Ames test (OECD 471)

The Ames test is recognized as the most relevant and frequently used in vitro assay for the detection of DNA reactivity, i.e., mutagenicity potential of chemical substances. It has a well-established method recommended in the OECD 471 guideline and is used for the safety assessment of chemicals in food contact materials (FCM) (EFSA 2016) and industrial chemicals (OECD 2016). The Ames test is known for its high sensitivity in detecting mutagens, with reported sensitivity values ranging from 93% to 97% when compared to rodent carcinogenicity assays. However, its specificity is generally lower, typically ranging from 55% to 86%, meaning it can produce some false positive results (Trejo-Martin et al., 2022). FCM extracts have been assessed using the Ames test but in general, the data obtained indicated evidence of the absence of mutagenicity/genotoxicity potential (Séverin et al. 2016). Very little literature is available on the capability of this test to detect low levels of mutagenic impurities in complex mixtures such as FCM migrates and the Limit of Detection (LOD) of this test is inadequate. It was observed that only very potent mutagens could potentially be detected by the standard AMES test at levels relevant for safety and regulatory purposes, i.e., TTC value of 0.15 ppb (Rainer et al., 2018). This highlighted the intrinsic limitations of the standard Ames test regarding the detection of mutagens at low concentrations in complex mixtures.

Ames Microplate Format (MPF) Assay

Ames Microplate Format (MPF) assay, a high-throughput version of the traditional Ames test was introduced for the mutagenicity assessment of the FCM extracts with low concentrations of NIAS. This is a miniaturized version of the plate agar Ames tests that takes advantage of a liquid microplate approach in 384-well plates with a color change-based readout. Ames MPF assay uses the same principle and tester strains (e.g., Salmonella typhimurium and Escherichia coli) as the standard Ames test but in a microplate format. Ames MPF assay enables simultaneous processing of multiple replicates and performs equally well as the standard Ames test. It requires smaller sample volumes, is fast, relatively simple and cost-effective and therefore can be a useful tool for screening of FCM migrates (Rainer B et al., 2019).

While some miniaturized testing versions are referred in the OECD 471 guideline, this guideline does not formally consider the miniaturized format sufficient and the regulatory acceptance of such approaches is still limited (OECD 2022; Spiliotopoulos and Koelbert, 2020). Consequently,  Ames MPF assay is not considered suitable for testing the pure substances, nevertheless can be used for the primary screening in early stages of product development, or to assess the impurities with limited quantity. The ICH guideline for evaluating the mutagenic potential of impurities in pharmaceuticals (ICH, 2023) identifies Ames MPF as a suitable alternative during the limited available quantity of the impurity. In the Detailed Review Paper (DRP) published by the OECD in 2022, a good agreement between the Ames MPF assay and standard assay was reported and when only a limited amount of testing material is available including NIAS migrating from FCMs, Ames MPF assay is then certainly acceptable and permitted (OECD 2022).

A particular challenge while conducting the Ames MPF assay is the extraction and concentration of the DNA-reactive genotoxic substances that are potentially present in the FCM extracts. It is important to ensure that the limit of detection of the Ames MPF assay is not compromised, leading to false negative results. In order to obtain higher substance concentrations for easier detection by the Ames MPF assay, suitable evaporation and concentration methods needs to be standardized depending on the physicochemical properties of the envisaged NIAS of the FCM extracts under investigation (Mayrhofer E et al., 2023). Hence, it is quite essential to standardize a suitable extraction method in advance prior to the initiation of the Ames MPF assay in order to obtain required limit of detection of the assay and reproducible study findings.

The positive and negative results obtained with the Ames MPF method should be interpreted in compliance with EFSA statement on genotoxicity assessment of chemical mixtures (EFSA, 2018). If the testing of the FCM extract provides a clear negative result, the mixture does not raise concern for DNA reactivity. If Ames MPF method provides positive result, it can be concluded that the FCM extract does raise a concern about DNA reactivity. Nevertheless, further analytical characterization of the FCM extract could elucidate the specific NIAS with DNA reactivity, which will enable the ink manufacturers to implement the specific technical measures to mitigate its formation/migration.

It should be noted that Ames MPF assay is considered as powerful tool to study genotoxic hazard assessment of FCM extracts and not the final indicator of potential risk to the consumer. Even in case of positive findings in the Ames MPF assay, the potential health concern due to genotoxic substances could still be mitigated as long as its migration is maintained with the applicable limit of 0.15 ppb for genotoxic compounds.

Use of the Ames MPF assay in recycling projects

Scientific projects like “PolyCycle” and “SafeCycle” were conducted by Fraunhofer Institute, Germany and Industry Association for Food Technology and Packaging, Germany for the comprehensive analysis of recyclates and their applicability for different packaging scenarios. The objectives of these projects are to develop and validate the testing strategy for carcinogens in recycled plastics and to clarify the origin of any systemic contamination. In these projects, it was found that the performance of the Ames MPF assay is in agreement with the standard agar based Ames assay while identifying the genotoxic components in post-consumer recycled (PCR) plastics/simulated PCRs.

Similar high-throughput, cost effective assays are under development to detect structural (clastogens) and numerical (aneugens) chromosomal damage of the FCM extracts with low concentrations of NIAS.