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How low can you go?
An analysis of the lowest
effective dose in the Ames test
Senior Database Scientist
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
threshold10 µg/kg limit
Experimental approach to address the challenge
Perform extraction and
migration testsEU 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 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
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
our larger
dataset, n=1,222:
11.3% (138/1,222) of 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)
0 50 100 200150 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?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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Thank you
- Matthew Tate
- Paul Rawlinson
- Richard Williams
