200% to 500% recovery
• 0% to 100% recovery only

Correct Answer: 50% to 200% recovery

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