Genetic Toxicology Studies - Study Design of In Vitro Mammalian Chromosomal Aberration Test (CHO cells) (OECD 473)
The in vitro mammalian chromosomal aberration test (CHO cells) is another key genotoxicity assay, outlined in the OECD Guideline 473, to assess the potential of a substance to induce structural changes in chromosomes of cultured mammalian cells. This test specifically utilizes Chinese hamster ovary (CHO) cells due to their well-defined karyotype and sensitivity to a wide range of clastogenic agents.
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
- Cell line: CHO cells are grown in culture media under controlled conditions.
- Metabolic activation: Similar to the Ames test, the study is conducted with and without metabolic activation, using an S9 mix from rat liver for the former.
- Dosing: The test substance is administered to the cells at various concentrations, typically spanning from non-toxic to potentially cytotoxic levels.
- Exposure and harvest: Cells are exposed to the substance for a defined time (typically 3-6 hours) and then allowed to recover for a specific period (e.g., 16-24 hours) to allow for cell division and chromosomal aberrations to manifest.
- Metaphase arrest and cell fixation: Cells are treated with a metaphase-arresting agent to capture dividing cells and then fixed for microscopic analysis.
- Slide preparation and scoring: Cell chromosomes are stained and visualized under a microscope. Aberrant chromosomes (e.g., breaks, gaps, exchanges) are counted and scored in a defined number of cells.
Endpoints
- Number of cells with chromosomal aberrations at each dose level.
- Frequency of different types of chromosomal aberrations (e.g., gaps, breaks, exchanges).
- Dose-response relationship for chromosomal aberrations.
- Mitotic index (percentage of dividing cells) as a measure of cytotoxicity.
- Clastogenic index (ratio of cells with aberrations to control cells).
- Classification of the test substance according to OECD 473 criteria (e.g., clastogenic, non-clastogenic).
Benefits of the CHO Cell Test
- Relatively simple and cost-effective.
- Sensitive in detecting structural chromosomal aberrations.
- Can be adapted to assess a wide range of chemicals.
- Used in a tiered testing strategy for genotoxicity assessment.
Limitations of the CHO Cell Test
- Cannot detect all types of genetic damage (e.g., point mutations).
- Does not directly assess mutagenic risk in humans.
- May not capture all potential clastogenic agents due to limitations in metabolic activation.
Additional Resources
- OECD Test Guideline
473:https://www.oecd.org/env/ehs/testing/test-no-473-in-vitro-mammalian-chromosomal-aberration-test-9789264264649-en.htm - National Toxicology Program information on CHO cell test:
https://ntp.niehs.nih.gov/whatwestudy/testpgm/genetic - In Vitro Mammalian Chromosomal Aberration Test: A Review:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2700603/
