HepGentox: a novel promising HepG2 reportergene-assay for the detection of genotoxic substances in complex mixtures.

BACKGROUND: In risk assessment, genotoxicity is a key factor to determine the safety for the consumer. Most in vitro genotoxicity assays were developed for the assessment of pure substances. However, in recent years more attention has been given to complex mixtures, where usually low amounts of a substance are present. For high-throughput screening, a toxicologically sensitive assay should be used, covering a broad range of genotoxic substances and detecting them at low concentrations. HepG2 cells have been recommended as one of the prime candidates for genotoxicity testing, as they are p53 competent, less prone towards cytotoxic effects and tend to have some metabolic activity. METHODS: A HepG2 liver cell line was characterized for its suitability for genotoxicity assessment. For this, a luciferase based reporter gene assay revolving around the p53 pathway was validated for the analysis of pure substances and of complex mixtures. Further, the cell's capability to detect genotoxins correctly with and without an exogenous metabolizing system, namely rat liver S9, was assessed. RESULTS: The assay proved to have a high toxicological sensitivity (87.5%) and specificity (94%). Further, the endogenous metabolizing system of the HepG2 cells was able to detect some genotoxins, which are known to depend on an enzymatic system. When complex mixtures were added this did not lead to any adverse effects concerning the assays performance and cytotoxicity was not an issue. DISCUSSION: The HepGentox proved to have a high toxicological sensitivity and specificity for the tested substances, with similar or even lower lowest effective concentration (LEC) values, compared to other regulatory mammalian assays. This combines some important aspects in one test system, while also being less time and material consuming and covering several genotoxicity endpoints. As the assay performs well with and without an exogenous metabolizing system, no animal liver fractions have to be used, which application is discussed controversially and is considered to be expensive and laborious in sample testing. Because of this, the HepGentox is suitable for a cost-efficient first screening approach to obtain important information with human cells for further approaches, with a relatively fast and easy method. Therefore, the HepGentox is a promising assay to detect genotoxic substances correctly in complex mixtures even at low concentrations, with the potential for a high throughput application. In a nutshell, as part of an in vitro bioassay test battery, this assay could provide valuable information for complex mixtures.

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

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/ß-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.

Evaluation of the Suitability of Mammalian In Vitro Assays to Assess the Genotoxic Potential of Food Contact Materials.

BACKGROUND: Non-targeted screening of food contact materials (FCM) for non-intentionally added substances (NIAS) reveals a great number of unknown and unidentified substances present at low concentrations. In the absence of toxicological data, the application of the threshold of toxicological concern (TTC) or of EU Regulation 10/2011 requires methods able to fulfill safety threshold criteria. In this review, mammalian in vitro genotoxicity assays are analyzed for their ability to detect DNA-damaging substances at limits of biological detection (LOBD) corresponding to the appropriate safety thresholds. RESULTS: The ability of the assays to detect genotoxic effects varies greatly between substance classes. Especially for direct-acting mutagens, the assays lacked the ability to detect most DNA reactive substances below the threshold of 10 ppb, making them unsuitable to pick up potential genotoxicants present in FCM migrates. However, suitability for the detection of chromosomal damage or investigation of other modes of action makes them a complementary tool as part of a standard test battery aimed at giving additional information to ensure safety. CONCLUSION: improvements are necessary to comply with regulatory thresholds to consider mammalian genotoxicity in vitro assays to assess FCM safety.

Value and limitation of in vitro bioassays to support the application of the threshold of toxicological concern to prioritise unidentified chemicals in food contact materials.

Some of the chemicals in materials used for packaging food may leak into the food, resulting in human exposure. These include so-called Non-intentionally Added Substances (NIAS), many of them being unidentified and toxicologically uncharacterized. This raises the question of how to address their safety. An approach consisting of identification and toxicologically testing all of them appears neither feasible nor necessary. Instead, it has been proposed to use the threshold of toxicological concern (TTC) Cramer class III to prioritise unknown NIAS on which further safety investigations should focus. Use of the Cramer class III TTC for this purpose would be appropriate if amongst others sufficient evidence were available that the unknown chemicals were not acetylcholinesterase inhibitors or direct DNA-reactive mutagens. While knowledge of the material and analytical chemistry may efficiently address the first concern, the second could not be addressed in this way. An alternative would be use of a bioassay capable of detecting DNA-reactive mutagens at very low levels. No fully satisfactory bioassay was identified. The Ames test appeared the most suitable since it specifically detects DNA-reactive mutagens and the limit of biological detection of highly potent genotoxic carcinogens is low. It is proposed that for a specific migrate, the evidence for absence of mutagenicity based on the Ames test, together with analytical chemistry and information on packaging manufacture could allow application of the Cramer class III TTC to prioritise unknown NIAS. Recommendations, as well as research proposals, have been developed on sample preparation and bioassay improvement with the ultimate aim of improving limits of biological detection of mutagens. Although research is still necessary, the proposed approach should bring significant benefits over the current practices used for safety evaluation of food contact materials.

Suitability of the Ames test to characterise genotoxicity of food contact material migrates.

Non-intentionally added substances (NIAS) are chemical impurities which can migrate from packaging materials (FCM) into food. Safety assessment of NIAS is required by European law, but currently there is no comprehensive testing strategy available. In this context, one key element is to get insight on the potential presence of genotoxic NIAS in FCM migrates. This raises questions about the limit at which genotoxins can be detected in complex mixtures such as FCM migrates, and if such limits of detection (LOD) would be compatible with safety. In this context, the present review assesses the suitability of the Ames assay to address genotoxicity of FCM migrates. Lowest effective concentrations of packaging-related and other chemicals in test media were retrieved from scientific literature and used as surrogates of LODs to be benchmarked against a value of 0.01 mg kg-1 (10 ppb) in migrates. This is a pragmatic threshold used in FCM safety evaluation to prioritise substances requiring proper identification and risk assessment. The analysis of the data shows that only potent genotoxins can theoretically be detectable at a level of 0.01 mg kg-1 in migrates or food. Only a minority (10%) of genotoxic chemicals reported to be associated with FCMs could be picked up at a level of 0.01 mg kg-1 or lower. Overall, this review shows that the Ames test in its present form cannot be used as standalone method for evaluating the genotoxic potential of FCM migrates, but must be used together with other information from analytical chemistry and FCM manufacturing.