Direct Comparison of the Lowest Effect Concentrations of Mutagenic Reference Substances in Two Ames Test Formats.

Bernhard Rainer,Lukas Prielinger,Manfred Tacker,Maricel Marin-Kuan,Elisabeth Pinter,Chiara Coppola,Silvia Apprich,Benoit Schilter

doi:10.3390/toxics9070152

Abstract

The Ames assay is the standard assay for identifying DNA-reactive genotoxic substances. Multiple formats are available and the correct choice of an assay protocol is essential for achieving optimal performance, including fit for purpose detection limits and required screening capacity. In the present study, a comparison of those parameters between two commonly used formats, the standard pre-incubation Ames test and the liquid-based Ames MPF™, was performed. For that purpose, twenty-one substances with various modes of action were chosen and tested for their lowest effect concentrations (LEC) with both tests. In addition, two sources of rat liver homogenate S9 fraction, Aroclor 1254-induced and phenobarbital/β-naphthoflavone induced, were compared in the Ames MPF™. Overall, the standard pre-incubation Ames and the Ames MPF™ assay showed high concordance (>90%) for mutagenic vs. non-mutagenic compound classification. The LEC values of the Ames MPF™ format were lower for 17 of the 21 of the selected test substances. The S9 source had no impact on the test results. This leads to the conclusion that the liquid-based Ames MPF™ assay format provides screening advantages when low concentrations are relevant, such as in the testing of complex mixtures.

Highlights

In multiple fields dealing with chemical safety, the Ames test plays an important role for the detection of DNA-reactive genotoxic substances and is recommended to be included as part of a battery of genetic toxicology tests by EFSA [1]
Together with a concentration factor that can be achieved during sample preparation, lowest effect concentration (LEC) values can be converted into limits of biological detection (LOBD) of the test procedure, which refers to the lowest concentration of a substance that can be detected in a sample [10]
Twenty substances classified as mutagenic were analysed for the comparison of the LEC values of the standard pre-incubation Petri dish agar-based Ames and the Ames MPFTM

Summary

Introduction

In multiple fields dealing with chemical safety, the Ames test plays an important role for the detection of DNA-reactive genotoxic substances (mutagens) and is recommended to be included as part of a battery of genetic toxicology tests by EFSA [1]. Further applications include food safety assessment [9], safety evaluation of packaging materials [10,11], testing of medical plant extracts [12] or testing materials of importance for the chemical industry such as mineral oils [13] Overall, those areas raise a common issue, which is the need to assess the mutagenicity of low-level contaminants potentially present in complex mixtures. It has to be low enough to meet regulatory/safety requirements and this in the presence of complex sample matrices, which may interfere with the test results In this context, the LEC refers to the lowest measured concentration of a mutagenic substance that causes a measurable effect on the test bacteria strains. Together with a (hypothetical) concentration factor that can be achieved during sample preparation, LEC values can be converted into limits of biological detection (LOBD) of the test procedure, which refers to the lowest concentration of a substance that can be detected in a sample [10]

Methods

Results

Conclusion