How low can you go? An analysis of the lowest effective dose in the Ames test Grace Kocks Senior Database Scientist grace.kocks@lhasalmited.org
Overview • Food contact material industry and the challenge. • Why are we interested in the lowest effective dose in the Ames test? • The story won’t end with the Ames test. • Conclusion - How low can you go?
Food contact materials Non-intentionally added Food contact materials substances (NIAS) (FCM) NIAS are chemicals that have Food contact materials are not been added for a technical materials that are intended to reason during the production be in contact with food. process. Often unknown & • Multiple types in 1 product unintentional materials. • Impurities from IAS components • Oligomers • Degradation products Intentionally added substances (IAS) Substances that are essential in the manufacturing process, these substances give the FCM its desired properties. • Antioxidants • Fillers • Additives
Potential source of genotoxic contaminants? Mertens et al., 2017 reviewed materials used in FCM coatings. We know there are potential genotoxic substances contained within FCM. If IAS can potentially be genotoxic, what about NIAS?
Experimental approach to address the challenge Detection of NIAS(s), possible quantification. Structural determination is unlikely. Hazard identification 10µg/kg limit and threshold of toxicological concern (TTC) 90 µg/person. OR Exposure threshold 10 µg/kg limit Perform extraction and migration tests EU plastics guidance on FCM Genotoxicity in vitro e.g. Ames test Concentrate Can the Ames test be used to assess genotoxic liability of detected NIAS?
Why are we interested in the lowest effective dose in the Ames test? Performing in vitro testing may prove difficult due to: • Insufficient sensitivity of the test methods at low doses. • The small amount of migrate produced. The Ames test is a sensitive assay and is one of the most common tests used for assessing the mutagenicity of impurities. Kenyon et al ., • Covered 454 Ames positive compounds – but there is a lot more data out there than that. • Focus was on looking at whether sensitivity was suitable to assess known concentrations of impurities, with known identities. Kenyon MO. 2007 An evaluation of the sensitivity of the Ames assay to discern low -level mutagenic impurities. Regulatory Toxicology and Pharmacology 48:75-86. [4]
What is the proposed safety cut-off for NIAS? • The European Food Safety Authority (EFSA) has produced guidance on NIAS in plastics – but this doesn’t apply to other FCMs yet. • The regulations on plastics state that any non-regulated substance migrating into food at a level at 0.01 mg/kg (10 ppb) needs to be risk assessed. Is the conventional Ames test sufficiently sensitive to detect toxicity at very low doses, 10ppb in migration sample?
Methodology • Dataset of 1,222 substances with at least one positive study. The lowest effective doses (LEDs) ranged from 0.001-20,000 µg/plate. • We later removed the ones that were only positive at >5,000 µg/plate.
Lowest effective dose distribution The Ames test can detect mutagens at very low doses. Minimum LED of 1 ng/plate was observed.
Turning doses into concentrations In line with Rainer et al., where they reported 10% of genotoxic FCMs being detected at 10ppb threshold (using a smaller µg/plate Converts 0.4 mg/L threshold limit 2.7 (top agar) units to µg/ml dataset, n=40). Using the Limit of detection of 10 ppb (0.01 mg/L), Accounts for the dilution in same methodology with and a 40-fold concentration factor the top agar of the plate. proposed for migrations in the Ames test, our larger this gives a threshold of 0.4 mg/L. dataset, n=1,222: 11.3% (138/1,222) CAS number Minimum µg/plate Minimum mg/L Below threshold of substances in the dataset 100-00-5 333.3 14.68 would be expected to be 100-01-6 333 14.67 positive. 100-02-7 333 14.67 100-14-1 5 0.22 <= 0.4 mg/L Rainer B. 2018. Suitability of the Ames test to characterise genotoxicity of food contact material migrates. Food Additives & Contaminants: Part A 35:2230-2243.
The story won’t end with just the Ames test OR Ames test Concentrated In vitro migrate could be? mammalian assay EFSA consistently requires assessment of genotoxicity rather than just mutagenicity, so typically an Ames test and in vitro Micronucleus assay are a minimum requirement. Work is ongoing to cross-reference our data with results from other assays.
Similar methodology to review the LED • Two datasets of in vitro mammalian assay data with positive results, standardised units and a LED stated by the study/author. • These were then compared to the original Ames dataset (n=1,222) with the calculated minimum LED.
Mammalian genotoxicity assay LED in vitro There are 81 substances in all three datasets, the box plots shows the data distribution. Mouse Lymphoma Mean = 76 µg/ml Assay (MLA) Chromosome Aberration and Micronucleus test Mean = 213 µg/ml Ames Mean = 69 µg/ml test 150 50 100 200 250 0 Minimum lowest effective concentration µg/ml The Ames test was the most sensitive based on the minimum LED (on average).
Transgenic rodent (TGR) • 62 substances in the Ames dataset (n=1,222) had TGR data. • Some of these Ames results were below the threshold calculated by Rainer et al ., 2018 methodology. Using 2.7 mL agar: TGR + TGR - < 10 ppb Threshold 13 *(93%) 1 Ames Positive > 10 ppb Threshold 32 *(67%) 16 * Percentage of substances TGR +, compared to TGR -
Conclusion - How low can you go? • Is the conventional Ames test sufficiently sensitive to detect toxicity at very low doses? o The Ames test can detect potent mutagens. • Is this sufficient for the safety studies of food contact materials? o The FCM industry could incorporate the Ames test and Ames test data to detect and/or confirm potential mutagenicity safety hazards. o The results of this study could assist with that decision making.
Conclusion - How low can you go? • Which in vitro mammalian genotoxicity assays are required for food safety assessment? o The MLA has been shown to have a similar average LED to the Ames test. o The MLA could be used to assess genotoxicity of different types of substance that the Ames test did not detect. • How does this relate to the most potent in vivo genotoxic substances? o The Ames positive results did correlate with the TGR positive results. 93% of substances below the theoretical threshold were also TGR positive.
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