How low can you go? An analysis of the lowest effective dose in the - - PowerPoint PPT Presentation

How low can you go?

An analysis of the lowest effective dose in the Ames test

Senior Database Scientist grace.kocks@lhasalmited.org Grace Kocks

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

Food contact materials (FCM)

Food contact materials are materials that are intended to be in contact with food.

• Multiple types in 1 product

Intentionally added substances (IAS)

Substances that are essential in the manufacturing process, these substances give the FCM its desired properties.

• Antioxidants
• Fillers
• Additives

Non-intentionally added substances (NIAS)

NIAS are chemicals that have not been added for a technical reason during the production process. Often unknown & unintentional materials.

• Impurities from IAS components
• Oligomers
• Degradation products

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?

Exposure threshold 10 µg/kg limit

Experimental approach to address the challenge

Perform extraction and migration tests

EU plastics guidance on FCM OR

Concentrate

Hazard identification 10µg/kg limit and threshold of toxicological concern (TTC) 90 µg/person.

Genotoxicity in vitro e.g. Ames test Detection of NIAS(s), possible quantification. Structural determination is unlikely. 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?

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.

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 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

• n

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

Rainer B. 2018. Suitability of the Ames test to characterise genotoxicity of food contact material migrates. Food Additives & Contaminants: Part A 35:2230-2243.

Converts units to µg/ml µg/plate 2.7 (top agar) 0.4 mg/L threshold limit

Limit of detection of 10 ppb (0.01 mg/L), and a 40-fold concentration factor proposed for migrations in the Ames test, this gives a threshold of 0.4 mg/L. Accounts for the dilution in the top agar of the plate.

CAS number Minimum µg/plate Minimum mg/L Below threshold 100-00-5 333.3 14.68 100-01-6 333 14.67 100-02-7 333 14.67 100-14-1 5 0.22 <= 0.4 mg/L

In line with Rainer et al., where they reported 10% of genotoxic FCMs being detected at 10ppb threshold (using a smaller dataset, n=40). Using the same methodology with

• ur larger

dataset, n=1,222: 11.3% (138/1,222)

• f substances in the dataset

would be expected to be positive.

The story won’t end with just the Ames test

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.

In vitro mammalian assay Ames test Concentrated migrate could be? OR

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.

Ames test

Chromosome Aberration and Micronucleus test

Mouse Lymphoma Assay (MLA) 50 100 200 150 250

Minimum lowest effective concentration µg/ml

Mean = 69 µg/ml Mean = 76 µg/ml Mean = 213 µg/ml The Ames test was the most sensitive based on the minimum LED (on average).

Mammalian genotoxicity assay LED in vitro

There are 81 substances in all three datasets, the box plots shows the data distribution.

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 - Ames Positive < 10 ppb Threshold 13 *(93%) 1 > 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?

• The Ames test can detect potent mutagens.
• Is this sufficient for the safety studies of food contact

materials?

• The FCM industry could incorporate the Ames test and Ames

test data to detect and/or confirm potential mutagenicity safety hazards.

• 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?

• The MLA has been shown to have a similar average LED to

the Ames test.

• 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?

• 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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Thank you

• Matthew Tate
• Paul Rawlinson
• Richard Williams