Mutagenicity Testing in Pharmaceutical Development

The Ames test has been used for decades in testing mutagenic property of chemicals, pharmaceutical candidates and food additives. This test is required as part of testing regiments before first-in-human studies, and most regulatory agencies, including the U.S. Food and Drug Administration (FDA) mandate a negative result in the Ames assay before continuing with candidate selection. In addition, intermediates, reagents, and reaction by-products inevitably end up in the final products as impurities during the manufacture of active pharmaceutical ingredients (APIs). The safety of a drug product is dependent not only on the toxicological properties of the active drug substance itself, but on the impurities it contains which necessitates stringent testing for these potential sources of genotoxicity. 

The Salmonella typhimurium reverse mutation assay or the Ames test remains the most widely employed test for assessing the mutagenic properties of drug candidates and impurities. This system is a microbial assay that uses a set of histidine or tryptophan-requiring strains of bacteria to detect frameshifts and base pair substitution mutations. Compound-induced mutations in the histidine or tryptophan operon can shift growth of the strains from a required to an independent phenotype. The change in the growth phenotype represents an indicator of mutagenic response and the Ames test has a sensitivity of 76–78% for detecting human carcinogens which supports its continued use in research and development. 

A copy of the FDA guidance document for genotoxicity screening of pharmaceuticals is provided here

References 

  1. Eastmond DA, Hartwig A, Anderson D, Anwarb WA, Cimino MC, Dobrev I, et al. (2009) Mutagenicity testing for chemical risk assessment: Update of the WHO/IPCS Harmonized Scheme. Mutagenesis, 24, 341-349
  2. Parasuraman, S. (2011). Toxicological screening. Journal of Pharmacology and Pharmacotherapeutics, 2, 74-78.
  3. https://www.fda.gov/downloads/drugs/guidances/ucm074931.pdf

 

OECD 471 Guideline for Testing of Chemicals

EBPI has developed a line of Ames Test kits that are designed to meet OECD 471 Guidelines; these include the Ames 384-ISO (6041S9-OECD) as well as our highly popular Ames MOD-ISO (5041S9-OECD).  

These OECD Ames Test kits allow users to run samples with and with a metabolic activation, in triplicates and 5 dilutions while still including all controls required (background, negative controls and positive controls).

Under the OECD 471 Guidelines, at least five strains of bacteria should be used and EBPI has chosen the following 

Strains:

  • TA1535
  • TA1537 or TA97a or TA97
  • TA98
  • TA100
  • E.coli WP2 strain  

(Note: if other strains are required, please advise and EBPI will include your required strains).

Controls:

  • Sodium azide [CAS no. 26628-22-8] TA1535 and TA100 
  • 2-Nitrofluorene [CAS no. 607-57-8] TA98  
  • 9-Aminoacridine [CAS no. 90-45-9] TA97a 
  • 4-nitroquinoline 1-oxide [CAS no. 56-57-5] WP2, WP2 uvrA and WP2 uvrA (pKM101)
  • 2-Aminoanthracene [CAS no. 613-13-8] (In-direct Positive Control)

(Note: If required, EBPI can include various other direct or in-direct controls such as B(a)P.  Please indicate when purchasing).

Reagents:

EBPI’s Ames 384-ISO (product no. 6041) along with our Ames MOD-ISO (Product no. 5061) meet these requirements.  Each set of reagents allows for 24 exposure points, ensuring that our Ames Test protocol for the OECD 471 guidelines meets your requirements and regulatory requirements.  The OECD 471 packages are designed for 1 sample, 5 sample or 10 sample kits allowing solvent controls, dilutions, replicates and positive controls to be tested in a simple, low cost and efficient manner.  EBPI’s OECD kits can be purchased with or without plastics depending on your laboratory requirements.

Product Descriptions are as follows: 

Ames 384-ISO (samples are tested in 384-well plates):

  • 6041S9-OECD-1S (5 Vials of Bacteria, 10 Sets of Reagents, S9 Activation for 1 Sample)
  • 6041S9-OECD-1SP (5 Vials of Bacteria, 10 Sets of Reagents, S9 Activation for 1 Sample and Plastics for 30 384-well plates)
  • 6041S9-OECD-5S (25 Vials of Bacteria, 50 Sets of Reagents, S9 Activation for 5 Samples)
  • 6041S9-OECD-5SP (25 Vials of Bacteria, 50 Sets of Reagents, S9 Activation for 5 Samples and Plastics for 150 384-well plates)
  • 6041S9-OECD-10S (50 Vials of Bacteria, 100 Sets of Reagents, S9 Activation for 5 Samples)
  • 6041S9-OECD-10SP (50 Vials of Bacteria, 100 Sets of Reagents, S9 Activation for 5 Samples and Plastics for 300 384-well plates)

Ames-MOD ISO (samples are tested in 96-well plates):

  • 5041S9-OECD-1S (5 Vials of Bacteria, 10 Sets of Reagents, S9 Activation for 1 Sample)
  • 5041S9-OECD-1SP (5 Vials of Bacteria, 10 Sets of Reagents, S9 Activation for 1 Sample and Plastics for 120 96-well plates)
  • 5041S9-OECD-5S (25 Vials of Bacteria, 50 Sets of Reagents, S9 Activation for 5 Samples)
  • 5041S9-OECD-5SP (25 Vials of Bacteria, 50 Sets of Reagents, S9 Activation for 5 Samples and Plastics for 600 96-well plates)
  • 5041S9-OECD-10S (50 Vials of Bacteria, 100 Sets of Reagents, S9 Activation for 5 Samples)
  • 5041S9-OECD-10SP (50 Vials of Bacteria, 100 Sets of Reagents, S9 Activation for 5 Samples and Plastics for 1200 96-well plates)

Please contact EBPI for pricing and shipping costs.

 

OECD 471 Guideline for Testing of Chemicals