OECDGenetic Toxicology Studies - Study Design of Bacterial Reverse Mutation Test (Ames Test) (OECD 471) 209
The Ames test, also known as the bacterial reverse mutation test, is a widely used in vitro genotoxicity assay employed to assess the potential of a substance to induce point mutations in bacterial DNA. It is standardized under the OECD Guideline 471 and plays a crucial role in ensuring the safety of chemicals, pharmaceuticals, food additives, and other agents before they reach the market.
Ames Test
An overview of the study design:
Objectives
- Determine if a test substance can induce mutations in specific genes of specially designed Salmonella typhimurium and/or Escherichia coli strains.
- Evaluate the mutagenic potency of the substance by assessing the dose-response relationship.
- Classify the substance according to its mutagenic potential for regulatory purposes.
Test Method
- Bacterial strains: The test employs specially-engineered Salmonella typhimurium and/or E. coli strains harboring mutations in genes responsible for histidine biosynthesis. These strains require histidine for growth and cannot survive on minimal media lacking histidine.
- Metabolic activation: Some chemicals require metabolic activation by enzymes (e.g., S9 mix from rat liver) to become mutagenic. The test is therefore conducted both with and without metabolic activation to cover a wider range of potential mutagens.
- Dosing: The test substance is administered to the bacteria at various concentrations, typically covering a range from non-toxic to potentially lethal.
- Exposure and incubation: The bacteria are exposed to the test substance for a specific time (usually 30 minutes) and then incubated in culture media for growth.
- Selection and scoring: After incubation, the bacteria are plated on minimal media lacking histidine. Histidine-independent revertant colonies, arising from mutations that restore the ability to synthesize histidine, are counted.
Endpoints
- Number of histidine-independent revertant colonies at each dose level.
- Dose-response relationship for mutagenicity.
- Mutagenic index (ratio of revertants per treated cell to revertants per control cell).
Classification of the test substance according to OECD 471 criteria (e.g., mutagenic, non-mutagenic).
Benefits of the Ames Test
- Relatively simple and cost-effective.
- Rapid and sensitive in detecting point mutations.
- Can be adapted to assess a wide range of chemicals.
- Used in a tiered testing strategy for genotoxicity assessment.
Limitations of the Ames Test
- Cannot detect all types of genetic damage (e.g., chromosomal aberrations).
- Does not directly assess mutagenic risk in humans.
- Relies on specific bacterial strains and may not capture all potential mutagens.
Additional Resources
- OECD Test Guideline 471:
https://www.oecd.org/chemicalsafety/risk-assessment/1948418.pdf - National Toxicology Program information on Ames Test:
https://ntp.niehs.nih.gov/whatwestudy/testpgm/genetic - The Ames Test: A Review
https://pubmed.ncbi.nlm.nih.gov/6341825/
