AMES Test – OECD 471

What Is the AMES Test?

The AMES test is a genotoxicity assay used to evaluate the potential of chemical substances to induce genetic mutations. Conducted in accordance with OECD Test Guideline 471, this internationally recognized test uses specific bacterial strains to determine whether a chemical causes DNA mutations.

The AMES test is widely applied as an early screening tool for carcinogenic potential, as mutagenic substances are often associated with carcinogenicity.

Purpose of the AMES Test According to OECD 471

1. Evaluation of Mutagenic Potential

Determines whether chemical substances can cause permanent genetic changes in DNA.

2. Ensuring Chemical Safety

Assesses genotoxicity risks associated with chemical, pharmaceutical, cosmetic, and industrial products.

3. Identification of Carcinogenic Potential

Since mutagenicity is often correlated with carcinogenicity, the AMES test plays a critical role in early carcinogenic risk assessment.

4. Regulatory Compliance

Ensures compliance with OECD guidelines and other international regulatory requirements.

AMES Test Procedure

1. Test Organisms (Bacterial Strains)

The AMES test is typically performed using genetically modified strains of Salmonella typhimurium and Escherichia coli. These strains are highly sensitive to specific genetic mutations and enable detection of the mutagenic activity of test substances.

2. Test Methodology

a. Sample Preparation and Test Substance Application

• Test chemicals are prepared at multiple dose levels.

• Substances are applied to bacterial cultures.

b. S9 Metabolic Activation System

Some chemicals exhibit mutagenic effects only after metabolic activation. Therefore:

• An S9 mix containing mammalian liver enzymes is used to simulate metabolic conversion.

• Tests are conducted both with and without S9 metabolic activation.

c. Reverse Mutation Assay

If the test substance is mutagenic, it induces genetic mutations that allow bacteria to grow without specific growth factors.

d. Agar Plate Cultures

• Bacteria and test substances are plated on agar media.

• Revertant colonies are formed by mutated bacteria.

• The number of colonies is counted to determine mutation frequency.

3. Control Groups

• Positive Control: A known mutagenic substance to confirm test validity.

• Negative Control: Untreated bacteria used to establish baseline mutation rates.

Evaluation Criteria

Colony Count

• The degree of mutation induced by the test substance is determined by the number of revertant colonies.

• A substance is considered mutagenic if it produces a statistically significant increase in colonies compared to the negative control.

Dose–Response Relationship

• Assessment of whether mutation frequency increases with increasing concentrations of the test substance.

Effect of S9 Metabolic Activation

• Comparison of mutation rates in the presence and absence of S9 enzymes.

Applications of the AMES Test

Cosmetic Products

• Evaluation of genotoxicity risks associated with cosmetic ingredients.

Pharmaceutical Products

• Assessment of genotoxic potential of active pharmaceutical ingredients during drug development.

Industrial Chemicals

• Safety evaluation of chemicals with potential human or environmental exposure.

Food Additives and Colorants

• Genotoxicity assessment of substances used in food-contact applications.

OECD 471 and Regulatory Framework

• OECD Test Guideline 471: International guideline for AMES test performance and reporting

• REACH Regulation: Requires genotoxicity testing for chemical substances in the European Union

• FDA Regulations: Mandate genotoxicity testing for pharmaceuticals and biological products

• EC Regulation No. 1223/2009 (Cosmetics Regulation): Requires safety evaluation, including genotoxicity, for cosmetic ingredients

Advantages of the AMES Test

Rapid and Cost-Effective

A fast and economical method for genotoxicity screening.

High Sensitivity

Provides reliable detection of mutagenic potential.

Broad Applicability

Suitable for evaluating a wide range of chemical classes.

Regulatory Acceptance

Ensures compliance with international safety and regulatory standards.