Genotoxicity testing: Ames test and in vitro assays MCQs With Answer

Introduction:

This quiz set focuses on genotoxicity testing with emphasis on the Ames test and complementary in vitro assays, tailored for M.Pharm students. It highlights principles, strains, metabolic activation (S9), assay designs, control requirements, and interpretation criteria used to detect gene mutations, DNA strand breaks, and chromosomal damage. Questions also cover commonly used mammalian in vitro assays such as the comet assay, micronucleus test, and gene mutation assays at HPRT/TK loci, along with OECD guideline references and practical limitations. The aim is to deepen conceptual understanding and prepare students for designing, analyzing, and critically evaluating genotoxicity data in regulatory and research settings.

Q1. What type of genetic damage is primarily detected by the Ames (bacterial reverse mutation) test?

• Large-scale chromosomal rearrangements
• Point mutations (base-pair substitutions and frameshifts) in bacteria
• Whole chromosome loss (aneuploidy)
• DNA methylation patterns

Correct Answer: Point mutations (base-pair substitutions and frameshifts) in bacteria

Q2. What is the main purpose of adding an S9 mix to an Ames test?

• To inhibit bacterial DNA repair enzymes
• To provide metabolic activation (enzymes) to simulate mammalian metabolism
• To change the pH of the medium for better colony growth
• To act as a positive control mutagen

Correct Answer: To provide metabolic activation (enzymes) to simulate mammalian metabolism

Q3. Which mutation type is the Salmonella strain TA98 primarily used to detect in the Ames assay?

• Base-pair substitutions
• Frameshift mutations
• Chromosomal translocations
• Large deletions

Correct Answer: Frameshift mutations

Q4. How does the pre-incubation modification of the Ames test differ functionally from the plate incorporation method?

• Pre-incubation uses a different bacterial strain
• Pre-incubation eliminates the need for S9 mix
• Pre-incubation allows longer contact between test compound and bacteria with S9, increasing sensitivity for some pro-mutagens
• Pre-incubation replaces minimal agar with nutrient-rich agar

Correct Answer: Pre-incubation allows longer contact between test compound and bacteria with S9, increasing sensitivity for some pro-mutagens

Q5. Which controls are essential for a valid Ames test?

• Only a vehicle (negative) control
• Only a positive control using a known mutagen
• Both vehicle (negative) and known mutagen (positive) controls
• No controls are required if strain is standardized

Correct Answer: Both vehicle (negative) and known mutagen (positive) controls

Q6. What indicates cytotoxicity to bacteria in an Ames plate assay?

• Increased number of revertant colonies above historical control
• Reduced background lawn and decreased bacterial growth
• No change in colony morphology
• Appearance of large, white colonies only

Correct Answer: Reduced background lawn and decreased bacterial growth

Q7. Which OECD Test Guideline covers the bacterial reverse mutation (Ames) test?

• OECD Test Guideline 476
• OECD Test Guideline 471
• OECD Test Guideline 487
• OECD Test Guideline 473

Correct Answer: OECD Test Guideline 471

Q8. What primary lesion does the alkaline comet assay detect in single cells?

• Micronuclei formation
• DNA strand breaks and alkali-labile sites at single-cell level
• Point mutations in specific genes
• Telomere shortening

Correct Answer: DNA strand breaks and alkali-labile sites at single-cell level

Q9. What does a positive result in an in vitro micronucleus assay generally indicate?

• Protein misfolding
• Chromosomal breakage (clastogenicity) or whole chromosome loss (aneugenicity)
• Mitochondrial DNA damage only
• Only point mutations

Correct Answer: Chromosomal breakage (clastogenicity) or whole chromosome loss (aneugenicity)

Q10. What is the role of cytochalasin B in the cytokinesis-block micronucleus (CBMN) assay?

• To induce DNA strand breaks for positive control
• To block cytokinesis and score binucleated cells
• To stain micronuclei for easier visualization
• To act as a metabolic activation enzyme

Correct Answer: To block cytokinesis and score binucleated cells

Q11. Which loci are commonly used in in vitro mammalian cell gene mutation assays?

• BRCA1 and BRCA2
• HPRT and TK (thymidine kinase)
• P53 and RAS oncogenes
• 18S and 28S rRNA genes

Correct Answer: HPRT and TK (thymidine kinase)

Q12. How do clastogens differ mechanistically from aneugens?

• Clastogens change epigenetic marks; aneugens cause base substitutions
• Clastogens cause DNA/chromosome breaks; aneugens interfere with spindle leading to whole chromosome loss
• Clastogens only act in bacteria; aneugens only act in mammals
• Clastogens cause oxidative stress; aneugens cause thymidine depletion

Correct Answer: Clastogens cause DNA/chromosome breaks; aneugens interfere with spindle leading to whole chromosome loss

Q13. Which limitation is most characteristic of the Ames test?

• It gives direct information on large animal reproductive toxicity
• It cannot detect chromosomal aberrations or aneugenic effects
• It requires primary human hepatocytes for activation
• It is insensitive to all small molecule mutagens

Correct Answer: It cannot detect chromosomal aberrations or aneugenic effects

Q14. From what source and induction method is S9 fraction commonly prepared for metabolic activation in genotoxicity assays?

• Human blood plasma treated with heat shock
• Phenobarbital/β‑naphthoflavone induced rat liver S9 fraction
• Uninduced mouse brain homogenate
• Synthetic enzyme mix without animal source

Correct Answer: Phenobarbital/β‑naphthoflavone induced rat liver S9 fraction

Q15. Sodium azide is commonly used as a positive control for which Salmonella strain in the Ames test?

• TA98
• TA100
• TA1537
• TA1535

Correct Answer: TA100

Q16. Which procedural description matches the plate incorporation method of the Ames test?

• Mix bacteria, test compound and S9 in top agar then pour onto minimal agar plate (plate incorporation)
• Pre-incubate bacteria overnight without agar before plating
• Grow bacteria on nutrient agar for 48 hours before exposure
• Use liquid culture only with spectrophotometric endpoint

Correct Answer: Mix bacteria, test compound and S9 in top agar then pour onto minimal agar plate (plate incorporation)

Q17. Which OECD guideline specifically addresses the in vitro mammalian cell micronucleus test?

• OECD Test Guideline 476
• OECD Test Guideline 473
• OECD Test Guideline 487
• OECD Test Guideline 471

Correct Answer: OECD Test Guideline 487

Q18. How is a positive mutagenic signal typically defined in the context of an Ames assay?

• A single plate with any colonies
• A reproducible, dose-related increase in revertant colonies not attributable to cytotoxicity indicates mutagenic potential
• Complete inhibition of all bacterial growth
• Only an increase in lawn density without colony formation

Correct Answer: A reproducible, dose-related increase in revertant colonies not attributable to cytotoxicity indicates mutagenic potential

Q19. Which cofactors are typically included in the S9 cofactor mix to support metabolic activity?

• ATP and DNA polymerase
• A cofactor mix including NADP and glucose-6-phosphate is required for S9 activity
• Hemoglobin and myoglobin
• Vitamin C and E only

Correct Answer: A cofactor mix including NADP and glucose-6-phosphate is required for S9 activity

Q20. Which quality control practice is important for interpreting variability in genotoxicity assays like Ames or micronucleus tests?

• Ignoring historical data when controls pass once
• Use of historical control ranges to interpret assay variability and acceptability
• Only running positive controls and no negative controls
• Running assays without replication to save resources

Correct Answer: Use of historical control ranges to interpret assay variability and acceptability

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