Analyzing the Lowest Effective Dosage in the Ames Test

Food contact materials come in a variety of different forms, be it cans or Styrofoam containers. Yes, FCMs do need to be assessed for safety, but due to the European regulatory framework, the food industry needs to determine the safety of non-intentionally added substances, known as NIAS, which could possibly migrate into food.

Typically, FCMs can protect food because they are stable materials – yet food could possibly be in contact with FCMs for a long time. In order to simulate the migration of intentionally added substances (IAS) and NIAS into food migration, Gentronix performs experiments under exaggerated conditions. The migrations can identify NIAS and IAS for safety assessments or can be used for bioassays on the migration as an element of the toxicology characterization.

Yet NIAS tends to occur in low concentrations and pinpointing them can be difficult. The NIAS risk assessment has been challenging, partially caused by small amounts of migrating available for testing and questions about the sensitivity of testing methods. A certain level of pragmatism is needed, as it is not possible to test every single substance of a migration. The industry has proposed applying the threshold of toxicological concern (TTC) Cramer class III threshold set at 90 ppb – unfortunately, this assumes less an absence of direct DNA reactivity of substances amongst the sample of migration.

 

The Utility of the Ames Test

The Ames test has been present in genotoxicity assessments for an extremely long time, and when it is combined with the in vitro micronucleus assay, you can pinpoint detect antigens, clastogens, and mutations. The Ames test uses many bacteria strains that have mutations of certain loci. When it grows grown on agar plates that have small quantities of amino acid histidine, it is only the bacteria that have mutated that will have redeveloped the ability to grow after the depletion of the histidine. Because of this, growth in the number of rising colonies implies the tested chemical has caused mutations within the testing system and should be used in further investigations investigated.

 

Typically speaking, the Ames test detects point mutations that are relevant to safety assessment as they often occur from directly interacting amongst DNA – meaning they could be potent due to having a linear dose-response.

 

Determining the Lowest Effective Dose in the Ames Test

 

Gentronix examined how sensitive the Ames test is and compared it to the possible threshold concentrations of relevance to NIAS safety assessment. The questions they wanted to get to the bottom of was, how many of the substances would be thought to be detected as positive in an Ames assay FCM migration sample if present in at regulated limits? How much confidence could be put on a negative Ames test of a migration sample?

 

For this to be done, their companion in this, Lhasa, thoroughly checked their Vitic database – this is an amalgamation of Ames test dose-response data that is available to the public. Different Ames tests are conducted in different manners, but a standardized approach to the test is given via OECD test guideline 471 which is meant to provide a robust study design that is fitting for a regulatory assessment. Using these aforementioned principles, they eliminated study records that didn’t match the quality criteria, including studies that didn’t use the standard tester strains when being conducted.

 

Conclusion

All details of the analysis cannot be given here as Gentronix is looking to include them in a scientific journal soon. However, some of the key conclusions can be shared for you today.

In total, more than 1,200 substances have been collated from the Vitic database. Data analytics have been used to find the lowest dose and the fold increase at which a mutagenic response can be detected. The lowest dose for an Ames positive test is 1 ng/plate for this data set, highlighting the Ames test can find potent mutagens. Using the 10ppb limit set by the food contact industry, 11.3% of the substances in the dataset were therefore detected via the Ames test. Gentronix also investigated the lowest effective doses in mammalian genotoxicity assays in vitro – no assay, however, was more sensitive than the Ames test, when judged by the lowest effective doses.

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